JP2005532064A - HCV fusion protein with modified NS3 domain - Google Patents

Abstract

The present invention provides an HCV fusion protein comprising a mutant NS3 protease domain fused to at least one other HCV epitope derived from another region of the HCV polypeptide. This fusion can be used in methods of stimulating a cellular immune response against HCV, such as activation of hepatitis C virus (HCV) specific T cells (including CD4 ⁺ T cells and CD8 ⁺ T cells). . This method can be used in model systems for developing HCV-specific immunogenic compositions and model systems for immunizing mammals against HCV.

Description

(Technical field)
The present invention relates to hepatitis C virus (HCV) constructs. More specifically, the present invention relates to an HCV fusion protein having a modified NS3 domain. The protein can stimulate a cell-mediated immune response, for example, to prime HCV-specific T cells and / or to activate HCV-specific T cells.

(Background of the Invention)
Hepatitis C virus (HCV) infection is a serious health problem, with approximately 1% of the world's population infected with this virus. Over 75% of acutely infected individuals can eventually progress to a chronic carrier state, resulting in cirrhosis, liver failure, and hepatocellular carcinoma. Alter et al. (1992) N. et al. Engl. J. et al. Med. 327: 1899-1905; Resnick and Koff. (1993) Arch. Intern. Med. 153: 1672-1677; Seeeff (1995) Gastrointest. Dis. 6: 20-27; Tong et al. (1995) N.R. Engl. J. et al. Med. 332: 1463-1466.

  HCV was first identified and characterized by Houghton et al. As the cause of NANBH. The viral genome sequence of HCV is known, and methods for obtaining this sequence are also known. See, for example, International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a single-stranded RNA genome that is 9.5 kb positive-sense and is a member of the Flavividae family of viruses. At least six distinct but related HCV genotypes have been identified based on phylogenetic analysis (Simmonds et al., J. Gen. Virol. (1993) 74: 2391-2399). This virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Science (1989) 244: 359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88. Han et al., Proc. Natl. Acad. Sci. USA (1991) 88: 1711-1715). This polyprotein is processed into both structural and non-structural (NS) proteins simultaneously with and after translation.

In particular, as shown in FIG. 1, some proteins are encoded by the HCV genome. Order and nomenclature of the cleavage products of the HCV polyprotein is as _{follows: NH 2 -C-E1-E2} -p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. The initial cleavage of the polyprotein is catalyzed by the host protease, three structural proteins (N-terminal nucleocapsid protein (referred to as “core”) and two envelope glycoproteins (“E1” (also known as E) and “ E2 "(also known as E2 / NS1)), as well as nonstructural (NS) proteins including viral enzymes. NS regions are referred to as NS2, NS3, NS4, and NS5. NS2 is a membrane-bound protein with proteolytic activity, and together with NS3 cleaves NS2-NS3 cissle bond and then generates NS3 N-terminus, and both serine protease activity and RNA helicase activity Releases large polyproteins containing NS3 protease serves to process the remaining polyproteins. In these reactions, NS3 releases the NS3 cofactor (NS4a), the two proteins (NS4b and NS5a), and the RNA-dependent RNA polymerase (NS5b). Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction catalyzed by the NS3 serine protease.

  Despite extensive progress in the development of medicines for specific viruses such as HIV, success in controlling acute and chronic HCV infections is limited (Hoofnagle and di Bisceglie (1997) N. Engl. Med. 336: 347-356). In particular, the generation of a cellular immune response (eg, a strong cytotoxic T lymphocyte (CTL) response) is considered important for the control and eradication of HCV infection. Accordingly, there is a need in the art for an effective method of stimulating a cellular immune response against HCV.

(Summary of the Invention)
It is an object of the present invention to provide reagents and methods for stimulating a cellular immune response against HCV (eg, pre-stimulating and / or activating T cells that recognize epitopes of HCV polypeptides). is there. This and other objects of the invention are provided by one or more embodiments described below.

  The present invention provides HCV fusion proteins useful for stimulating such responses. One embodiment of the present invention relates to an HCV fusion protein comprising an NS3 polypeptide that has been modified to inhibit protease activity and consequently inhibit fusion cleavage. This fusion protein includes, in addition to the modified NS3 polypeptide, one or more polypeptides derived from other regions of the HCV polyprotein described in detail below. These polypeptides are derived from the same HCV isolate as the NS3 polypeptide, or different strains and isolates (any variety of HCV) to provide increased protection against a wide range of HCV genotypes. Including isolates having a genotype).

  In certain embodiments, the NS3 modification comprises an amino acid substitution corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the full-length HCV-1 polyprotein.

  In further embodiments, the protein comprises a modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide, and optionally a core polypeptide.

  In further embodiments, the protein further comprises an NS5b polypeptide and optionally a core polypeptide.

  In still further embodiments, the protein further comprises an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core polypeptide.

  In further embodiments, the protein further comprises an El polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, and optionally a core polypeptide.

  In further embodiments, the protein further comprises an E2 polypeptide and optionally a core polypeptide.

  In still 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 further embodiments, the protein comprises an El polypeptide, an E2 polypeptide, a modified NS3 polypeptide, and optionally a core polypeptide.

  Another embodiment provides a fusion protein consisting essentially of an HCV modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide, and optionally a core polypeptide. In certain embodiments, NS5b polypeptides are also present.

  In the above embodiments, the various regions in the fusion protein need not be in the order in which they occur naturally in the native HCV polyprotein. Thus, for example, the core polypeptide, if present, can be the N-terminus and / or C-terminus of this fusion.

In still further embodiments, the invention consists essentially of from the amino terminus to the carboxy terminus:
(A) a modified NS3 comprising substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the full length HCV-1 polyprotein so that protease activity is inhibited A polypeptide, NS4 polypeptide, and NS5a polypeptide;
(B) Modified NS3 comprising substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the full-length HCV-1 polyprotein so that protease activity is inhibited A polypeptide, NS4 polypeptide, NS5a polypeptide and NS5b polypeptide;
(C) substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, and NS5a polypeptides that result in inhibition of protease activity;
(D) of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165 numbered with respect to E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, and NS5a polypeptides that contain substitutions that result in inhibition of protease activity;
(E) substitutions of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, NS5a polypeptides and NS5b polypeptides that result in inhibition of protease activity;
(F) amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, NS5a polypeptides and NS5b polypeptides, wherein the protease activity is inhibited;
(G) including substitutions of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the E2 polypeptide, and full length HCV-1 polyprotein, resulting in inhibition of protease activity A modified NS3 polypeptide;
(H) substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for E1 polypeptide, E2 polypeptide, and full-length HCV-1 polyprotein, and results A modified NS3 polypeptide that inhibits protease activity;
(I) substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for E2 polypeptide, p7 polypeptide, NS2 polypeptide, and full-length HCV-1 polyprotein. Or a modified NS3 polypeptide that results in inhibition of protease activity; or (j) numbered with respect to E1, E2, E7, p7, NS2, and full-length HCV-1 polypeptide A modified NS3 polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105, and / or Ser-1165, such that protease activity is inhibited;
An immunogenic fusion protein consisting of

In another embodiment, the invention consists essentially of the following from the amino terminus to the carboxy terminus:
(A) a modified NS3 comprising substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the full length HCV-1 polyprotein so that protease activity is inhibited A polypeptide, NS4 polypeptide, NS5a polypeptide, and core polypeptide;
(B) Modified NS3 comprising substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the full-length HCV-1 polyprotein so that protease activity is inhibited A polypeptide, NS4 polypeptide, NS5a polypeptide, NS5b polypeptide and core polypeptide;
(C) substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, NS5a polypeptides and core polypeptides, resulting in inhibition of protease activity;
(D) amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, NS5a polypeptides and core polypeptides, which contain a substitution of
(E) substitutions of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, NS5a polypeptides, NS5b polypeptides and core polypeptides, which result in inhibition of protease activity;
(F) amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein Modified NS3 polypeptides, NS4 polypeptides, NS5a polypeptides, NS5b polypeptides, and core polypeptides, wherein the protease activity is inhibited, thereby inhibiting protease activity;
(G) including substitutions of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered for the E2 polypeptide, full length HCV-1 polyprotein, resulting in inhibition of protease activity. Modified NS3 polypeptides and core polypeptides;
(H) substitutions of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E1 polypeptide, E2 polypeptide, full length HCV-1 polyprotein, and thus protease A modified NS3 polypeptide whose activity is inhibited, and a core polypeptide;
(I) substitution of amino acids corresponding to His-1083, Asp-1105, and / or Ser-1165 numbered with respect to E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein A modified NS3 polypeptide and a core polypeptide that results in inhibition of protease activity; or (j) a number with respect to an E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, full length HCV-1 polyprotein A modified NS3 polypeptide comprising a substitution of an amino acid corresponding to the attached His-1083, Asp-1105, and / or Ser-1165, such that protease activity is inhibited, and a core polypeptide;
An immunogenic fusion protein consisting of

  In still further embodiments, the invention relates to a modified NS3 polypeptide, which corresponds to His-1083, Asp-1105, and / or Ser-1165 numbered for the full length HCV-1 polyprotein. Including an amino acid substitution so that protease activity is inhibited when this modified NS3 polypeptide is present in an HCV fusion protein.

  Yet another embodiment of the invention provides an isolated polynucleotide encoding any of the proteins detailed above, a recombinant vector comprising the same, a host cell transformed with the vector, and a fusion protein. A method for recombinant production is provided.

  The invention also provides a composition comprising any of these fusion proteins, a polynucleotide encoding these fusions, or a recombinant vector comprising these polynucleotides, and a pharmaceutically acceptable carrier.

  Yet another embodiment of the invention provides a method of stimulating a cellular immune response in a vertebrate subject by administering a composition as described herein. In certain embodiments, the composition primes and / or activates T cells that recognize an epitope of HCV polypeptide. T cells are contacted with a fusion protein comprising a modified NS3 polypeptide and at least one additional HCV polypeptide. Activated T cell populations recognize this NS3 polypeptide and / or additional HCV polypeptides.

  Accordingly, the present invention provides methods and reagents for stimulating a cellular immune response against HCV (eg, pre-stimulating and / or activating T cells that recognize an epitope of HCV polypeptide). These methods and reagents are particularly advantageous for identifying epitopes of HCV polypeptides that are associated with strong CTL responses and for immunizing mammals (including humans) against HCV.

(Detailed description of the invention)
The practice of the present invention uses conventional methods of chemistry, biochemistry, recombinant DNA technology and immunology within the skill of the art, unless otherwise indicated. Such techniques are explained fully in the literature. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition); Methods In Enzymology (Edited by S. Cowick and N. Kaplan, Academic Press, Inc.); I and II (D.N. Glover edition); Oligonucleotide Synthesis (M.J. Gait edition); Nucleic Acid Hybridization (B.D. Hames & S.J.Higgins edition); Animal Cell. Ed.); Perbal, B .; , A Practical Guide to Molecular Cloning.

  As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Must be noted. Thus, for example, reference to “an antigen” includes a mixture of two or more antigens, and the like.

The following amino acid abbreviations are used throughout this specification:
Alanine: Ala (A) Arginine: Arg (R)
Asparagine: Asn (N) 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 (W)
Tyrosine: Tyr (Y) Valine: Val (V)
(I. Definition)
In describing the present invention, the following terms are used 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 product length. Thus, peptides, oligopeptides, dimers, multimers, etc. are encompassed by this definition. Both full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of the polypeptide (eg, glucosylation, acetylation, phosphorylation, etc.). Furthermore, for the purposes of the present invention, a “polypeptide” is a modification (eg, deletion, addition and substitution (naturally conservative in nature)) to a native sequence as long as the protein maintains the desired activity. A protein containing These modifications may be intentional, such as through site-directed mutagenesis, or may be incidental, such as through errors due to mutations in the host producing this protein or PCR amplification. .

  An HCV polypeptide is a polypeptide derived from an HCV polyprotein as described above. The polypeptide need not be physically derived from HCV, but can be produced synthetically or recombinantly. In addition, the polypeptide is isolated from various HCV strains and isolates (eg, isolates having the six genotypes of HCV described in Simmonds et al., J. Gen. Virol. (1993) 74: 2391-1399). Derived from any of the newly identified isolates and subtypes of these isolates (eg, HCV1a, HCV1b, etc.) obtain. Many conserved and variable regions are known between these strains, and in general, the amino acid sequences of epitopes derived from these regions have a high degree of sequence homology (e.g., when the two sequences are aligned) , Greater than 30%, preferably greater than 40% amino acid sequence homology). Thus, for example, the term “NS4” polypeptide refers to native NS4 from any of a variety of HCV strains, as well as NS4 analogs, muteins and immunogenic fragments as further defined below.

  The terms “analog” and “mutein” refer to biologically active derivatives of reference molecules or fragments of such derivatives, such as the ability to stimulate a cell-mediated immune response as defined below. Maintain desired activity. In the case of modified NS3, “analog” or “mutein” refers to an NS3 molecule that lacks its native proteolytic activity. In general, the term “analog” refers to one or more amino acid additions, substitutions (generally active in the case of NS3 that are conservative or modified in nature) to the native molecule, unless the modification destroys the immunogenic activity. A compound having a native polypeptide sequence and structure with a proteolytic active site (non-conservative in nature) and / or having a deletion. The term “mutein” refers to peptides having one or more peptidomimetics (“peptoids”), for example, as described in International Publication No. WO 91/04282. Preferably, the analog or mutein has at least the same immune activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are further described below.

  As explained above, analogs generally include substitutions that are conservative in nature (ie, those substitutions that occur within a family of amino acids that are related in their side chains). Specifically, amino acids are generally classified into four families: (1) acidic—aspartic acid and glutamic acid; (2) basic—lysine, arginine, histidine; (3) nonpolar—alanine. , Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged and polar--glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are often classified as aromatic amino acids. For example, leucine isoleucine or valine, aspartic acid glutamic acid, threonine with serine alone or similar conservative substitution of amino acids structurally related to amino acids is It is reasonably predictable that there will be no major effects. For example, the polypeptide of interest can have up to about 5-10 conservative or non-conservative amino acid substitutions, or up to about 15-25 conservative amino acid substitutions, or so long as the desired function of the molecule remains intact. It may comprise non-conservative amino acid substitutions, or any integer between 5 and 25 conservative amino acid substitutions or non-conservative amino acid substitutions. One skilled in the art can readily determine the region of the molecule of interest that can tolerate changes by referring to Hopp / Woods and Kyte-Doolittle plots well known in the art.

  “Modified NS3” means an NS3 polypeptide having a modification such that the protease activity of the NS3 polypeptide is removed. The modification may include one or more amino acid additions, substitutions (generally non-conservative in nature) and / or deletions relative to the native molecule, removing the protease activity of the NS3 polypeptide. Methods for measuring protease activity are further discussed below.

  By “fragment” is intended a polypeptide consisting of only part of an intact full-length polypeptide sequence and structure. This fragment may contain a C-terminal deletion and / or an N-terminal deletion of the native polypeptide. An “immunogenic fragment” of a particular HCV protein generally has 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 Contains at least about 20-50 or more contiguous amino acid residues of the full-length molecule (which define an epitope), or any integer number of contiguous amino acid residues between the five amino acids and the full-length sequence. Including, however, the fragment of interest retains immunogenic activity as measured by the assays described herein.

  As used herein, the term “epitope” refers to a sequence of at least about 3-5, preferably about 5-10 or 15, and about 1000 amino acids or less (or any integer number of amino acids in between). This defines a sequence that binds to an antibody produced in response to such a sequence, either by itself or as part of a larger sequence. There is no critical upper limit on the length of the fragment, and this fragment may comprise a nearly full-length protein sequence or even a fusion protein comprising two or more epitopes derived from HCV polyprotein. Epitopes for use in the present invention are not limited to polypeptides having the exact sequence of the portion of the parent protein from which they are derived. In fact, the viral genome contains several variable regions that are in a constant flux and show a relatively high degree of variability between isolates. Thus, the term “epitope” encompasses sequences identical to the native sequence, as well as modifications to the native sequence (eg, deletions, additions and substitutions (generally conservative in nature)).

  A region of a given polypeptide containing an epitope can be identified using any number of epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66 (Edited by Glenn E. Morris, 1996) Humana Press, Totowa, New Jersey. For example, a linear epitope can be synthesized, for example, by simultaneously synthesizing a number of peptides (these peptides correspond to a portion of a protein molecule) on a solid support and still attaching these peptides to the solid support. Can be determined by reacting these peptides with antibodies. Such techniques are known in the art and are described below: see, eg, US Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; Geysen et al. (1986) Molec. Immunol. 23: 709-715. Similarly, conformational epitopes are readily determined by determining the spatial conformation of amino acids, eg, by X-ray crystallography and two-dimensional nuclear magnetic resonance. For example, see Epitope Mapping Protocols (supra). The antigenic region of the protein is also used using standard antigenic and hydrophobic hydrophilicity index plots (eg, those calculated using the Omega version 1.0 software program available from the Oxford Molecular Group). Can be identified. This computer program includes the Hopp / Woods method for determining antigenicity profiles (Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78: 3824-3828), and Kyte for hydrophobic hydrophilicity index plots. Use the Doolittle technology (Kyte et al., J. Mol. Biol. (1982) 157: 105-132).

  For a description of various HCV epitopes, see, eg, Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89: 1001-10015; Chien et al., J, Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D .; Y. , International Publication No. WO 94/01778; and US Pat. Nos. 6,280,927 and 6,150,087.

  As used herein, the term “T cell epitope” refers to a property of a peptide structure that can induce T cell immunity with respect to the peptide structure or related hapten. T cell epitopes generally include linear peptide determinants that assume an extended conformation within the peptide binding interval of MHC molecules (Unanue et al., Science (1987) 236: 551-557). Conversion of a polypeptide to an MHC II type-related linear peptide determinant (generally 5-14 amino acids long) refers to “antigen processing” performed by antigen presenting cells (APC). More specifically, T cell epitopes are defined by the local properties of the short peptide structure, so that the nature of the main amino acid sequence is secondary, independent of charge and hydrophobicity, and overall polypeptide folding. Includes a specific type of structure (eg, helicity). In addition, short peptides that can be recognized by helper T cells are amphipathic structures that generally include a hydrophobic side (for interaction with MHC molecules) and a hydrophilic side (for interaction with T cell receptors) ( Margalit et al., Computer Prediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. GC Woodrow et al. (See, for example, Spouge et al., J. Immunol. (1987) 138: 204-212; Berkouer et al., J. Immunol. (1986) 136: 2498-2503). The

  Therefore, protein segments containing T cell epitopes can be easily predicted using a number of computer programs (see, eg, Margalit et al., Computer Prediction of T-cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc., ed. See GC Woodrow et al. (1990) pp. 109-116). Such programs generally compare the amino acid sequence of the peptide with a sequence known to induce a T cell response and search for patterns of amino acids that may require a T cell epitope.

  An “immunological response” to an HCV antigen (including both polypeptides and polynucleotides encoding polypeptides expressed in vivo) or humoral immunity of a subject against molecules present in the composition of interest Occurrence in response and / or cellular immune response. For the purposes of the present invention, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is mediated by T lymphocytes and / or other leukocytes. Is. One important aspect of cellular immunity relates to antigen-specific responses by cytolytic T cells (“CTL”). CTLs are present in association with proteins encoded by the major histocompatibility complex (MHC) and have specificity for peptide antigens expressed on the cell surface. CTLs help induce and promote intracellular destruction of intracellular microorganisms or lysis of cells infected with such microorganisms. Both CD8 + T cells and CD4 + T cells can kill HCV infected cells. Another aspect of cellular immunity relates to antigen-specific responses by helper T cells. Helper T cells act to stimulate function on cells that present peptide antigens bound to MHC molecules on their surface and help focus the activity of non-specific effector cells on this cell. . A “cellular immune response” also includes antiviral cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocytes, including those derived from CD4 + T cells and CD8 + T cells. Refers to the production of (including but not limited to IFN-γ and TNF-α).

  A composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of an antigen bound to an MHC molecule on the cell surface. This cell-mediated immune response is directed to or near cells that present the antigen on the surface of the cell. In addition, antigen-specific T lymphocytes can be generated to allow future protection of the immunized host.

  The ability of a particular antigen to stimulate a cell-mediated immunological response can be sensitized by a number of assays (eg, by a lymphocyte proliferation (lymphocyte activation) assay, a CTL cytotoxic cell assay, or by By assaying for T lymphocytes specific for antigen in a subject). Such assays are well known in the art. For example, Erickson et al. Immunol. (1993) 151: 4189-4199; Doe et al., Eur. J. et al. Immunol. (1994) 24: 2369-2376; and the examples below.

  Thus, an immunological response as used herein can stimulate CTL production and / or helper T cell production or activation. The antigen of interest can also elicit an antibody-mediated immune response. Thus, an immunological response can include one or more of the following effects: production of antibodies by B cells; and / or suppressor T cells specifically directed against an antigen present in the composition or vaccine of interest. And / or activation of γδ T cells. These responses neutralize infectivity and / or mediate antibody-complement or antibody-dependent cytotoxicity (ADCC) to provide protection or reduction of symptoms to the immunized host Can work. Such a response can be determined using standard immunoassays and neutralization assays that are well known in the art.

  “Equivalent antigenic determinant” means an antigenic determinant derived from a different subspecies or strain of HCV (eg, derived from HCV strain 1, strain 2 or strain 3), and the antigenic determinant is a sequence variation. Are not equally required due to, but occur in equivalent positions in the HCV sequences in question. In general, the amino acid sequences of equivalent antigenic determinants have a high degree of sequence homology (eg, greater than 30%, usually greater than 40%, greater than 60%, still 80 when the two sequences are aligned. Amino acid sequence homology greater than ~ 90%).

  A “coding sequence” or a sequence that “encodes” a selected polypeptide is transcribed in vitro or in vivo (in the case of DNA) and translated into a polypeptide when placed under the control of appropriate regulatory sequences. It is a nucleic acid molecule (in the case of mRNA). The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and by 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-stranded and single-stranded sequences, including cDNA from viruses, mRNA from prokaryotes or eukaryotes, viruses (eg, DNA viruses and retroviruses). ) Derived genomic DNA sequences or prokaryotic DNA, especially synthetic DNA sequences, but not limited thereto. The term also includes sequences that include any of the known base analogs of DNA and RNA.

  “Operably linked” refers to an arrangement of elements such that the components so described are configured to perform their desired function. Thus, a given promoter operably linked to a coding sequence can result in the expression of the coding sequence if appropriate transcription factors and the like are present. A promoter need not be contiguous with a coding sequence, so long as it functions to direct the expression of the coding sequence. Thus, for example, intervening, untranslated but transcribed sequences can exist between the promoter sequence and the coding sequence (introns can be transcribed), and the promoter sequence can still be “operably linked to the coding sequence. Can be considered.

  As used herein to describe a nucleic acid molecule, a “recombinant” is a genomic, cDNA that is not accompanied by all or part of a naturally associated polynucleotide, depending on its origin or manipulation. Means a polynucleotide of viral, semi-synthetic, or synthetic origin. The term “recombinant” when used in reference 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 the transformed organism as described further below. A host organism expresses a foreign gene under expression conditions to produce a protein.

  “Control element” refers to a polynucleotide sequence that aids in the expression of a coding sequence to which it is linked. The term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, untranslated regions (including 5'-UTR and 3'-UTR), and, where appropriate, leader sequences and enhancers (which are collectively Provides transcription and translation of the coding sequence in the host cell).

  As used herein, a “promoter” is a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3 ′ direction) coding sequence operably linked thereto. It is. For the purposes of the present invention, a promoter sequence contains the minimum number of bases or elements necessary to initiate transcription of the gene of interest at a detectable level above background. The promoter sequence has a transcription initiation site, as well as a protein binding domain (consensus sequence) responsible for binding of RNA polymerase. Eukaryotic promoters often include a "TATA" box and a "CAT" box, but not always.

  The control sequence “directs transcription” of the coding sequence in the cell when RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which is then translated into a polypeptide encoded by the coding sequence. Is done.

  “Expression cassette” or “expression construct” refers to an assembly capable of directing expression of a sequence or gene of interest. This expression cassette contains control elements as described above (eg, a promoter operably linked to the sequence or gene of interest (to direct their transcription) and often also polyadenylation sequences including. In certain embodiments of the invention, the expression cassette described herein can be included in a plasmid construct. In addition to the components of the expression cassette, the plasmid construct also includes at least one selectable marker, a signal that allows the plasmid construct to be present as single stranded DNA (eg, the origin of replication of M13), at least one. It may include multiple cloning sites and “mammalian” origins of replication (eg, SV40 origin of replication or adenovirus origin).

  As used herein, “transformation” refers to the host of an exogenous polynucleotide, regardless of the method used for insertion (eg, transformation by direct uptake, transfection, infection, etc.). Refers to insertion into a cell. See further below for specific methods of transfection. Exogenous polynucleotides can be maintained as non-integrated vectors (eg, episomes) or can be integrated into the host genome.

  A “host cell” is a cell that has been transformed or can be transformed by an exogenous DNA sequence.

  “Isolated” when referring to a polypeptide is that the indicated molecule is separated from the whole organism in which the molecule is found in nature and is separate or other biological species of the same type. Means present in the substantial absence of the molecule. With respect to a polynucleotide, the term “isolated” refers to a nucleic acid molecule that lacks all or part of the sequence normally associated with the polynucleotide in nature; or has a heterologous sequence that exists in nature but is associated with that sequence. Sequence; or a molecule dissociated from a chromosome.

  The term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably at least 95% by weight, most preferably at least 98% by weight. % Of the same type of biological macromolecules.

  “Homology” refers to the percent identity between two polynucleotide portions or two polypeptide portions. Two DNA or two polypeptide sequences, wherein these sequences are at least about 50%, preferably at least about 75%, more preferably at least about 80% to 85%, preferably over a molecule of defined length They are “substantially homologous” to each other if they exhibit at least about 90%, and most preferably at least about 95% to 98% or more sequence identity. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a particular DNA or polypeptide sequence.

  In general, “identity” refers to the exact nucleotide-nucleotide correspondence or amino acid-amino acid correspondence of two polynucleotide sequences or polypeptide sequences, respectively. The percent identity is 2 by aligning their 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. It can be determined by direct comparison of sequence information between two molecules. Easily available computer programs (eg, ALIGN, Dayhoff, M.O., Atlas of Protein Sequence and Structure M. O. Dayhoff, Addendum 5, 3: 353-358, National biomedical Research, DC This fits the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2: 482-489, 1981) for peptide analysis)) can be used to aid the analysis. Programs for determining nucleotide sequence identity, such as the BESTFIT, FASTA, and GAP programs (which also depend on the Smith and Waterman algorithms), are available from the Wisconsin Sequence Analysis Package, Version 8 (Genetics Computer Group, Available from WI). These programs are readily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referenced above. For example, the percent identity of a particular nucleotide sequence relative to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and gap penalty of 6 nucleotide positions.

  Another method of establishing percent identity in the context of the present invention is copyrighted by the University of Edinburgh, and John F. Collins and Shane S. Developed by Sturrok, IntelliGenetics, Inc. Using the MPSRCH package of the program distributed by (Mountain View, CA). From this set of packages, the Smith-Waterman algorithm can be used, where the default parameters are used for the scoring table (eg, 12 gap open penalties, 1 gap extension penalties, and 6 gap). From the generated data, the “match” value reflects “sequence identity”. Other suitable programs for calculating percent identity or percent homology between sequences are generally known in the art, for example, another alignment program is BLAST used with default parameters. is there. For example, BLASTN and BLASTP can be used using the following default parameters: gene code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; (Descriptions) = 50 sequences; Sort = HIGH SCORE; Database (Database) = no degeneracy, 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 is the hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand specific nuclease, and determination of the size of the digested fragment. Can be determined by DNA sequences that are substantially homologous can be identified in Southern hybridization experiments, eg, under stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, Sambrook et al., Supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

  By “nucleic acid immunization” is meant the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell for expression of the immunogen (s) in vivo. Nucleic acid molecules can be introduced directly into a recipient subject, for example, by infusion administration, inhalation administration, oral administration, intranasal administration, and mucosal administration, or in cells removed from the host ex vivo. Can be introduced. In the latter case, the transformed cells can be reintroduced into the subject and initiate an immune response against the antigen encoded by the nucleic acid molecule in the subject.

  As used herein, “treatment” refers to (i) prevention of infection or reinfection, as in traditional vaccines, (ii) reduction or elimination of symptoms, and (iii) of the pathogen in question. Refers to either substantial removal or complete removal. Treatment can be achieved either prophylactic (pre-infection) or therapeutic (post-infection).

  "Vertebrate subject" means any member of the Cordata subgenus, including but not limited to: humans and other primates (chimpanzees and other ape species and monkeys) (Including non-human primates such as monkey species); farm animals (eg, cattle, sheep, pigs, goats and horses); domestic animals (eg, dogs and cats); laboratory animals (mouse, Including rodents such as rats and guinea pigs); birds (including domestic, wild and hunting birds such as chickens, turkeys and other poultry, ducks, geese, etc.). The term does not denote a particular age. Thus, it is intended to cover both adult and newborn individuals. Since all these vertebrate immune systems operate similarly, the invention described herein is intended for use in any of the above vertebrate species.

(II. Mode for carrying out the invention)
Before describing the present invention in detail, it should be understood that the present invention is of course not limited to any particular formulation or process parameter, as the specific formulation and process parameters may vary. is there. 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.

  Numerous compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, and preferred materials and methods are described herein.

  The present invention relates to fusion proteins and polynucleotides encoding them, which include a modified NS3 polypeptide and at least one other HCV polypeptide derived from an HCV polyprotein. The fusion proteins of the invention may be used to stimulate cellular immune responses (eg, to activate HCV-specific T cells (ie, T cells that recognize epitopes of these polypeptides) and / or to helper T cells. To induce production and / or to stimulate production of antiviral cytokines, chemokines, etc.). Activation of HCV-specific T cells by such a fusion protein will allow both in vitro and in vivo model systems for the development of HCV vaccines (especially for identifying HCV polypeptide epitopes associated with responses). provide. The fusion protein also generates an immune response (eg, a CTL response) against HCV in a mammal and / or primes CD8 + T cells and CD4 + T cells to either anti-viral agents for either therapeutic or prophylactic purposes Can be used to make.

  For a further understanding of the present invention, further detailed discussion is provided below with respect to fusion proteins for use in the compositions of the present invention, as well as production of proteins, compositions containing them and methods of using the proteins. The

(Fusion protein)
The genome of the HCV strain contains a single open reading frame of about 9,000 to 12,000 nucleotides, which is transcribed into a polyprotein. As shown in FIG. 1 and Table 1, HCV polyproteins, upon cleavage, are at least in the order NH ₂ -core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. 10 different products are produced. This core polypeptide is located at positions 1 to 191 numbered for HCV-1 (for the HCV-1 genome, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451- 2455). This polypeptide is further processed to produce an HCV polypeptide comprising about amino acids 1 to 173. The envelope polypeptides (E1 and E2) are present at about positions 192 to 383 and about 384 to 746, respectively. The P7 domain is found from about position 747 to about position 809. NS2 is a complete membrane protein with proteolytic properties and is found at about positions 810 to about 1026 of this polyprotein. NS2 either alone or in combination with NS3 (found at about position 1027 to about position 1657) cleaves NS2-NS3 single bond and then generates NS3 N-terminus And release large polyproteins containing both serine protease activity and RNA helicase activity. This NS3 protease (found at about position 1027 to about position 1207) serves to process the rest of the polyprotein. Helicase activity is found at about position 1193 to about position 1657. NS3 is an NS3 cofactor (NS4a found in about 1658 to about 1711), two proteins (NS4b found in about 1712 to about 1972, and NS5a found in about 1973 to about 2420) As well as RNA-dependent RNA polymerase (NS5b found at about position 2421 to about position 3011). Completion of polyprotein maturation begins with autocatalytic cleavage of NS3-NS4a binding, catalyzed by NS3 serine protease.

^＊ＨＣＶ−１に対して番号付けられている。Ｃｈｏｏら（１９９１）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ８８：２４５１−２４５５を参照のこと。

^* Numbered for HCV-1. Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451-2455.

  The fusion proteins of the invention include NS3 polypeptides that have been modified to inhibit protease activity, such that further cleavage of the fusion is inhibited. NS3 polypeptides can be modified by deletion of all or part of the NS3 protease domain. Alternatively, proteolytic activity can be inhibited by substitution of amino acids in the active region of the protease domain. Finally, the addition of amino acids to the active region of this domain such that the catalytic site is modified also serves to inhibit proteolytic activity.

  As explained above, this protease activity is found around amino acid positions 1027-1207 and is a full-length HCV-1 polyprotein (Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88: 2451. (See 2455), numbered at position 2-182 in FIG. 2 (SEQ ID NO: 4). The structure and active site of NS3 protease are known. See, for example, De Francesco et al., Antivir. Ther. (1998) 3: 99-109; Koch et al., Biochemistry (2001) 40: 631-640. Thus, native sequence deletions and modifications typically occur at or near the active site of the molecule. In particular, it modifies one or more amino acids present at position 1-182 or 2-182, preferably 1-170 or 2-170 or 1-155 or 2-155 in FIG. 2 (SEQ ID NO: 4). It is desirable to create a deletion. Preferred modifications are to the catalytic triad, ie H, D and / or S residues at the active site of the protease in order to inactivate the protease. These residues are present at positions 1083, 1105 and 1165, respectively, and are numbered relative to the full length HCV polyprotein (positions 58, 80 and 140 in FIG. 2 (SEQ ID NO: 4)). Such modifications suppress proteolytic cleavage while retaining T cell epitopes.

  One skilled in the art can readily determine the portion of the NS3 protease that should be deleted to interfere with activity. The presence or absence of activity can be determined using methods known to those skilled in the art.

  For example, protease activity or its deletion can be determined using the procedures described at the bottom of the Examples, as well as using assays well known in the art. For example, Takeshita et al., Anal. Biochem. (1997) 247: 242-246; Kakiuchi et al., J. Biol. Biochem. (1997) 122: 749-755; Sai et al., Biochemistry (1998) 37: 3392-3401; Cho et al., J. Biol. Virol. Meth. (1988) 72: 109-115; Cerretani et al., Anal. Biochem. (1999) 266: 192-197; Zhang et al., Anal. Biochem. (1999) 270: 268-275; Kakiuchi et al., J. Biol. Virol. Meth. (1999) 80: 77-84; Fowler et al., J. Biol. Biomolp. Screen. (2000) 5: 153-158; and Kim et al., Anal. Biochem. (2000) 284: 42-48.

  The fusion proteins of the invention include one or more polypeptides from one or more other regions of the HCV polyprotein in addition to the modified NS3 polypeptide. In practice, the fusion may include all parts of the HCV polyprotein. These polypeptides can be derived from the same HCV isolate as the NS3 polypeptide or from isolates including isolates with different strains or any of a variety of HCV genotypes, with a wide range of HCV genotypes Increase protection against. In addition, polypeptides can be selected based on a particular viral clade edema that is unique in a particular geographic region, where a vaccine composition comprising the fusion is used. It is readily apparent that the fusions of the present invention provide an effective means of treating HCV infection in a broad context.

In certain embodiments, the fusion protein comprises a modified NS3 (also referred to herein as NS3 ^* ), NS4 (NS4a and NS4b), NS5a, and optionally an HCV core polypeptide (NS3 ^* NS4NS5a Or NS3 ^* NS4NS5aCore fusion protein, also referred to herein as “NS3 ^* 45a” and “NS3 ^* 45aCore”). These regions need not be in the order they naturally occur in the native HCV polyprotein. Thus, for example, the core polypeptide can be the N-terminus and / or C-terminus of the fusion.

Another embodiment is NS3 ^* , NS4, NS5a, NS5b and optionally the core polypeptide of HCV (NS3 ^* NS4NS5aNS5b or NS3 ^* NS4 ^* NS5bCore fusion proteins, herein referred to as “NS3 ^* 45ab” and “ NS3 ^* 45abCore ") is also provided. These regions need not be in the order in which they occur naturally in the native HCV polyprotein. Thus, for example, the core polypeptide can be the N-terminus and / or C-terminus of the fusion.

Yet another embodiment is NS3 ^* combined with NS2, NS3 ^* combined with NS2, p7 and E1, NS3 ^* combined with NS2, p7 and NS3 ^* combined with E1 and E2, NS3 ^* , E2 combined with E1 and NS3 * combined, relates to a fusion protein comprising an NS3 * combined with ^E1 and E2 ^(or may not have have all of these core polypeptide). As with the fusion above, these regions need not be in the order in which they occur in nature. In addition, each of these regions can be derived from the same HCV isolate or a different HCV isolate.

  FIG. 3 (SEQ ID NOs: 5-6) shows a typical modified fusion protein, which lacks the NS3 protease domain from the N-terminus and contains amino acids 1-121 of the core on the C-terminus. Including.

  The various HCV polypeptides present in the various fusions described above can be either full-length polypeptides or portions thereof. The portion of the HCV polypeptide that constitutes the fusion protein contains at least one epitope, which is recognized by the T cell receptor on activated T cells, eg, 2152-HEYPVGSQL-2160 (SEQ ID NO: 1) and / or Or 2224-AELIEANLLWRQEMG-2238 (SEQ ID NO: 2). The epitopes of NS2, p7, E1, E2, NS3, NS4 (NS4a and NS4b), NS5a, NS5b, NS3NS4NS5a, and NS3NS4NS5aNS5b can be identified by various methods. For example, individual polypeptides or fusion proteins comprising any combination of the above can be isolated, for example, by immunoaffinity purification using monoclonal antibodies against the polypeptides or proteins. The isolated protein sequence can then be screened by preparing a series of short peptides by proteolytic cleavage of the purified protein over the entire protein sequence. For example, by starting with a 100-mer polypeptide, each polypeptide can be tested for the presence of an epitope recognized by a T cell receptor on HCV activated T cells, and progressively smaller and overlapping fragments are then Can be tested from the identified 100-mer to map the epitope of interest.

Epitopes recognized by T cell receptors on HCV activated T cells can be identified, for example, by ⁵¹ Cr release assay (eg, Example 4) or by lymphoproliferation assay (eg, Example 6). ^{In a 51} Cr release assay, a target cell can be obtained by cloning a polypeptide encoding the epitope into an expression vector, and transforming the expression vector into the target cell, so that the target cell displaying the epitope of interest is Can be built. HCV-specific CD8 ⁺ T cells lyse target cells that represent one or more epitopes from one or more regions of the HCV polyprotein found in, for example, a fusion, and cells that do not display such epitopes. Does not dissolve. In a lymphoproliferation assay, HCV activated CD4 ⁺ T cells proliferate if cultured with one or more epitopes, eg, from one or more regions of HCV polyprotein found in the fusion, but not HCV epitopes. It does not grow in the absence of sex peptides.

  The various HCV polypeptides can be present in any order in the fusion protein. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of the one or more polypeptides may be present in the fusion protein. There are multiple HCV virus strains and any of these strains of HCV polypeptides can be used in the fusion protein.

  Nucleic acid and amino acid sequences of many HCV strains and isolates (nucleic acid sequences of various regions of the HCV polyprotein, including core, NS2, p7, E1, E2, NS3, NS4, NS5a, NS5b genes and polypeptides And amino acid sequence) were determined. For example, isolated HCV J1.1 is described in Kubo et al. (1989) Japan. Nucl. Acids Res. 17: 10367-10372; Takeuchi et al. (1990) Gene 91: 287-291; Takeuchi et al. (1990) J. MoI. Gen. Virol. 71: 3072-3033; and Takeuchi et al. (1990) Nucl. Acids Res. 18: 4626. The complete coding sequences of two independent isolates, HCV-J and BK are described in Kato et al. (1990) Proc. Natl. Acad. Sci. USA 87: 9524-9528 and Takamizawa et al. (1991) J. MoI. Virol. 65: 1105-1113, respectively.

  Publications describing HCV-1 isolates include Choo et al. (1990) Brit. Med. Bull. 46: 423-441; Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2451-1455 and Han et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1711-1715. HCV isolates HC-J1 and HC-J4 have been described by Okamoto et al. (1991) Japan J. MoI. Exp. Med. 60: 167-177. HCV isolates HCT18-, HCT23, Th, HCT27, EC1 and EC10 are described in Weiner et al. (1991) Virol. 180: 842-848. HCV isolates Pt-1, HCV-K1 and HCV-K2 were obtained from Enomoto et al. (1990) Biochem. Biophys. Res. Commun. 170: 1021-1025. HCV isolates A, C, D, and E are described in Tsukiyama-Kohara et al. (1991) Virus Genes 5: 243-254.

  Each component of the fusion protein can be obtained from the same HCV strain or isolate or from different HCV strains or isolates. Fusion proteins, including HCV polypeptides, eg, derived from NS3 polypeptide, can be obtained from a first strain of HCV, and other HCV polypeptides present can be obtained from a second strain of HCV. . Alternatively, one or more other HCV polypeptides (eg, NS2, NS4, core, p7, E1 and / or E2 if present) can be obtained from a first strain of HCV and the remaining HCV polypeptide Can be obtained from a second strain of HCV. Furthermore, each HCV polypeptide present can be obtained from a different HCV strain.

  As explained above, it may be desirable to include a polypeptide obtained from the core region of the HCV polyprotein in the fusion of the invention. This region is present at amino acid positions 1-191 of the HCV polyprotein, numbered relative to HCV-1. Full length protein, fragments thereof (eg amino acids 1-160, eg amino acids 1-150, 1-140, 1-130, 1-120, eg amino acids 1-121, 1-122, 1-123 ... 1. ˜151), or a smaller fragment containing an epitope of this full length protein (eg, amino acids 10-53, amino acids 10-45, amino acids 67-88, amino acids 120-130, or eg Houghton et al., US Pat. No. 5, , 350, 671; Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89: 1001-10015; Number WO93 / 00365; C ien, DY, International Publication No. WO 94/01778; and US Pat. Nos. 6,280,927 and 6,150,087, epitopes found between any core epitopes) It can be used in the fusion of the present invention. In addition, proteins resulting from a frameshift of the core region of the polyprotein (eg, as described in International Publication No. WO 99/63941) can be used.

  If a core polypeptide is present, it can be at the N-terminus, C-terminus and / or within the fusion. Particularly preferred are core polypeptides on the C-terminus that allow formation of complexes with certain adjuvants (eg, ISCOM, further described below).

  As described above, polypeptides useful in HCV fusions include T cell epitopes derived from any of a variety of regions of the polyprotein. In this regard, E1, E2, p7 and NS2 contain one or more human cell epitopes (both CD4 + and CD8 +) that serve to increase vaccine efficacy and increase the level of protection against multiple HCV genotypes. It is known to contain these epitopes. Furthermore, multiple copies of specific, conserved T cell epitopes can also be used in fusions (eg, a composite of epitopes from different genotypes).

  For example, polypeptides from the HCV E1 and / or E2 region can be used in the fusions of the invention. E2 is a multi-species species (Spaete et al., Virol. (1992) 188: 819-830; Selby et al., J. Virol. (1996) 70: 5177-5182; Grakoui et al., J. Virol. (1993) 67: 1385. -1395; Tomei et al., J. Virol. (1993) 67: 4017-4026), and clipping and proteolysis can occur at the N-terminus and C-terminus of the E2 polypeptide. Accordingly, E2 polypeptides for use herein are amino acids 405 to 661 (eg, 400, 401, 402... 661) of the HCV polyprotein, numbered relative to the full length HCV-1 polyprotein. (E.g., 383 or 384 to 661, 383 or 384 to 715, 383 or 384 to 746, 383 or 384 to 749, or 383 or 384 to 809 or 383 or any C-terminus between 384 and 661 to 809) May be included. Similarly, E1 polypeptides for use herein are amino acids 192 to 326, 192 to 330, 192 to 333, 192 to 360, 192 to 363, 192 to 383 or 192 to 326 to HCV polyprotein. Any C-terminus between 383 may be included.

  Immunogenic fragments of E1 and / or E2 that contain an epitope can be used in the fusions of the invention. For example, a fragment of an E1 polypeptide can be about 5 to about the full length of a molecule (eg, 6, 10, 25, 50, 75, 100, 125, 150, 175, 185 or more amino acids of an E1 polypeptide) or stated. Any integer between the given numbers can be included. Similarly, an E2 polypeptide fragment may comprise 6, 10, 25, 50, 75, 100, 150, 200, 250, 300 or 350 amino acids of the E2 polypeptide or any integer between the stated numbers. .

  For example, an epitope derived from the hypervariable region of E2 (such as a region spanning amino acids 384-410 or 390-410) can be included in the fusion. A particularly effective E2 epitope incorporated into the E2 polypeptide sequence is a consensus sequence derived from this region (for example, the consensus sequence Gly-Ser-Ala-Ala-Arg- representing the consensus sequence of amino acids 390-410 of the HCV type 1 genome). Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-Lys-Gln-Asn (SEQ ID NO: 7)). Additional epitopes E1 and E2 are known and are described, for example, in Chien et al., International Publication No. WO 93/00365.

  Furthermore, the E1 polypeptide and / or E2 polypeptide may lack all or part of the membrane spanning the domain. For E1, most polypeptides that terminate at amino acid positions above about 370 (based on HCV-1 E1 numbering) are retained by the ER and are therefore not secreted into the growth medium. For E2, a polypeptide that terminates at amino acid position 731 and above (which is also based on the numbering of the HCV-1 E2 sequence) is retained by the ER and not secreted. (See, for example, International Publication No. WO 96/04301, published February 15, 1996). It should be noted that these amino acid positions are not critical and can vary to some extent. Accordingly, the present invention lacks all or part of a transmembrane binding domain and a transmembrane binding domain comprising an E1 polypeptide (terminating at about amino acid position 369 or less) and an E2 polypeptide (terminating at amino acid position 730 or less). The use of E1 and / or E2 polypeptides that retain the polypeptide is contemplated. Furthermore, the C-terminal truncation can extend through the membrane across the domain towards the N-terminus. Thus, for example, E1 truncation present at a position below, for example, 360 and E2 truncation present at, for example, a position below 715 are also included in the present invention. All that is necessary is that the truncated E1 and E2 polypeptides retain functionality for their intended purpose. However, particularly preferred shortened E1 constructs are those that do not extend beyond about 300 amino acids. Most preferred is termination at position 360. Preferred shortened E2 constructs are those that have a C-terminal truncation that does not extend beyond about amino acid position 715. Particularly preferred E2 truncations are molecules that are truncated after any amino acid 715-730 (eg, 725).

  For a description of various HCV epitopes derived from these and other HCV regions, see, eg, Chien et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10011-10015; Chien et al. Gastroent. Hepatol. (1993) 8: S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D. et al. Y. , International Publication No. WO 94/01778; and US Pat. Nos. 6,280,927 and 6,150,087.

  Preferably, the fusion proteins described above and the individual components of these proteins are produced recombinantly. Polynucleotides encoding these proteins are introduced into expression vectors, which can be expressed in suitable expression systems. A variety of bacterial, yeast, mammalian and insect expression systems are available in the art, and any such expression system can be used. If desired, the polynucleotides encoding these proteins can be translated in a cell-free translation system. Such methods are well known in the art. This protein can also be constructed by solid phase protein synthesis.

  If desired, the fusion proteins or individual components of these proteins may also be useful in protein purification such as other amino acid sequences (eg, amino acid linkers or signal sequences, and glutathione-S-transferase and staphylococcal protein A). Ligand).

(Polynucleotide encoding fusion protein)
As described above, the polynucleotide may comprise the less than complete HCV genome, or alternatively may comprise the complete polyprotein sequence having a mutated NS3 domain. The polynucleotide can be RNA or single-stranded or double-stranded DNA. Preferably, the polynucleotide is isolated free of other components (eg, proteins and lipids). The polynucleotide encodes the fusion protein described above, and thus at least one other HCV polypeptide (eg NS2, p7, E1, E2, NS4, NS5a, NS5b from a different region of NS3 ^* and HCV polyprotein). A polypeptide encoding a core, etc.). The polynucleotides of the present invention also include other nucleotide sequences, such as those encoding linkers, signal sequences, or ligands (eg, glutathione-S-transferase, and staphylococcal protein A) useful in protein purification. obtain.

In order to increase the expression yield, it may be desirable to split the polyprotein into fragments for expression. These fragments can be used in combination in the compositions as described herein. Alternatively, these fragments can be combined after expression. Thus, for example, NS3 ^* NS4Core can be expressed as one construct and NS5aNS5bCore can be expressed as a second construct. Similarly, NS3 ^* NS4NS5a can be expressed as one construct, and for example, a second construct with NS3 ^* NS4NS5aCore can be expressed as a second construct. For example, NS2p7E2NS3 ^* NS4 can be expressed as one construct, and NS3 (unmodified) NS4NS5b can be expressed as a further construct for use in the compositions of the invention. It should be understood that the above combinations are only representative and that any combination of fusions can be expressed separately.

  Polynucleotides encoding various HCV polypeptides can be isolated from genomic libraries derived from nucleic acid sequences present, for example, in plasma, serum, or liver homogenates of individuals infected with HCV, or, for example, automated synthesis It can be synthesized in the laboratory using a machine. Amplification methods such as PCR can be used to amplify the polynucleotide from either HCV genomic DNA or the cDNA encoding it.

  Polynucleotides can include coding sequences for these polypeptides that can be naturally occurring coding sequences for these polypeptides or non-naturally occurring artificial sequences. These polynucleotides can be ligated using standard molecular biology techniques to form the coding sequence for the fusion protein. If desired, the polynucleotide can be cloned into an expression vector and transformed into, for example, bacterial, yeast, or mammalian cells, such that the fusion protein of the invention can be expressed in cell culture. Can be isolated from this.

  The expression constructs of the present invention, including individual fusion constructs comprising the desired fusions or individual components of these fusions, are used for nucleic acid immunization and are used for cellular immune responses using standard gene delivery protocols. Can irritate. Methods for gene delivery are known in the art. See, for example, U.S. Pat. Nos. 5,399,346, 5,580,859, and 5,589,466. The gene can be delivered directly to a vertebrate subject, or alternatively, can be delivered ex vivo to a cell derived from the subject, and the cell can be reimplanted into the subject. For example, the construct can be delivered, for example, as plasmid DNA contained within a plasmid (eg, pBR322, pUC, or ColE1).

  Further, the expression construct can be encapsulated in liposomes before being delivered into the cell. Lipid encapsulation is generally achieved using liposomes that can stably bind or trap and maintain nucleic acids. The ratio of aggregated DNA to lipid preparation can vary, but is generally about 1: 1 (mg DNA: polymer lipid) or more lipid. For a review of the use of liposomes as carriers for the delivery of nucleic acids, see Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097: 1-17; Straubinger et al., Methods of Enzymology (1983), Vol. 101, pp. See 512-527.

  Liposome preparations for use in the present invention include cationic (positively charged) preparations, anionic (negatively charged) preparations, and neutral preparations, with cationic liposomes being particularly preferred. Cationic liposomes are readily available. For example, N [1-2,3-dioleorroxy) propyl] -N, N, N-triethyl-ammonium (DOTMA) liposomes are commercially available from GIBCO BRL (Grand Island, NY.), A registered trademark of Lipofectin. (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416). Other commercially available lipids include transfectase (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. For example, for a description of the synthesis of DOTAP (1,2-bis (oleyloxy) -3- (trimethylammonio) propane) liposomes, see Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75: 4194-4198; see PCT application publication number WO 90/11092. Various liposome-nucleic acid complexes are prepared using methods known in the art. For example, Straubinger et al., METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75: 4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394: 483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443: 629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76: 836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76: 3348; Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76: 145; Fraley et al. , J .; Biol. Chem. (1980) 255: 10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75: 145; and Schaefer-Ridder et al., Science (1982) 215: 166.

  DNA is also described in Papahadjopoulos et al., Biochem. Biophys. Acta. (1975) 394: 483-491 can be delivered in a similar lipid composition. See also U.S. Pat. Nos. 4,663,161 and 4,871,488.

  Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses (eg, mouse sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and rous sarcoma virus) provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and encapsulated in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and then delivered to the cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (US Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7: 980-990; Miller, AD, Human Gene Therapy (1990) 1 Scarpa et al., Virology (1991) 180: 849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90: 8033-8037; Genet.Develop. (1993) 3: 102-109). Briefly, the retroviral gene delivery vehicles of the present invention include, for example, a wide variety of retroviruses, including B, C and D retroviruses, and FIV, HIV, HIV-1, HIV-2 and SIV. (See RNA Tumor Viruses, 2nd Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses can be obtained from depository authorities or collection authorities such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, VA 201110-2209), or using commonly available techniques. Can be isolated from known sources.

  A number of adenoviral vectors have also been described, such as adenovirus type 2 and type 5 vectors. Unlike retroviruses that integrate into the host genome, adenoviruses remain extrachromosomal, thus minimizing the risk associated with insertional mutations (Haj-Ahmad and Graham, J. Virol. (1986) 57: 267-274. Bett et al., J Virol. (1993) 67: 5911-5921; Mittereder et al., Human Gene Therapy (1994) 5: 717-729; Seth et al., J Virol. Therapy (1994) 1: 51-58; Berkner, KL BioTechniques (1988) 6: 616-629; and Rich et al., Human Gene Therapy (1993) 4: 461-476)).

  Michael et al., J. MoI. Biol. Chem. (1993) 268: 6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. Molecular conjugate vectors such as the adenovirus chimeric vectors described in 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 Sindbis virus and Semliki Forest virus, VEE also find use as viral vectors for delivery of genes of interest. For a description of Sindbis virus-derived vectors useful for the practice of the methods of the invention, see Dubensky et al., J. MoI. Virol. (1996) 70: 508-519; and International Publication Nos. WO 95/07995 and WO 96/17072.

  Other vectors can be used, and other vectors include, but are not limited to, simian virus 40 and cytomegalovirus. Salmonella ssp. Yersinia enterocolitica, Shigella spp. Bacterial vectors such as, Vibrio cholerae, Mycobacterium BCG strain, and Listeria monocytogenes can be used. Minichromosomes such as MC and MC1, bacteriophages, cosmids (plasmids into which the λ phage cos site has been inserted) and replicons (genetic elements that allow replication in the cell under its own control) Can also be used.

  The expression construct can also be encapsulated, adsorbed or bound to a particle carrier. Such a carrier presents many copies of the selected molecule to the immune system, facilitating the capture and maintenance of the molecule in the local lymph node. The particles can be phagocytosed by macrophages and can increase antigen presentation through cytokine release. Examples of particle carriers include particle carriers derived from polymethylmethacrylate polymers, and fine particles derived from poly (lactide) and poly (lactide-coglycolide) (known as PLG). See, for example, Jeffery et al., Pharm. Res. (1993) 10: 362-368; and McGee et al. Microencap. (1996).

  A wide variety of other methods can be used to deliver the expression construct to the cells. Such methods include DEAE dextran mediated transfection, calcium phosphate precipitation, polylysine mediated transfection or polyornithine mediated transfection, or other insoluble inorganic salts such as strontium phosphate, aluminum silicate (bentonite and kaolin And precipitation using chromium oxide, magnesium silicate, talc, etc.). Other useful transfection methods include electroporation, sonovolation, protoplast fusion, liposomes, peptoid delivery, or microinjection. For example, see Sambrook et al. (Supra) for a discussion of techniques for transforming cells of interest; and for a review of delivery systems useful for gene transfer, see Felgner, P. et al. L. , Advanced Drug Delivery Reviews (1990) 5: 163-187. A particularly effective method of delivering DNA using electroporation is described in International Publication No. WO / 0045823.

  Moreover, particle gun delivery systems that use particle carriers such as gold and tungsten are particularly useful for delivering the expression constructs of the present invention. The particles are coated with the construct to be delivered and accelerated to high speed, typically under a reduced pressure atmosphere, using gun powder fired from a “gene gun”. For a description of such techniques and devices useful therefor, see, for example, U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; No. 5,021,015; No. 5,371,015; and No. 5,478,744.

(Composition containing fusion protein or fusion polynucleotide)
The present invention also provides compositions containing the fusion protein or polynucleotide. The composition can contain one or more fusions, one of the fusions as described herein comprising a mutated NS3 domain. The composition of the present invention may also contain a pharmaceutically acceptable carrier. This carrier should not itself induce the production of antibodies that are harmful to the host. Pharmaceutically acceptable carriers are well known to those skilled in the art. Such carriers include proteins, polysaccharides (eg latex functionalized sepharose, agarose, cellulose, cellulose beads etc.), polylactic acid, polyglycolic acid, multimeric amino acids (eg polyglutamic acid, polylysine etc.), amino acid copolymers And large, slowly metabolized macromolecules such as, but not limited to, inactive virus particles.

  Pharmaceutically acceptable salts can also be used in the compositions of the invention. For example, inorganic salts such as hydrochloride, hydrobromide, phosphate or sulfate, and salts of organic acids such as acetate, propionate, malonate, or benzoate. Particularly useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin, and other proteins well known to those skilled in the art. The compositions of the present invention can also be used alone or in combination with liquids or excipients such as water, saline, glycerol, dextrose, ethanol, and the like, and substances such as wetting agents, emulsifying agents, or pH buffering agents. contains. The proteins of the present invention can also be adsorbed to, entrapped or otherwise bound to particle carriers such as liposomes and PLG. Liposomes and other specific carriers are described above.

If desired, costimulatory molecules that improve immune presentation to lymphocytes, such as B7-1 or B7-2, or cytokines, lymphokines, and chemokines can be included in the composition. The chemokines include cytokines such as IL-2, modified IL-2 (cys125 to ser125), GM-CSF, IL-12, γ-interferon, IP-10, MIP1β, FLP-3, ribavirin and RANTES. For example, but not limited to. If desired, adjuvants can also be included in the composition. Adjuvants that 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 ( Contains or does not contain other specific immunostimulants such as muramyl peptides (see below) or bacterial cell wall components) {eg: (a) (contains various amounts of MTP-PE as required) MF59 containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (PCT Publication No. WO 90/14837) (Model 110Y microfluidizer (Microfluidics, Newton, Mass.) (B) 10 formulated into submicron particles using a fluidizer % Squalene, 0.4% Tween 80, 5% Pluronic Block Polymer L121 and thr-MDP (see below) containing SAF (microfluidized into submicron emulsions or vortexed to produce larger grain size emulsions) And (c) one or more from the group consisting of 2% squalene, 0.2% Tween 80, monophospholipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS) bacterial cell wall components ^{(preferably, MPL + CWS (Detox TM)} ) and Ribi ^TM adjuvant system comprising (RAS) (Ribi Immunochem, Hamilton , MT)}; (3) QS21 or Stimulon ^TM (Cambridge Bioscience, Worc saponin adjuvants such as ster, MA) may be used or particles such as ISCOMs (immunostimulatory fusions) made therefrom, which ISCOMs may have no additional surfactant (See, eg, International Publication No. WO 00/07621); (4) Freunds Complete Adjuvant (CFA) and Freunds Incomplete Adjuvant (IFA); (5) Cytokines {eg, IL-1, IL-2, IL -4, interleukins such as IL-5, IL-6, IL-7, IL-12, etc. (see, eg, International Publication No. WO99 / 44636), interferons such as γ interferon, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.}; (6) Cholera toxin ( CT), detoxified mutants of bacterial ADP-ribosylating toxins such as pertussis toxin (PT) or E. coli heat-labile toxin (LT), in particular LT-K63 (where the wild type amino acid at position 63 is replaced with lysine) LT-R72 (where the wild type amino acid at position 72 is replaced with arginine), CT-S109 (where the wild type amino acid at position 109 is replaced with serine), and PT -K9 / G129 (where the wild type amino acid at position 9 is replaced by lysine and the wild type amino acid at position 129 is replaced by glycine) (see eg, International Publication Nos. WO 93/13202 and WO 92/19265) (7) Monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL), optionally in the substantial absence of alum. (See, for example, GB 2220221; EPA 0869454), (see, eg, International Publication No. WO 00/56358); (8) a combination of 3dMPL with, for example, QS21 and / or an oil-in-water emulsion (eg, EPA 0835318; EPA 0735898; see EPA 0762311); (9) polyoxyethylene ethers or polyoxyethylene esters (see, eg, International Publication No. WO 99/52549); (10) immune stimuli such as CpG oligonucleotides Or immunostimulatory oligonucleotides such as saponin + CpG oligonucleotides (see, eg, International Publication No. WO 00/62800); (11) immunostimulants and metal salt particles (eg, (See open number WO 00/23105); (12) saponins and water-in-oil emulsions (see eg, International Publication No. WO 99/11241); (13) saponins (eg, QS21) + 3dMPL + IL-12 (see (Optional + sterol) (eg, International Publication No. WO 98/57659); (14) MPL derivative RC529; and (15) Other substrates that act as immunostimulants and enhance the usefulness of the composition. Alum and MF59 are preferred.

  As described above, muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), -acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637 (nor- Called MDP)), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP) 19835A (referred to as MTP-PE)), and the like, but is not limited thereto.

  Furthermore, the fusion protein can be adsorbed to ISCOM or captured within ISCOM. A classical ISCOM is formed by a combination of cholesterol, saponin, phospholipid, and an immunogen (eg, a viral envelope protein). Generally, an immunogen (usually having a hydrophobic region) is solubilized in a surfactant and added to the reaction mixture, whereby ISCOM forms the immunogen incorporated therein. Is done. ISCOM matrix compositions are similarly formed but do not contain viral proteins. Proteins with a high positive charge can be electrostatically bound into the ISCOM particles rather than via hydrophobic forces. For a more detailed general discussion of saponins and ISCOMs and methods for formulating ISCOMs, see Barr et al. (1998) Adv. See Drug Delivery Reviews 32: 247-271 (1998).

  ISCOMs for use with the present invention are generated using standard techniques well known in the art and are described, for example, in U.S. Pat. Nos. 4,981,684, 5,178,860, 5, 679,354 and 6,027,732; European Patent Application Publication Nos. EPA 109,942; 180,564 and 231,039; Coulter et al. (1998) Vaccine 16: 1243. Typically, the term “ISCOM” refers to an immunogenic complex formed between a glycoside (eg, a triterpenoid saponin (particularly Quil A)) and an antigen that includes a hydrophobic region. See, for example, European Patent Application Publication Nos. EPA 109,942 and 180,564. In this embodiment, the HCV fusion (usually a fusion with a hydrophobic region) is solubilized in a surfactant and added to the reaction mixture so that ISCOM incorporates the fusion therein. Formed with. The HCV polypeptide ISCOM is readily produced using HCV polypeptides that exhibit amphiphilic properties. However, proteins and peptides that lack desirable hydrophobic properties can be incorporated into immunogenic complexes after conjugation with peptides having hydrophobic amino acids, fatty acid radicals, alkyl radicals, and the like.

  As explained in European Patent Application Publication No. EPA 231,039, the presence of an antigen is not necessary to form a basic ISCOM structure (referred to as a matrix or ISCOMATRIX). This structure can be formed from sterols (eg, cholesterol), phospholipids (eg, phosphatidylethanolamine), and glycosides (eg, Quil A). Thus, the desired HCV fusion is not incorporated into the matrix, but is present outside the matrix and is adsorbed to the matrix, for example, via electrostatic interactions. For example, HCV fusions with a high positive charge can be electrostatically bound to ISCOM particles rather than via hydrophobic forces. For a more detailed general discussion of saponins and ISCOMs and how to formulate ISCOMs, see Barr et al. (1998) Adv. See Drug Delivery Reviews 32: 247-271 (1998).

  The ISCOM matrix can be prepared, for example, by mixing together soluble sterols, glycosides, and (optionally) phospholipids. If no phospholipid is used, a two-dimensional structure is formed. See, for example, European Patent Application Publication No. EPA 231,039. The term “ISCOM matrix” is used to refer to both three-dimensional and two-dimensional structures. The glycosides to be used are generally glycosides that exhibit amphiphilic properties and contain hydrophobic and hydrophilic regions in the molecule. Preferably, saponalbin (eg saponin extract from Quillaja saponaria (Molina and Quil A)) is used. Other preferred saponins are aescine from Aesculus hippocastanum (Patt et al. (1960) Arznemitelforschuung 10: 273-275) and saponin from Gypsophila struthium (Vochten et al. 226).

  To prepare ISCOMs, glycosides are used at least at critical micelle forming concentrations. In the case of Quil A, this concentration is about 0.03% by weight. The sterols used to produce ISCOMs can be known sterols of animal or plant origin (eg, cholesterol, lanosterol, lumisterol, stigmasterol, and sitosterol). Suitable phospholipids include phosphatidylcholine and phosphatidylethanolamine. In general, the molar ratio of glycoside (especially when it is Quil A), sterol (especially when it is cholesterol) and phospholipid is 1: 1: 0 to 1, with ± 20% (preferably ± 10) for each number. % Or less). This is equivalent to a weight ratio of about 5: 1 for Quil A: cholesterol.

A solubilizer may also be present, which may be, for example, a surfactant, urea, or guanidine. In general, nonionic surfactants, ionic surfactants, or zwitterionic surfactants, or cholic acid-based surfactants (eg, sodium dezoxycholate, cholate, and CTAB (cetyltriammonium bromide) )) Can be used for this purpose. Examples of suitable surfactants include octyl glucoside, nonyl N-methyl glucamide, or decanoyl N-methyl glucamide, alkylphenyl polyoxyethylene ethers (eg, polyethylene glycol p having 9-10 oxyethylene groups). -Isooctyl-phenyl ether (sold under the trade name TRITON X-100R ^TM ) Acyl polyoxyethylene esters (for example, acyl polyoxyethylene sorbitan esters (sold under the trade names TWEEN 20 ^™ , TWEEN 80 ^™, etc.) The solubilizers are generally removed for the formation of ISCOMs by, for example, ultrafiltration, dialysis, ultracentrifugation or chromatography, although not limited to In certain ways, this The process is unnecessary (see, eg, US Pat. No. 4,981,684).

  In general, the weight ratio of glycoside (eg Quil A) to HCV fusion is in the range of 5: 1 to 0.5: 1. Preferably, this weight ratio is about 3: 1 to 1: 1, more preferably this ratio is 2: 1.

  Once ISCOM is formed, it can be formulated into a composition and administered to an animal as described herein. If desired, the resulting solution of the immunogenic complex can be lyophilized and then reconstituted prior to use.

(Method of generating HCV-specific antibody)
HCV fusion proteins can be used to generate HCV specific polyclonal antibodies and HCV specific monoclonal antibodies. HCV-specific polyclonal antibodies and HCV-specific monoclonal antibodies specifically bind to HCV antigens. Polyclonal antibodies can be generated by administering this fusion protein to a mammal (eg, a mouse, rabbit, goat, or horse). Serum from the immunized animal is collected and the antibodies are purified from plasma by, for example, precipitation with ammonium sulfate followed by chromatography (preferably affinity chromatography). Techniques for generating and processing polyclonal antisera are known in the art.

  Monoclonal antibodies against HCV specific epitopes present in the fusion protein can also be readily generated. Normal B cells from a mammal (eg, a mouse) immunized with an HCV fusion protein can be fused with, for example, HAT-sensitive mouse myeloma cells to generate 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 screen.

  Antibodies against HCV epitopes (either monoclonal or polyclonal) are particularly useful for detecting the presence of HCV or HCV antigen (eg, a serum sample from a human infected with HCV) in a sample. Immunoassays for HCV antigens can utilize one antibody or several antibodies. Immunoassays for HCV antigens include, for example, monoclonal antibodies against HCV epitopes, combinations of monoclonal antibodies against multiple epitopes of one HCV polypeptide, monoclonal antibodies against multiple epitopes of various HCV polypeptides, polyclonal antibodies against the same HCV antigen Polyclonal antibodies against various HCV antigens or a combination of monoclonal and polyclonal antibodies can be used. The immunoassay protocol can be based on, for example, a competitive assay, a direct reaction assay, or a sandwich type assay, for example, using a labeled antibody. The label can be, for example, a fluorescent label, a chemiluminescent label, or a radioactive label.

  Polyclonal or monoclonal antibodies can be further used to isolate HCV particles or HCV antigens by immunoaffinity columns. The antibody can be immobilized to a solid support, for example, by adsorption or covalently, so that the antibody retains its immunoselective activity. If desired, a spacer group can be included so that the antigen binding site of the antibody remains accessible. The immobilized antibody can then be used to bind to HCV particles or HCV antigens from a biological sample (eg, blood or plasma). Bound HCV particles or bound HCV antigen is recovered from the column matrix, for example, by a change in pH.

(HCV specific T cells)
NS3 ^* NS4NS5a fusion protein (with or without core polypeptide) or NS3 ^* NS4NS5aNS5b fusion protein, as well as various other fusions described herein, expressed in vivo or in vitro HCV-specific T cells activated by HCV polypeptides (e.g., NS2 polypeptide, p7 polypeptide, E1 polypeptide, E2 polypeptide, NS3 polypeptide, NS4 polypeptide, NS5a polypeptide, Or an NS5b polypeptide) epitope (which includes an epitope of a fusion of one or more of these peptides with 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 kill HCV-infected cells presenting any of these epitopes complexed with MHC class I molecules. Can be. HCV specific CD8 ⁺ T cells can be detected, for example, by a ⁵¹ Cr release assay (see Example 4). ^{The 51} Cr release assay measures the ability of HCV-specific CD8 ⁺ T cells to lyse target cells presenting one or more of these epitopes. Also contemplated herein are HCV-specific CD8 ⁺ T cells that express an antiviral agent (eg, IFN-γ), which is also preferably expressed by immunological methods, preferably the HCV polypeptide (eg, , NS3 polypeptide, NS4 polypeptide, NS5a polypeptide, or NS5b polypeptide) but after in vitro stimulation with IFN-γ or similar cytokines Can be detected by staining (see Example 5).

HCV activated by the fusions described above (including but not limited to NS3 ^* NS4NS5a fusion protein or NS3 ^* NS4NS5aNS5b fusion protein), expressed in vivo or in vitro Specific CD4 ⁺ cells are preferably HCV polypeptides (eg, NS2 polypeptide, p7 polypeptide, E1 polypeptide, E2 polypeptide, NS3 polypeptide, NS4 polypeptide) bound to MHC class II molecules on HCV infected cells. An epitope of a peptide, NS5a polypeptide, or NS5b polypeptide, including but not limited to a fusion thereof (eg, NS3NS4NS5a fusion protein or NS3NS4NS5aNS5b fusion protein) The epitope of this HCV polypeptide is bound to an MHC class II molecule on HCV-infected cells and, for example, with or without a core polypeptide In response to stimulation with NS3 ^* NS4NS5a peptide or NS3 ^* NS4NS5aNS5b peptide.

HCV specific CD4 ⁺ T cells can be detected by a lymphoproliferation assay (see Example 6). The lymphoproliferation assay measures the ability of HCV specific CD4 ⁺ T cells to proliferate in response to, eg, NS2 epitope, p7 epitope, E1 epitope, E2 epitope, NS3 epitope, NS4 epitope, NS5a epitope, and / or NS5b epitope To do.

(Method for activating HCV-specific T cells)
HCV fusion proteins or polynucleotides can be used to activate HCV specific T cells either in vitro or in vivo. Activation of HCV-specific T cells can be used, in particular, to provide a model system that optimizes the CTL response to HCV and to provide prophylactic or therapeutic treatment against HCV infection. For in vitro activation, proteins are preferably supplied to T cells via plasmids or viral vectors (eg, adenoviral vectors) as described above.

  The polyclonal population of T cells can be derived from blood of mammals infected with HCV, and preferably from peripheral lymphoid organs (eg, lymph nodes), spleen, or thymus. Preferred mammals include mice, chimpanzees, baboons, and humans. HCV acts to expand the number of activated HCV-specific T cells in mammals. Thereafter, the HCV-specific T cell from the mammal is an HCV fusion protein described herein (eg, an HCV NS3NS4NS5a epitope peptide or an HCV NS3NS4NS5aNS5b epitope peptide with or without a core polypeptide, (But not limited to) can be restimulated in vitro by addition to T cells. HCV-specific T cells can then be tested in vitro, particularly for proliferation, production of IFN-γ, and the ability to lyse target cells presenting, for example, the NS3NS4NS5a epitope or NS3NS4NS5aNS5b epitope.

In a lymphoproliferation assay (see Example 6), HCV activated CD4 ⁺ T cells are HCV polypeptides (eg, NS3 epitope peptide, NS4 epitope peptide, NS5a epitope peptide, NS5b epitope peptide, NS3NS4NS5a epitope peptide, or NS3NS4NS5aNS5b epitope peptide, but not limited to, when grown with, but not in the absence of the epitope peptide. Thus, certain HCV epitopes recognized by HCV-specific CD4 ⁺ T cells (eg, NS2, p7, E1, E2, NS3, NS4, NS5a, NS5b, and fusions of these epitopes (eg, NS3NS4NS5a epitope or NS3NS4NS5aNS5b The epitope) can be identified using a lymphoproliferation assay.

Similarly, detecting IFN-γ in HCV-specific CD4 ⁺ T cells and / or CD8 ⁺ T cells after in vitro stimulation using the fusion protein described above, for example, CD4 ⁺ T to produce IFN-γ. Fusion protein epitopes (eg, NS2, p7, E1, E2, NS3, NS4, NS5a, NS5b, and fusions of these epitopes (eg, such as those that are particularly effective in stimulating cells and / or CD8 ⁺ T cells) NS3NS4NS5a epitope or NS3NS4NS5aNS5b epitope)))) can be used to identify.

In addition, ⁵¹ Cr release assays are useful for determining the level of CTL response to HCV. See Cooper et al., Immunity 10: 439-449. For example, HCV-specific CD8 ⁺ T cells can be derived from the liver of a mammal infected with HCV. These T cells can be tested, for example, in a ⁵¹ Cr release assay against target cells presenting the NS3NS4NS5a epitope or NS3NS4NS5aNS5b epitope. Several target cell populations expressing different NS3NS4NS5a epitopes or NS3NS4NS5aNS5b epitopes can be constructed such that each target cell population presents a different epitope NS3NS4NS5a or NS3NS4NS5aNS5b. HCV specific CD8 ⁺ cells can be assayed against each of these target cell populations. ^{The 51} Cr release assay results can be used to determine which of NS3NS4NS5a or NS3NS4NS5aNS5b is responsible for the strongest CTL response to HCV. Subsequently, NS3 ^* NS4NS5a fusion protein or NS3 ^* NS4NS5aNS5b fusion protein (with or without core polypeptide) containing the epitope responsible for the strongest CTL response was used using information derived from the ⁵¹ Cr release assay. Can be built.

  An HCV fusion protein as described above, or a polynucleotide encoding such a fusion protein, can be used in mammals (eg, mice, baboons, chimpanzees, or humans) to activate HCV-specific T cells in vivo. Can be administered. Administration is as described above by any means known in the art, including parenteral, nasal, intramuscular, or subcutaneous injection (including infusion using a biological microparticle gun (“gene gun”)). Including).

  Preferably, injection of HCV polynucleotides is used to activate T cells. In addition to the practical advantage of simplifying construction and modification, injection of this polynucleotide results in the synthesis of the fusion protein in the host. Accordingly, these immunogens are presented to the host immune system with natural post-translational modifications, structures, and conformations. The polypeptide is preferably administered to a large mammal (eg, human) at a dose of 0.5 mg, 0.75 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 5 mg or 10 mg per kg. And injected intramuscularly.

Compositions of the invention comprising HCV fusion proteins or polynucleotides are measured in a manner compatible with the particular composition used and, inter alia, by ⁵¹ Cr release assay, lymphocyte proliferation assay or IFN-γ Is administered in an amount effective against active HCV-specific T cells as determined by intracellular staining for. The protein and / or polynucleotide can be administered to either a mammal not infected with HCV or to a mammal infected with HCV. The particular dose of polynucleotide or fusion protein in the composition will depend on many factors, including but not limited to the species, age, and general condition of the mammal being administered, and the mode of administration of the composition. . An effective amount of the composition of the present invention can be readily determined using only routine experimentation. The above in vitro and in vivo models can be used to identify appropriate doses. The amount of polynucleotide used in the examples described below provides general guidance that can be used to optimize activation of HCV-specific T cells either in vivo or in vitro. In general, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5, or 10 mg of HCV fusion protein or polynucleotide (may or may not include the core polypeptide) ) Is administered to large animals such as baboons, chimpanzees, or humans. If desired, costimulatory molecules or adjuvants can also be provided before, after, or simultaneously with the composition.

  The mammalian immune response (including activation of HCV-specific T cells) generated by delivery of the compositions of the invention can be enhanced by altering the dose, route of administration, or boosting regimen. . The compositions of the invention may be given in a single dose schedule, or preferably in a multiple dose schedule. In a multi-dose schedule, the initial vaccination includes 1-10 separate doses, with other doses being required for subsequent time intervals required to maintain and / or enhance the immune response (eg, the second The dose is given 1-4 months), and if necessary, subsequent doses are given after several months.

(III. Experiment)
The following are examples of specific embodiments for carrying out the present invention. This example is provided for illustrative purposes only, and is not intended to limit the scope of the invention in any way. Those skilled in the art will readily appreciate that the present invention may be implemented in various ways, once given the teachings of the present disclosure.

  Efforts are being made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should, of course, be allowed for.

Example 1: Generation of NS3 ^* NS4NS5a core polynucleotide
In the following examples, NS3 ^* represents a modified NS3 molecule. A polynucleotide encoding NS3NS4NS5a (amino acids about 1027 to 2399 when referred to by number for HCV-1) (also referred to herein as NS345a) is isolated from HCV. The NS3 portion of this molecule is mutagenized by mutating the coding sequence for the His, Asp, and Ser residues found in the protease active site. As a result, the resulting molecules encode amino acids other than His, Asp, and Ser in these portions and lack NS3 protease activity. This construct is fused to a polynucleotide encoding a core polypeptide comprising amino acids 1-122 of the full length polypeptide. The polynucleotide sequence encoding the core is fused downstream from the NS5a coding portion of this construct, so that the resulting fusion protein comprises a core polypeptide at its C-terminus. This construct is cloned into plasmids, vaccinia viruses, and adenoviral vectors. In addition, this construct is inserted into a recombinant expression vector and used to transform host cells producing the NS3 ^* NS4NS5a core fusion protein.

  Protease enzyme activity is determined as follows. NS4A peptide (KKGSVVIVGRIVLSGPKPAIPIPKK) (SEQ ID NO: 8) and its fusion protein were mixed with 90 μl of reaction buffer (25 mM Tris, pH 7.5, 0.15 M NaCl, 0.5 mM EDTA, 10% glycerol, 0.05 n-dodecyl). (BD-maltoside, 5 mM DTT) and mix for 30 minutes at room temperature. 90 μl of this mixture is added to a microtiter plate (Costar Inc., Corning, NY) and 10 μl of HCV substrate (AnaSpec, Inc., San Jose CA) is added. The plate is mixed and read with a Fluostar plate reader. Results are expressed in relative fluorescence units (RFU) per minute.

(Example 2: Priming of HCV-specific CTL in vaccinated animals)
The HCV fusion protein NS3 ^* NS4NS5a core produced as described above is used as follows to produce HCV fusion-ISCOM. This fusion-ISCOM formulation is prepared by mixing the fusion protein and preformed ISCOMATRIX (empty ISCOM) using ionic interactions to maximize association between antigen and adjuvant. . ISCOMATRIX is essentially the same as Coulter et al. (1998) Vaccine 16: 1243.

Rhesus macaques are immunized under anesthesia. Animals are divided into two groups. The first group, 2 × 10 ⁸ plaque forming units (pfu) (percutaneously 1 × 10 ^8, scarification (Scarification) by 1 × 10 ⁸⁾ to infect rVVC / E1 (Part October). This group is used as a positive control for CTL priming. A second group of animals is immunized by intramuscular (IM) injection in the left quadriceps using 25-100 μg HCV fusion polypeptide adsorbed to ISCOM as described above (0, 1, February and June). Cytotoxic activity has been demonstrated by Pallaard et al. (2000) AIDS Res. Hum. Assay in a standard ⁵¹ Cr release assay, as described in Retroviruses 16: 273.

(Example 3: Immunization with NS3 ^* NS4NS5a core polynucleotide)
In one immunization protocol, animals are immunized by intramuscular injection into the anterior tibialis muscle with 50-250 μg of plasmid DNA encoding NS3 ^* NS4NS5a core fusion protein. Booster injections of vaccinia virus (VV) -NS5a 10 ⁷ pfu (intraperitoneal) or plasmid control 50-250 μg (intramuscular) are provided 6 weeks later.

In another immunization protocol, the anterior tibial muscle of the animal is injected intramuscularly with adenoviral particles 10 ¹⁰ encoding NS3 ^* NS4NS5a core fusion protein. An intraperitoneal booster injection of VV-NS5a 10 ⁷ pfu or an intramuscular booster injection of adenovirus 10 ¹⁰ encoding NS3 ^* NS4NS5a core is provided after 6 weeks.

(Example 4: Activation of HCV-specific CD8 + T cells)
⁵¹ Cr release assay The ⁵¹ Cr release assay is used to measure the ability of HCV-specific T cells to lyse target cells presenting the NS5a epitope. Spleen cells are pooled from the immunized animal. These cells are restimulated in vitro for 6 days with CTL epitope peptide p214K9 (2152-HEYPVGSQL-2160; SEQ ID NO: 1) from HCV NS5a in the presence of IL-2. The spleen cells were then cultured on a Weiss (1980) J. Biol. Chem. 255: 9912-9917, assayed for cytotoxic activity against peptide-sensitized target cells (L929) that express class I MHC molecules but not class II MHC molecules in a standard ⁵¹ Cr release assay. . Test 60: 1, 20: 1, and 7: 1 effector (T cell) to target (B cell) ratios. Percent specific lysis is calculated for each effector to target ratio.

(Example 5: Activation of HCV-specific CD8 + T cells expressing IFN-γ)
Intracellular staining for interferon gamma (IFN-γ) Intracellular staining of IFN-γ is used to identify CD8 + T cells that secrete IFN-γ following in vitro stimulation with NS5a epitope p214K9. Spleen cells of individual immunized animals are restimulated in vitro for 6-12 hours with either p214K9 or a non-specific peptide in the presence of IL-2 and monensin. The cells are then stained for surface CD8 and intracellular IFN-γ and analyzed by flow cytometry. The percentage of CD8 + T cells that are also positive for IFN-γ is then calculated.

(Example 6: Proliferation of HCV-specific CD4 + T cells)
Lymphocyte proliferation assay Spleen cells from pooled immunized animals were depleted of CD8 + T cells using magnetic beads and NS5a-epitope peptide derived from p222D, HCV-NS5a (2224-AELIIANLLWRQEMG-2238; SEQ ID NO: 2) Or culture with either medium alone (triplicate). After 72 hours, cells are pulsed with 1 μl Ci / well ³ H-thymidine and harvested 6-8 hours later. Radioactive uptake is measured after collection. Average cpm is calculated.

(Example 7: CTL prime ability of NS3 * 45a core-coded DNA vaccine formulation)
The animals are treated with 10-250 μg of plasmid DNA encoding NS3 * 45a core fusion protein as described in Example 3, PLG-linked DNA encoding NS3 * 45a core (see below), or NS3 * 45a Any of the DNA encoding the core is used to immunize by delivery through electroporation (see, eg, International Publication No. WO / 0045823 for this delivery technique). This immunization is followed by a booster injection 6 weeks after the plasmid DNA encoding NS3 * 45a core.

  (DNA Delivered by PLG) Polylactide-co-glycolide (PLG) polymer was obtained from Boehringer Ingelheim, U.S. Pat. S. A. Get from. The PLG polymer is RG505 with a 50/50 copolymer ratio and a molecular weight of 65 kDa (manufacturer data). Cationic microparticles adsorbed with DNA are essentially made up of Singh et al. , Proc. Natl. Acad. Sci. Prepared using a modified solvent evaporation process as described in USA (2000) 97: 811-816. Briefly, microparticles are prepared by emulsifying 10 ml of a 5% w / v polymer solution in methylene chloride and 1 ml of PBS at high speed using an IKA homogenizer. This primary emulsion is then added to 50 ml of distilled water containing (0.5% w / v) cetyltrimethylammonium bromide (CTAB). This gives a w / o / w emulsion formulation which is stirred at 6000 rpm for 12 hours at room temperature to evaporate the methylene chloride. The resulting microparticles are washed twice with distilled water by centrifuging at 10,000 g and lyophilized. After preparation, washing, and collection, DNA is adsorbed to the microparticles by incubating 100 mg of cationic microparticles in a 1 mg / ml DNA solution at 4 ° C. for 6 hours. The microparticles are then separated by centrifugation, the pellet is washed with TE buffer and the microparticles are lyophilized.

CTL activity and IFN-γ expression are measured by ⁵¹ Cr release assay or intracellular staining as described in the examples above.

Example 8: Replicon particles SINCR (DC +) encoding immune pathway and NS3 * 45a core
Alphavirus replicon particles (eg, SINCR (DC +)) are obtained from Polo et al. , Proc. Natl. Acad. Sci. USA (1999) 96: 4598-4603. Animals are injected intramuscularly (IM) as described in Example 3 with SINCR (DC +) replicon particles 5 × 10 ⁶ IU encoding NS3 * 45a core, or at the base of the tail (BoT) and paw Either a subcutaneous (S / C) injection in the pad (FP) or a combination that delivers 2/3 DNA via IM administration and 1/3 DNA via the BoT route is used. This immunization is followed by a booster injection with vaccinia virus encoding NS5a as described in Example 3. IFN-γ expression is measured by intracellular staining as described in Example 5.

Example 9: Alphavirus replicon priming followed by various boost regimens
Alphavirus replicon particles (eg, SINCR (DC +)) are obtained from Polo et al. , Proc. Natl. Acad. Sci. USA (1999) 96: 4598-4603. Animals were primed by intramuscular injection of replicon particles 1.5 × 10 ⁶ IU encoding SINCR (DC +), NS345a core into the tibialis anterior muscle, followed by 10-100 μg of plasmid DNA encoding NS5a, NS3 ^* Booster of either 10 ¹⁰ adenoviral particles encoding 45a core, NS3 ^* SINCR (DC +) replicon particle 1.5 × 10 ⁶ IU encoding 45a core, or vaccinia virus 10 ⁷ pfu encoding NS5a Used for week 6. IFN-γ expression is measured by intracellular staining as described in Example 5.

Example 10: Alphavirus expressing NS3 ^* 45a core
Alphavirus replicon particles (e.g., SINCR (DC +) and SINCR (LP)) are prepared according to Polo et al. , Proc. Natl. Acad. Sci. USA (1999) 96: 4598-4603. Animals are combined with delivery routes (2/3 in IM and 1/3 in S / C) using a SINCR (DC +) replicon 1 × 10 ² to 1 × 10 ⁶ IU encoding NS3 ^* 45a core. Through a combination of delivery routes (2/3 in IM) using SINCR (LP) replicon 1 × 10 ² to 1 × 10 ⁶ IU encoding NS3 ^* 45a core , 1/3 at S / C) and by S / C alone. This immunization is followed by a booster injection of the vaccinia virus 10 ⁷ pfu encoding NS5a in the sixth week. IFN-γ expression is measured by intracellular staining as described in Example 5.

  Thus, HCV fusion polypeptides for stimulating cell-mediated immune responses are disclosed. While preferred embodiments of the invention have been described in some detail, it will be understood that obvious changes can be made without departing from the spirit and scope of the invention as defined herein.

FIG. 1 is a diagrammatic representation of the HCV genome, showing various regions of the HCV polyprotein. FIG. 2 shows representative native unmodified NS3 protease domain DNA and corresponding amino acid sequences (SEQ ID NOs: 3-4). FIG. 3 shows representative modified fusion protein DNA and corresponding amino acid sequences (SEQ ID NOs: 5-6) in which the NS3 protease domain is deleted from the N-terminus and contains the core amino acids 1-121 on the C-terminus. Show.

[Sequence Listing]

Claims (24)

Less than:
(A) at least one modified NS3 polypeptide comprising an amino acid substitution such that protease activity is inhibited in the HCV NS3 region; and (b) at least one poly derived from a region of the HCV polyprotein other than the NS3 region. An immunogenic fusion protein comprising a peptide. 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 in relation to the full length HCV-1 polyprotein. The fusion protein according to any one of claims 1 or 2, wherein the protein comprises a modified NS3 polypeptide, NS4 polypeptide, NS5 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. 4. 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. 4. 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. 4. The fusion protein of claim 3, wherein the protein further comprises an E2 polypeptide and optionally a core polypeptide. 4. The fusion protein of claim 3, wherein the protein further comprises an E1 polypeptide, an E2 polypeptide and optionally a core polypeptide. The fusion protein of claim 1, wherein the protein further comprises an E2 polypeptide, a modified NS3 polypeptide and optionally a core polypeptide. 2. The fusion protein of claim 1, wherein the protein further comprises an E1 polypeptide, an E2 polypeptide, a modified NS3 polypeptide and optionally a core polypeptide. The fusion protein according to any one of claims 1 to 10, wherein the polypeptides of (a) and (b) are derived from the same HCV isolate. 11. A fusion protein according to any one of claims 1 to 10, wherein at least one of the polypeptides present in the fusion protein is derived from a different isolate that is a modified NS3 polypeptide. From the amino terminus to the carboxy terminus, the following:
(A) a modified NS3 polypeptide, NS4 polypeptide, and NS5a polypeptide, wherein the modified NS3 polypeptide is numbered relative to the full-length HCV-1 polyprotein, His-1083, Asp A polypeptide comprising a substitution of amino acids corresponding to -1105 and / or Ser-1165, such that protease activity is inhibited;
(B) a modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide and NS5b polypeptide, wherein the modified NS3 polypeptide is numbered in relation to the full-length HCV-1 polyprotein, A polypeptide comprising a substitution of an amino acid corresponding to 1083, Asp-1105 and / or Ser-1165, such that protease activity is inhibited;
(C) E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, and NS5a polypeptide, wherein the modified NS3 polypeptide is a full-length HCV-1 polypeptide Polypeptides comprising amino acid substitutions corresponding to His-1083, Asp-1105 and / or Ser-1165, resulting in inhibition of protease activity, as numbered in association;
(D) E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, and NS5a polypeptide, wherein the modified NS3 polypeptide is a full-length HCV- A polypeptide comprising substitutions of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to one polyprotein, such that protease activity is inhibited;
(E) E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide and NS5b polypeptide, wherein the modified NS3 polypeptide has a full-length HCV-1 A polypeptide comprising substitutions of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the polyprotein, such that protease activity is inhibited;
(F) E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide and NS5b polypeptide, wherein the modified NS3 polypeptide is: A polypeptide comprising substitutions of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the full length HCV-1 polyprotein, so that protease activity is inhibited;
(G) an E2 polypeptide and a modified NS3 polypeptide, wherein the modified NS3 polypeptide is numbered relative to the full-length HCV-1 polyprotein, His-1083, Asp-1105 and / or A polypeptide comprising a substitution of an amino acid corresponding to Ser-1165 so that the protease activity is inhibited;
(H) an El polypeptide, an E2 polypeptide and a modified NS3 polypeptide, wherein the modified NS3 polypeptide is numbered relative to the full-length HCV-1 polyprotein, His-1083, Asp- A polypeptide comprising a substitution of an amino acid corresponding to 1105 and / or Ser-1165, such that protease activity is inhibited;
(I) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide and a modified NS3 polypeptide, wherein the modified NS3 polypeptide is numbered in relation to the full-length HCV-1 polyprotein, A polypeptide comprising a substitution of an amino acid corresponding to 1083, Asp-1105 and / or Ser-1165, such that protease activity is inhibited; or (j) an E1 polypeptide, an E2 polypeptide, a p7 polypeptide, an NS2 poly Peptides and modified NS3 polypeptides, wherein the modified NS3 polypeptide corresponds to His-1083, Asp-1105 and / or Ser-1165 numbered in relation to the full length HCV-1 polyprotein Including the substitution of amino acids resulting in proteases An immunogenic fusion protein consisting essentially of a polypeptide whose activity is inhibited. From the amino terminus to the carboxy terminus, the following:
(A) a modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide and core polypeptide, wherein the modified NS3 polypeptide is numbered in relation to the full-length HCV-1 polyprotein, A polypeptide comprising a substitution of an amino acid corresponding to 1083, Asp-1105 and / or Ser-1165, such that protease activity is inhibited;
(B) Modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide, NS5b polypeptide and core polypeptide, wherein the modified NS3 polypeptide is numbered relative to the full length HCV-1 polyprotein A polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and / or Ser-1165, such that protease activity is inhibited;
(C) E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide and core polypeptide, wherein the modified NS3 polypeptide comprises full-length HCV-1 A polypeptide comprising substitutions of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the polyprotein, such that protease activity is inhibited;
(D) E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide and core polypeptide, wherein the modified NS3 polypeptide comprises: A polypeptide comprising substitutions of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the full length HCV-1 polyprotein, so that protease activity is inhibited;
(E) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide, an NS4 polypeptide, an NS5a polypeptide, an NS5b polypeptide and a core polypeptide, wherein the modified NS3 polypeptide comprises: A polypeptide comprising substitutions of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the full length HCV-1 polyprotein, so that protease activity is inhibited;
(F) E1 polypeptide, E2 polypeptide, p7 polypeptide, NS2 polypeptide, modified NS3 polypeptide, NS4 polypeptide, NS5a polypeptide, NS5b polypeptide and core polypeptide, wherein the modified NS3 The polypeptide comprises a substitution of amino acids corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the full length HCV-1 polyprotein so that the protease activity is inhibited. peptide;
(G) an E2 polypeptide, a modified NS3 polypeptide and a core polypeptide, wherein the modified NS3 polypeptide is numbered relative to the full-length HCV-1 polyprotein, His-1083, Asp- A polypeptide comprising a substitution of an amino acid corresponding to 1105 and / or Ser-1165, such that protease activity is inhibited;
(H) an E1 polypeptide, an E2 polypeptide, a modified NS3 polypeptide and a core polypeptide, wherein the modified NS3 polypeptide is numbered in relation to the full-length HCV-1 polyprotein, A polypeptide comprising a substitution of an amino acid corresponding to 1083, Asp-1105 and / or Ser-1165, such that protease activity is inhibited;
(I) an E2 polypeptide, a p7 polypeptide, an NS2 polypeptide, a modified NS3 polypeptide and a core polypeptide, wherein the modified NS3 polypeptide is numbered relative to the full-length HCV-1 polyprotein A polypeptide comprising a substitution of an amino acid corresponding to His-1083, Asp-1105 and / or Ser-1165, so that protease activity is inhibited; or (j) an E1 polypeptide, an E2 polypeptide, a p7 poly Peptides, NS2 polypeptides, modified NS3 polypeptides and core polypeptides, wherein the modified NS3 polypeptides are numbered in relation to the full length HCV-1 polyprotein, His-1083, Asp-1105 And / or amino corresponding to Ser-1165 An immunogenic fusion protein consisting essentially of a polypeptide, comprising an acid substitution, so that the protease activity is inhibited. A modified NS3 polypeptide, wherein the modified NS3 polypeptide is an amino acid corresponding to His-1083, Asp-1105 and / or Ser-1165, numbered in relation to the full length HCV-1 polyprotein Wherein the protease activity is inhibited when a modified NS3 polypeptide is present in the HCV fusion protein. 15. A composition comprising the immunogenic fusion protein of any one of claims 1-14 or the modified NS3 polypeptide of claim 15 in combination with a pharmaceutically acceptable excipient. 17. A method of stimulating a cellular immune response in a vertebrate subject, the method comprising administering a therapeutically effective amount of the composition of claim 16. 16. Use of the composition of claim 15 in a method of stimulating a cellular immune response in a vertebrate subject. 16. The immunogenic fusion protein of any one of claims 1-14 or the modified NS3 poly of claim 15 in the manufacture of a medicament for stimulating a cellular immune response in a vertebrate subject. Use of peptides. A composition comprising an immunogenic fusion protein according to any one of claims 1 to 14 or a modified NS3 polypeptide according to claim 15 in combination with a pharmaceutically acceptable excipient. How to manufacture. A polynucleotide comprising a coding sequence encoding the fusion protein of any one of claims 1-14 or the modified NS3 polypeptide of claim 15. Less than:
(A) the polynucleotide of claim 21; and (b) at least one control element operably linked to the polynucleotide;
A recombinant vector comprising: wherein the coding sequence can be transcribed and translated in a host cell. A host cell comprising the recombinant vector according to claim 22. 24. A method of producing an immunofusion protein or modified polypeptide, the method comprising culturing a host cell population of claim 23 under conditions for producing the protein. .

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