WO2008107400A1 - Produit de construction de polyépitope hcv et ses utilisations - Google Patents

Produit de construction de polyépitope hcv et ses utilisations Download PDF

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Publication number
WO2008107400A1
WO2008107400A1 PCT/EP2008/052517 EP2008052517W WO2008107400A1 WO 2008107400 A1 WO2008107400 A1 WO 2008107400A1 EP 2008052517 W EP2008052517 W EP 2008052517W WO 2008107400 A1 WO2008107400 A1 WO 2008107400A1
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seq
epitopes
polypeptide
hcv
vector
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PCT/EP2008/052517
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English (en)
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Marie-Ange Buyse
Denise Baker
Gert Verheyden
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Genimmune N.V.
Pharmexa Inc.
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Application filed by Genimmune N.V., Pharmexa Inc. filed Critical Genimmune N.V.
Publication of WO2008107400A1 publication Critical patent/WO2008107400A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is directed to a Hepatitis C virus (HCV) polyepitope construct and the use thereof for the prevention and/or treatment of HCV infection.
  • HCV Hepatitis C virus
  • HCV is the major cause of non-A, non-B hepatitis worldwide. Acute infection with HCV (20% of all acute hepatitis infections) frequently leads to chronic hepatitis (70% of all chronic hepatitis cases) and end-stage cirrhosis. It is estimated that up to 20% of chronic HCV carriers may develop cirrhosis over a time period of about 20 years and that of those with cirrhosis between 1 to 4%/year is at risk to develop liver carcinoma (Lauer & Walker 2001, Shiftman 1999). An option to increase the life-span of HCV-caused end- stage liver disease is liver transplantation (30% of all liver transplantations world-wide are due to HCV-infection).
  • HLA human leukocyte antigen
  • CTL HLA class Il-restricted helper T lymphocytes
  • CTL HLA class Il-restricted helper T lymphocytes
  • MHC Major Histocompatibility Complex
  • MHC class II molecules are expressed primarily on activated antigen-presenting cells and lymphocytes.
  • CD4+ T lymphocytes helper T lymphocytes or HTLs
  • CD4+ T lymphocytes proliferate and secrete cytokines that either support an antibody-mediated response through the production of IL-4 and IL-IO or support a cell-mediated response through the production of IL-2 and IFN-gamma.
  • T lymphocytes recognize an antigen in the form of a peptide fragment bound to the MHC class I or class II molecule rather than the intact foreign antigen itself.
  • An antigen presented by a MHC class I molecule is typically one that is endogenously synthesized by the cell (e.g., an intracellular pathogen).
  • the resulting cytoplasmic antigens are degraded into small fragments in the cytoplasm, usually by the proteasome (Niedermann et al., 1995).
  • Antigens presented by MHC class II molecules are usually soluble antigens that enter the antigen presenting cell via phagocytosis, pinocytosis, or receptor-mediated endocytosis. Once in the cell, the antigen is partially degraded by acid-dependent proteases in endosomes (Blum et al., 1997; Arndt et al., 1997).
  • immunodominance (Yewdell et al., 1997). More simply, immunodominance describes the phenomenon whereby immunization or exposure to a whole native antigen results in an immune response directed to one or a few "dominant" epitopes of the antigen rather than every epitope that the native antigen contains. Immunodominance is influenced by a variety of factors that include MHC-peptide affinity, antigen processing and T-cell receptor recognition.
  • minigene vaccines composed of approximately ten MHC Class I epitopes in which a plurality of epitopes were immunogenic and/or antigenic have been reported.
  • minigene vaccines composed of 9 EBV (Thomson et al, Proc, 1995), 7 HIV (Woodbe ⁇ y et al, 1999), 10 murine (Thomson et al, (1998) and 10 tumor-derived (Mateo et al., 1999) epitopes have been shown to be active.
  • minigenes comprising multiple epitopes have been described.
  • a possible model of a HCV minigene construct is presented in WOO 1/21189 (Pharmexa Inc). The selection of the epitopes is based on the presence of a supermotif and on the binding affinity.
  • WOO 1/47541 (Pharmexa Inc.) addresses the problem of junctional epitopes and discloses the immunogenicity of two HCV-derived minigenes (same epitopes, different order) in HLA transgenic mice.
  • WO04/007556 (CSL Ltd et al.) describes the design of polyepitope polypeptides based on the hydrophobicity of the individual peptides.
  • a polyepitope sequence comprising 26 HCV CTL epitopes is studied. Furthermore, a DNA construct comprising 13 HCV supertype CTL epitopes is disclosed in WO06/004362 (Mogam Biotechnology Research Institute), whereby cellular immunity was enhanced by using co-stimulatory molecules.
  • minigene vaccines containing multiple MHC class I restricted epitopes possibly combined with class II (i.e., CTL and possibly HTL) epitopes can be designed, and presentation and recognition can be obtained for a plurality of epitopes.
  • class II i.e., CTL and possibly HTL
  • the immunogenicity of polyepitope constructs appears to be strongly influenced by a number of variables. More specific, the immunogenicity of the same epitope expressed in the context of different vaccine constructs can vary over several orders of magnitude. Antigen-processing is an important factor to consider with the epitope-based approach to vaccine development.
  • epitope selection should include identification of epitopes retaining their immunogenic properties in different microenvironments.
  • HCV vaccine development Another important factor to be considered in HCV vaccine development is the existence of viral escape mutants. Immune evasion by viral escape mutations in certain CTL epitopes that result in either loss of the epitope or perturbed T cell recognition has been described for different viral diseases including HCV (Chang KM et al, 1997; Guglietta S et al, 2005). The thus far identified HCV T cell epitopes that are sensitive to viral escape are a few well-known epitopes that are not well-conserved. This is at least in part due to the identification of escape variants using a population-based approach (Gaudieri et al., 2006).
  • HCV chronically infected HCV patients with a T cell vaccine can be prone to viral escape and the effectiveness of the vaccine will highly depend on the ability of the virus to escape the immune pressure.
  • HCV is less likely to evade multi-specific CTL responses if induced by a therapeutic vaccine, containing different, highly conserved epitopes for which there are no indications of viral immune escape. Therefore, several conserved, immunogenic epitopes need to be identified. It is considered that regions of the HCV proteins in which little variation is observed might either reflect as yet unknown structurally or functionally relevant sites or lack of immune pressure.
  • CTL escape mutations are constrained by a fitness cost to replication that varies between epitopes (S ⁇ derholm et al., 2006). Furthermore, it has been described that epitopes with potentially high associated fitness cost revert to wild-type sequence upon transmission (Timm et al., 2004; Friedrich et al., 2004).
  • the present invention provides strategies to optimize antigenicity and immunogenicity of polyepitope vaccines encompassing a large number of relevant epitopes, and provides optimized polyepitope vaccines, particularly HCV polyepitope constructs.
  • the present invention is directed to a polypeptide, a polynucleotide, a vector or a composition comprising a polyepitope construct comprising specifically selected epitopes derived from the Core, El, E2, NS3, NS4 (NS4A and NS4B) and/or NS5 (NS5A and NS5B) protein of the Hepatitis C Virus (HCV).
  • the epitopes are those which elicit a HLA class I- and/or class II- restricted T lymphocyte response in an immunized host, which are immunogenic when used in a construct and/or which are not prone to HLA-related viral escape.
  • the current invention relates to a polypeptide comprising or consisting of a poly epitope construct comprising the following HCV CTL epitopes: SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, and SEQ ID NO 78, and wherein the construct does not comprise a full-length protein from HCV.
  • the polyepitope construct of the invention further comprises at least one CTL and/or HTL epitope.
  • the epitopes are isolated. More preferably, the HTL epitope is a PADRE® epitope. Even more preferably, the CTL and/or HTL epitope is derived from HCV.
  • the invention encompasses a polypeptide comprising or consisting of a polyepitope construct comprising the HCV CTL epitopes represented by SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, SEQ ID NO 78, and at least one of the epitopes selected from the group consisting of: SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 20, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 43, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 59, SEQ ID NO 65, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 76, SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 92, SEQ ID NO 122, and SEQ ID NO
  • the epitopes in the polyepitope construct are linked to each other by one or more spacer amino acids.
  • the one or more spacer amino acids are, independently from each other, selected from the group consisting of: K, R, N, Q, G, A, S, C, G, P, and T.
  • the CTL and/or HTL epitopes comprised in the polyepitope construct are sorted to minimize the number of CTL and/or HTL junctional epitopes.
  • the polypeptide of the present invention comprises a polyepitope construct consisting of the amino acid sequence selected from the group consisting of: SEQ ID NO 128, SEQ ID NO 130, SEQ ID NO 132, SEQ ID NO 134, SEQ ID NO 136, SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 142, SEQ ID NO 144, SEQ ID NO 146, and SEQ ID NO 147.
  • the polypeptide of the present invention comprises a polyepitope construct comprised in the amino acid sequence selected from the group consisting of: SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 115, and SEQ ID NO 116.
  • the current invention also relates to a polynucleotide encoding the polypeptide as described herein.
  • the polynucleotide of the present invention further comprises one or more regulatory sequences.
  • said regulatory sequence is an internal ribosome binding site (IRES).
  • the polynucleotide of the present invention further comprises one or more promoters.
  • the promoter is a CMV promoter.
  • the polynucleotide of the invention comprises one or more MHC class I and/or MHC class II-targeting sequences.
  • the targeting sequence is selected from the group consisting of: Ig kappa signal sequence, tissue plasminogen activator signal sequence, insulin signal sequence, endoplasmic reticulum signal sequence, LAMP-I lysosomal targeting sequence, LAMP-2 lysosomal targeting sequence, HLA-DM lysosomal targeting sequence, HLA-DM-association sequences of HLA-DO, Ig-alpha cytoplasmic domain, Ig-beta cytoplasmic domain, Ii protein, influenza matrix protein, HBV surface antigen, HBV core antigen, and yeast Ty protein.
  • the polynucleotide comprises a polyepitope construct consisting of the nucleotide sequence selected from the group consisting of: SEQ ID NO 129, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 135, SEQ ID NO 137, SEQ ID NO 139, SEQ ID NO 141, SEQ ID NO 143, SEQ ID NO 145, SEQ ID NO 148 and SEQ ID NO 149.
  • the polynucleotide comprises a polyepitope construct comprised in the nucleotide sequence selected from the group consisting of: SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 103, SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO 111, SEQ ID NO 118, and SEQ ID NO 119.
  • the invention encompasses a vector comprising the polynucleotide as described herein.
  • the vector is an expression vector. More preferably, the vector is a plasmid, a viral or bacterial vector.
  • the viral vector is a pox virus.
  • the pox virus is a vaccinia virus. Even more preferably, the vaccinia virus is MVA.
  • the current invention also relates to a composition comprising the polypeptide, the polynucleotide, or the vector as described herein, or any combination thereof.
  • the composition further comprises a pharmaceutical acceptable excipient.
  • the composition is a vaccine.
  • the present invention relates to the use of the polynucleotide, the vector, the polypeptide, or the composition as described herein, as a medicament. Further, the invention encompasses the polynucleotide, the vector, the polypeptide, or the composition, as described herein, for use as a medicament
  • the invention includes the use of the composition, the polynucleotide, the vector or the polypeptide for the preparation of a medicament for treating and/or preventing hepatitis C. Furthermore, the invention includes the composition, the polynucleotide, the vector or the polypeptide as described herein for use as a medicament, and more particular, for use in treating and/or preventing hepatitis C.
  • the invention encompasses the use of the polypeptide as described herein, or the composition comprising it, as a priming agent in a heterologuous prime boost treatment regimen.
  • the invention envisages the use of the polypeptide, or composition comprising it, for the manufacture of a medicament for inducing a T cell response against HCV in a prime boost treatment regimen, wherein the prime boost treatment regimen comprises the steps of: a. administering the polypeptide, or composition comprising it, as a priming agent; and b. subsequently administering a boosting agent comprising a vector encoding one or more CTL epitopes of the HCV target antigen, including at least one CTL epitope which is the same as a CTL epitope of the priming agent.
  • the invention envisages the polypeptide, or composition comprising it, for use in inducing a T cell response against HCV in a prime boost treatment regimen, wherein the prime boost treatment regimen comprises the steps of: c. administering the polypeptide, or composition comprising it, as a priming agent; and d. subsequently administering a boosting agent comprising a vector encoding one or more CTL epitopes of the HCV target antigen, including at least one CTL epitope which is the same as a CTL epitope of the priming agent.
  • the T cell response comprises a Cytotoxic T Lymphocyte (CTL) response and/or a T Helper (HTL) response.
  • CTL Cytotoxic T Lymphocyte
  • HTL T Helper
  • the CTL response is a CD8+ T cell response
  • the HTL response is a CD4+ T cell response.
  • the epitopes encoded by the vector are the same as the epitopes comprised in the polypeptide.
  • the vector for use as a boosting agent is a plasmid, a viral vector, a bacterial vector or a yeast vector.
  • the invention envisages the use of the polypeptide, or composition comprising it, for the manufacture of a medicament prepared for inducing a T cell response against HCV in a prime boost treatment regimen.
  • the present invention includes a cell comprising the polypeptide, the polynucleotide, or the vector as described herein.
  • the invention relates to a method of inducing an immune response against HCV in an individual, comprising administering the polypeptide, the polynucleotide, the vector, the composition, or the cell as described herein, to said individual.
  • the immune response comprises or consists of a T cell response. More particular, the T cell response is a CTL response and/or a HTL response. Even more specific, the CTL response is a CD8+ T cell response and the HTL response is a CD4+ T cell response.
  • the invention covers a method of making the polypeptide, the polynucleotide, the vector, the composition, or the cell as described herein.
  • FIG. 1 HCV Epitope sequence data obtained from 63 chronic HCV patients for typical examples of conserved and non-conserved epitopes. " ": same amino acid (AA) as within the epitope sequence shown at the top of the table;
  • a Tyrosine is sequenced at position 3 of the epitope instead of the AA found in the epitope as shown at the top of the table;
  • Figure 2 Organization of polyepitope-encoding insert.
  • Figure 3 Construct ICCG5754: A. Amino acid sequence of the signal sequence and the poly epitope,
  • FIG. 13 A. Amino acid sequence of the HCV poly epitope protein (CTL-HTL) with N-terminal translation initiator Met (M),
  • HCV-CTL HCV polyepitope protein
  • M N-terminal translation initiator Met
  • C. Amino acid sequence of the HCV polyepitope protein (CTL-HTL) is C. Amino acid sequence of the HCV polyepitope protein (CTL-HTL),
  • HCV-CTL Amino acid sequence of the HCV polyepitope protein
  • Figure 14 A. Nucleic acid sequence with start codon encoding the HCV polyepitope protein (CTL-HTL) and tag, B. Nucleic acid sequence encoding the HCV polyepitope protein (CTL-HTL).
  • Figure 15 A. Nucleic acid sequence with start codon encoding the HCV polyepitope protein
  • HCV-CTL Nucleic acid sequence encoding the HCV polyepitope protein
  • Figure 16 Restriction map of plasmid pAcI (ICCG1396).
  • Figure 17 Nucleic acid sequence of the plasmid pAcI (1-4947 bps).
  • Figure 18 Restriction map of the plasmid pcI857 (ICCG167).
  • Figure 19 Nucleic acid sequence of the plasmid pcI857 (1-4182 bps).
  • Figure 20 SDS-PAGE and Silver staining (a) + Western blot analysis (b) on IMAC samples of the HCV polyepitope construct (HTL-CTL), expressed in E.
  • strain Figure 2 IA and B CTL responses after immunization of HLA- A24/K b or HLA-A 11 /K b transgenic x Balb/C mice with the HCV polyepitope protein as prime and plasmid DNA as boost. Cumulative amount of specific IFNg spots in CD8 + spleen cells direct ex vivo, after stimulation with resp.
  • HLA-A24-restricted HCV epitopes loaded on LCL721.22 IHLA- A24/H-2K b cells (figure 21A) and HLA A-11 -restricted HCV epitopes loaded on LCL721.221HLA-A1 l/H-2K b cells (figure 21B).
  • the number of specific spots (delta) is given by the bars.
  • Figure 22 HTL type 1 response after immunization of HLA-A24/K b (group 1) or HLA- Al 1/K b transgenic x Balb/C (group 3) mice with the HCV polyepitope protein as prime and plasmid DNA as boost.
  • Cumulative amount of specific IFNg spots in CD4 + spleen cells direct ex vivo, after stimulation with HLA-DR-restricted HCV epitopes and PADRE, presented by irradiated syngeneic spleen cells. The number of specific spots (delta) is given by the bars.
  • the present invention is directed to a polypeptide or polynucleotide comprising a polyepitope construct comprising epitopes derived from the Core, El, E2, NS3, NS4 (NS4A and NS4B) and/or NS5 (NS5A and NS5B) protein of the Hepatitis C Virus (HCV).
  • the epitopes are those which elicit a HLA class I- and/or class II- restricted T-lymphocyte response in an immunized host.
  • the present invention describes highly optimized and effective polyepitope constructs characterized by efficient processing and comprising highly immunogenic and/or conserved epitopes that are not prone to HLA-related viral escape.
  • HCV genotype 1- derived CTL epitopes for the most prominent HLA-A, -B and -C alleles were identified using different prediction algorithms. Further selection was made based on their affinity. Binding characteristics of the epitopes were evaluated using molecular and/or cellular assays.
  • Binding epitopes were evaluated for their induction of (ex vivo) recall T cell responses using PBMC from HCV patients.
  • PBMC from subjects were cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen-presenting cells (APC to allow activation of "memory" T cells, as compared to "naive” T cells).
  • APC antigen-presenting cells
  • T cell activity was detected using assays such as 51 Cr release involving peptide-loaded target cells, T cell proliferation, or cytokine release.
  • a subset of binding epitopes was evaluated for their immunogenicity in different HLA transgenic mice.
  • polyepitope constructs were designed comprising candidate therapeutic epitopes.
  • the epitopes in the constructs were sorted and optimized using the method as described in WO04/031210 (Pharmexa Inc. et al.; incorporated herein by reference).
  • Epitopes were included in one or more constructs.
  • the constructs were subsequently tested in HLA transgenic mice and immunogenicity was measured for the encoded epitopes.
  • the present invention provides a specific selection of epitopes for an optimal design of a polyepitope construct in order to obtain a highly immunogenic and conserved polyepitope T cell vaccine for HCV infection.
  • the construct of the invention contains epitopes that are immunogenic in all constructs tested, with a minimum of 4 different polyepitope constructs evaluated for these epitopes.
  • said epitopes are not prone to viral escape.
  • the present invention relates to a polypeptide comprising or consisting of a polyepitope construct comprising the following HCV cytotoxic T lymphocyte (CTL) epitopes: SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, and SEQ ID NO 78.
  • CTL cytotoxic T lymphocyte
  • the present invention also encompasses a polynucleotide encoding the polypeptide as described herein.
  • the polynucleotide construct thus comprises nucleic acids encoding the following HCV CTL epitopes: SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, and SEQ ID NO 78.
  • the epitopes of the poly epitope construct are directly or indirectly linked to one another in the same reading frame.
  • construct generally denotes a composition that does not occur in nature.
  • the polynucleotide construct of the present invention does not encode a wild- type full-length protein from HCV but encodes a chimeric protein containing isolated, viral epitopes from at least one HCV protein not necessarily in the same sequential order as in nature.
  • a construct may be a "polynucleotide construct” or a “polypeptide construct”. Said polynucleotides or peptides are "isolated” or “biologically pure”.
  • isolated refers to material that is substantially free from components that normally accompany it as found in its naturally occurring environment.
  • isolated polynucleotide or peptide of the present invention might comprise heterologous cell components or a label and the like.
  • An "isolated" epitope refers to an epitope that does not include the neighboring amino acids of the whole sequence of the antigen or polypeptide from which the epitope was derived.
  • the present invention relates to a polypeptide comprising or consisting of a polyepitope construct comprising the following isolated HCV CTL epitopes: SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, and SEQ ID NO 78.
  • the construct of the present invention comprises isolated epitopes that are not embedded in the naturally occurring full length protein from HCV.
  • a construct can be produced by synthetic technologies, e.g. recombinant DNA preparation and expression or chemical synthetic techniques for nucleic acids and amino acids.
  • a construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form.
  • an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) molecules.
  • MHC Major Histocompatibility Complex
  • nucleic acid epitope is a set of nucleic acids that encode for a particular amino acid sequence that forms an epitope.
  • an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or MHC molecule. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention or polynucleotides encoding these molecules, are still within the bounds of the invention.
  • polypeptide is used interchangeably with “oligopeptide” and designates a series of amino acids, connected one to the other, typically by peptide bonds between the amino and carboxyl groups of adjacent amino acids.
  • polyepitope construct when referring to nucleic acids and polynucleotides can be used interchangeably with the terms “minigene” and “polyepitope nucleic acid” and other equivalent phrases, and comprises multiple nucleic acid epitopes that encode peptides of any length that can bind to a molecule functioning in the immune system, preferably a HLA class I or a HLA class II and a T-cell receptor. All disclosures herein with regard to epitopes comprised in an amino acid construct apply mutatis mutandis to the nucleic acid epitopes comprised in a polynucleotide construct.
  • the epitopes in a polyepitope construct can be HLA class I epitopes and/or HLA class II epitopes.
  • HLA class I epitopes are referred to as CTL epitopes
  • HLA class II epitopes are referred to as HTL epitopes.
  • Some polyepitope constructs can have a subset of HLA class I epitopes and another subset of HLA class II epitopes.
  • a CTL epitope usually consists of 13 or less amino acid residues in length, 12 or less amino acids in length, or 11 or less amino acids in length, preferably from 8 to 13 amino acids in length, more preferably from 8 to 11 amino acids in length (i.e.
  • a HTL epitope consists of 50 or less amino acid residues in length, and usually from 6 to 30 residues, more usually from 12 to 25, and preferably consists of 15 to 20 (i.e. 15, 16, 17, 18, 19, or 20) amino acids in length.
  • the polyepitope construct described herein preferably includes 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, and up till 150 epitopes, preferably up till 100 and more preferably up till 80 epitopes. More specific, the polyepitope construct comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60 or more epitopes.
  • the polyepitope construct of the present invention further comprises at least one CTL and/or HTL epitope.
  • Said "further" CTL and/or HTL epitope to be used in combination with the epitopes of the present invention can be derived from HCV or from a foreign antigen or organism (non-HCV).
  • the present invention encompasses a polypeptide comprising or consisting of a polyepitope construct comprising the HCV CTL epitopes represented by SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, SEQ ID NO 78, and at least one CTL and/or HTL epitope, and wherein the construct does not comprise a full-length protein from HCV.
  • the at least one CTL and/or HTL epitope is derived from HCV.
  • the polyepitope construct of the present invention further comprises at least one HCV CTL epitope selected from the group consisting of: SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 20, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 43, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 59, SEQ ID NO 65, SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO 76, SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 92, SEQ ID NO 122, and SEQ ID NO 125.
  • said epitopes are characterized in that they are efficiently processed and immunogenic when administered within the context of polyepitope plasmid DNA. Specifically, said epitopes are immunogenic in
  • HLA class I or class II epitopes present in a polyepitope construct can be derived from the same antigen, or from different antigens.
  • a polyepitope construct can contain one or more HLA epitopes than can be derived from two different antigens of the same virus, or from two different antigens of different viruses.
  • the epitopes can be derived from any desired antigen of interest, e.g. a viral antigen, a tumor antigen or any pathogen.
  • the epitopes of the present invention are derived from HCV. There is no limitation on the length of said further epitopes, these can have a length of e.g.
  • the "at least one" can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more epitopes.
  • the polyepitope construct of the present invention further comprises the universal T cell epitope called PADRE ® (Pharmexa A/G, H ⁇ rsholm); described for example in US 5 736 142 or US 6 413 935 or International Application WO95/07707 or WO97/26784, which are enclosed herein by reference).
  • PADRE ® the universal T cell epitope
  • a "PanDR binding epitope or PADRE ® epitope” is a member of a family of molecules that binds more that one HLA class II DR molecule. The pattern that defines the PADRE ® family of molecules can be thought of as an HLA Class II supermotif.
  • PADRE ® binds to most HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses.
  • HTL epitopes can be used from universally used vaccines such as tetanos toxoid.
  • the aim of the present invention is to provide strategies to optimize antigenicity and immunogenicity of poly epitope vaccines encompassing a large number of relevant epitopes, and to provide optimized polyepitope vaccines, particularly HCV polyepitope constructs. Examples of such constructs are depicted in Figures 3 to 11 and 13 to 15. Said constructs comprise a plurality of HCV- specific epitopes that are efficiently processed, and thus highly immunogenic, and a plurality of epitopes that are not prone to viral escape.
  • the present invention is directed to a polypeptide comprising or consisting of a polyepitope construct consisting of the amino acid sequence selected from the group consisting of: SEQ ID NO 128, SEQ ID NO 130, SEQ ID NO 132, SEQ ID NO 134, SEQ ID NO 136, SEQ ID NO 138, SEQ ID NO 140, SEQ ID NO 142, SEQ ID NO 144, SEQ ID NO 146, and SEQ ID NO 147, or comprised in the amino acid sequence selected from the group consisting of: SEQ ID NO 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 102, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 108, SEQ ID NO 110, SEQ ID NO 115, and SEQ ID NO 116.
  • the present invention is directed to a polynucleotide comprising or consisting of a polyepitope construct consisting of the nucleotide sequence selected from the group consisting of: SEQ ID NO 129, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 135, SEQ ID NO 137, SEQ ID NO 139, SEQ ID NO 141, SEQ ID NO 143, SEQ ID NO 145, SEQ ID NO 148 and SEQ ID NO 149, or comprised in the nucleotide sequence selected from the group consisting of: SEQ ID NO 95, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 103, SEQ ID NO 105, SEQ ID NO 107, SEQ ID NO 109, SEQ ID NO 111, SEQ ID NO 118, and SEQ ID NO 119.
  • immunogenic or “immunogenicity” as used herein is the ability to evoke an immune response.
  • Immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high affinity binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides that bind with intermediate affinity (Sette et al., 1994; Alexander et al., 2003). Moreover, higher binding affinity peptides lead to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Various strategies can be utilized to evaluate immunogenicity, including but not limited to:
  • T cell cultures from normal individuals (see, e. g., Wentworth et al., 1995; Celis et al., 1994; Tsai et al., 1997; Kawashima et al., 1998).
  • This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen-presenting cells in vitro over a period of several weeks.
  • T cells specific for the peptide become activated during this time and are detected using, e.g., a 51 Cr-release assay involving peptide sensitized target cells.
  • HLA transgenic mice see, e.g., Wentworth et al., 1996; Wentworth et al., 1996a; Alexander et al., 1997) or surrogate mice.
  • peptides e.g. formulated in incomplete Freund's adjuvant
  • splenocytes are removed.
  • Cells are either cultured in vitro in the presence of test peptide for approximately one week.
  • Peptide-specific T cells are detected using, e.g., a 51 Cr-release assay involving peptide- sensitized target cells and/or target cells expressing endogenously generated antigen.
  • cells are incubated overnight together with peptide-loaded APC in the IFNg ELISPOT assay for the quantitation of peptide-specific single T cells releasing mouse interferon gamma upon stimulation.
  • recall T cell responses are detected by culturing PBL from subjects that have been naturally exposed to the HCV antigen, for instance through infection, and thus have generated an immune response "naturally", or from patients who were vaccinated with a vaccine comprising the epitope of interest.
  • PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells.
  • a given epitope is stated to be immunogenic if T cell reactivity can be shown to target cells sensitized with that peptide.
  • Immunogenicity for a given epitope can further be described by the number of individuals in a group of HLA matched infected or vaccinated subjects (e.g. humans, primates, transgenic mice, surrogate mice) that show T cell reactivity to that particular epitope, or e.g. by the number of spots detected in an ELISPOT assay, as described in Examples 3 and 4.
  • the term “conserved” or “stable” refers to epitopes that have a given amino acid sequence in at least 80% of HCV sequences of genotype Ib as obtained from the Los Alamos database (see Table A for the set of HCV genotype Ib sequences used to calculate the conservancy), and that the frequency of mutations is not statistically different (two-tailed Fisher's exact test, p ⁇ 0.05) between samples from HLA-matched patients and the total set of patient samples.
  • the epitopes of the polyepitope construct are directly or indirectly linked to one another in the same reading frame. More specific, the epitopes are either contiguous or are separated by a linker or a spacer nucleic acid encoding a spacer amino acid or spacer peptide.
  • Link or “join” refers to any method known in the art for functionally connecting peptides
  • the polyepitope construct of the present invention further comprises one or a plurality of spacer nucleic acids, linked in the same reading frame to the CTL and/or HTL epitope nucleic acids.
  • a reading frame is a contiguous and non-overlapping set of three- nucleotide codons in DNA or RNA. There are 3 possible reading frames in a strand and six in a double stranded DNA molecule. "In the same reading frame" means that there is no shift from one frame to another that could lead to different genes/proteins.
  • said epitopes can be sorted and optimized using a computer program or, for fewer epitopes, not using a computer program.
  • “Sorting epitopes” refers to determining or designing an order of the epitopes in a polyepitope construct.
  • Optimizing refers to increasing the antigenicity of a polyepitope construct having at least one epitope pair by sorting epitopes to minimize the occurrence of junctional epitopes, and inserting a spacer residue (as described herein) to further prevent the occurrence of junctional epitopes or to provide a flanking residue.
  • a "flanking residue” is a residue that is positioned next to an epitope. A flanking residue can be introduced or inserted at a position adjacent to the N-terminus (N+ 1) or the C-terminus (C+ 1) of an epitope.
  • An increase in immunogenicity or antigenicity of an optimized polyepitope construct is measured relative to a polyepitope construct that has not been constructed based on the optimization parameters by using assays known to those skilled in the art, e.g. assessment of immunogenicity in HLA transgenic mice, ELISPOT, tetramer staining, 51 Cr release assays, and presentation on antigen presenting cells in the context of MHC molecules.
  • the process of optimizing polyepitope constructs is given e.g. in WO01/47541 and WO04/031210 (Pharmexa Inc. et al.; incorporated herein by reference).
  • the polyepitope construct of the present invention is optimized for CTL and/or HTL epitope processing. More particular, the optimization comprises the introduction of one or more spacers. More preferred, the polyepitope construct as described herein comprises 0, 3, 6, 9, 12, 15, 18, or more spacer nucleic acids or 0, 1, 2, 3, 4, 5, 6, or more spacer amino acids between two epitopes.
  • a "spacer” refers to a sequence that is inserted between two epitopes in a polyepitope construct to prevent the occurrence of junctional epitopes, or to facilitate cleavage between epitopes and thereby enhance epitope presentation.
  • Junctional epitopes refer to epitopes recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes.
  • a spacer nucleic acid may encode one or more amino acids.
  • a spacer nucleic acid flanking a HLA class I epitope in a polyepitope construct encodes preferably 1 to 9, and more preferably 1 to 5 amino acids, i.e. 1, 2, 3, 4 or 5 amino acids.
  • a spacer nucleic acid flanking a HLA class II epitope in a polyepitope construct encodes preferably 5, 6, 7, or more amino acids, and more preferably 5 or 6 amino acids.
  • a spacer nucleic acid separating a HLA class I epitope and a class II epitope in a polyepitope construct encodes preferably 1 to 9, and more preferably 1 to 5 amino acids, i.e. 1, 2, 3, 4 or 5 amino acids.
  • the number of spacers in a construct, the number of amino acids in a spacer, and the amino acid composition of a spacer can be selected to optimize epitope processing and/or minimize junctional epitopes.
  • spacers are selected by concomitantly optimizing epitope processing and preventing junctional motifs.
  • the "spacer amino acid” or “spacer peptide” is typically comprised of one or more relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • spacers flanking HLA class II epitopes preferably include G (GIy), P (Pro), and/or N (Asn) residues.
  • a particularly preferred spacer for flanking a HLA class II epitope includes alternating G and P residues, for example, (GP)n, (PG)n, (GP)nG, (PG)nP, and so forth, where n is an integer between 1 and 10, preferably 2 or 3, and where a specific example of such a spacer is GPGPG (SEQ ID NO 113).
  • the spacers are typically selected from, e.g., A (Ala), N (Asn), K (Lys), G (GIy), L (Leu), I (lie), R (Arg), Q (GIn), S (Ser), C (Cys), P (Pro), T (Thr), or other neutral spacers of nonpolar amino acids or neutral polar amino acids, though polar residues could also be present.
  • a preferred spacer, particularly for HLA class I epitopes comprises 1, 2, 3 or more consecutive alanine (A) residues, or a combination of K (Lys) and A (Ala) residues, e.g.
  • KA, KAA or KAAA or a combination of N (Asn) and A (Ala) residues, e.g. NA, NAA or NAAA.
  • the present invention is thus directed to a polypeptide comprising a polyepitope construct as described herein, and wherein the epitopes in the construct are separated by one or more spacer amino acids.
  • the one or more spacer amino acids are, independently from each other, selected from the group consisting of: K, R, N, Q, G, A, S, C, G, P and T.
  • each spacer nucleic acid encodes the same amino acid sequence. In other polyepitope constructs, one or more of the spacer nucleic acids may encode different amino acid sequences.
  • polypeptides of the present invention can be in their natural (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications. Also included in the definition are peptides modified by additional substituents attached to the amino acids side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains, such as oxidation of sulfhydryl groups.
  • polypeptide or its equivalent terms is intended to include the appropriate amino acid sequence referenced, and may be subject to those of the foregoing modifications as long as its functionality is not destroyed.
  • the present invention also contemplates a polyepitope construct comprising or consisting of multiple repeats or combinations of any of the epitopes of the present invention.
  • the polyepitope construct can exist as a homopolymer comprising multiple copies of the same (combination of) peptide(s), or as a heteropolymer of various peptides.
  • Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce HTL 's and/or CTLs that react with different antigenic determinants of the pathogenic organism targeted for an immune response.
  • the present invention also encompasses a method of making a polyepitope construct.
  • Polynucleotides or nucleic acids that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, 1981, using an automated synthesizer, or as described in Van Devanter et al., 1984. Purification of polynucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, 1983. Other purification methods are reversed phase separation and hydroxyapatite and are well known to the skilled person. Chemically synthesized and purified polynucleotides can be assembled into longer polynucleotides by PCR-based methods (Stemmer et al., 1995; Kriegler et al., 1991).
  • the epitopes of the polyepitope constructs are typically subcloned into an expression vector that contains a promoter to direct transcription, as well as other regulatory sequences such as enhancers and polyadenylation sites. Additional elements of the vector are e.g. signal or target sequences, translational initiation and termination sequences, 5' and 3' untranslated regions and introns, required for expression of the polyepitope construct in host cells.
  • Polyepitope constructs can for example be prepared according to the methods set forth in Ishioka et al., 1999; Velders et al., 2001; or as described in WO04/031210 - Pharmexa Inc. (all incorporated herein by reference).
  • a polyepitopic polypeptide or the polypeptide comprising the polyepitope construct can be generated synthetically or recombinantly.
  • the polyepitopic polypeptide can be expressed as one protein.
  • eukaryotic cells including yeast
  • cultured vertebrate hosts such as Chinese Hamster Ovary (CHO), Vero cells, RK13, COSl, BHK, and MDCK cells, or invertebrate hosts such as insect cells
  • transformation of an appropriate cellular host with a recombinant vector or by means of adenoviruses, influenza viruses, BCG, and any other live carrier systems, in which a nucleotide sequence coding for one of the polypeptides of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host or of the live carrier system and in the case of a prokaryotic host,
  • polyepitopic polypeptide can be purified by methods well known to the person skilled in the art.
  • the polyepitope construct of the invention can be expressed by vectors.
  • the present invention thus also relates to a vector comprising the polynucleotide of the present invention.
  • the term "vector” may comprise a plasmid, a cosmid, a prokaryotic organism, a phage, a virus or an eukaryotic organism such as an animal or human cell or a yeast cell.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the polyepitope construct in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the polyepitope construct and signals required for efficient polyadenylation of the transcript. Additional elements of the cassette may include enhancers and introns with functional splice donor and acceptor sites.
  • Suitable promoters are well known in the art and described, e.g., in Sambrook et al, Molecular cloning, A Laboratory Manual (2 nd ed. 1989) and in Ausubel et al, Current Protocols in Molecular Biology (1994). Eukaryotic expression systems for mammalian cells are well known in the art and are commercially available. Such promoter elements include, for example, cytomegalovirus (CMV), Rous sarcoma virus long terminal repeats (RSV LTR) and Simian Virus 40 (SV40). See, e.g. US 5 580 859 and US 5 589 466 (Vical Inc.; incorporated by reference) for other suitable promoter sequences.
  • CMV cytomegalovirus
  • RSV LTR Rous sarcoma virus long terminal repeats
  • SV40 Simian Virus 40
  • the expression cassette can also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the polynucleotide of the present invention further comprises one or more regulatory sequences.
  • regulatory sequence is meant a polynucleotide sequence that contributes to or is necessary for the expression of an operably associated nucleic acid or nucleic acid construct in a particular host organism.
  • the regulatory sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and an internal ribosome binding site (IRES).
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • a promoter may be a CMV promoter or other promoter described herein or known in the art.
  • Regulatory sequences include IRESs. Other specific examples of regulatory sequences are described herein and otherwise known in the art.
  • the polynucleotide of the present invention further comprises one or more MHC class I and/or MHC class II "targeting nucleic acids” or “targeting sequences”.
  • MHC targeting sequence enhances the immune response to an antigen, relative to delivery of antigen alone, by directing the peptides to the site of MHC molecule assembly and transport to the cell surface, thereby providing an increased number of MHC molecule- peptides complexes available for binding to and activation of T cells.
  • Examples of possible targeting sequences are well known to the skilled person and are described e.g. in
  • the epitopes of polyepitope construct of the present invention are operably linked to a nucleic acid encoding a targeting sequence selected from the group consisting of: Ig kappa signal sequence, tissue plasminogen activator signal sequence, insulin signal sequence, endoplasmic reticulum signal sequence, LAMP-I lysosomal targeting sequence, LAMP-2 lysosomal targeting sequence, HLA-DM lysosomal targeting sequence, HLA-DM-association sequences of HLA-DO, Ig-alpha cytoplasmic domain, Ig-beta cytoplasmic domain, Ii protein, influenza matrix protein, HBV surface antigen, HBV core antigen, and yeast Ty protein.
  • a targeting sequence selected from the group consisting of: Ig kappa signal sequence, tissue plasminogen activator signal sequence, insulin signal sequence, endoplasmic reticulum signal sequence, LAMP-I lysosomal targeting sequence, LAMP-2 lysosomal targeting sequence, HLA-DM
  • operably linked refers to a linkage in which a nucleotide sequence is connected to another nucleotide sequence (or sequences) in such a way as to be capable of altering the functioning of the sequence (or sequences).
  • a nucleic acid or polyepitope nucleic acid construct that is operably linked to a regulatory sequence, such as a promoter/operator, places expression of the nucleic acid or construct under the influence or control of the regulatory sequence.
  • Two nucleotide sequences are said to be operably linked if induction of promoter function results in the transcription of the protein encoding sequence mRNA and if the nature of the linkage between the two nucleotide sequences does not (1) result in the introduction of a frame-shift mutation nor (2) prevent the expression regulatory sequences to direct the expression of the mRNA or protein.
  • a promoter region would be operably linked to a nucleotide sequence if the promoter were capable of effecting transcription of that nucleotide sequence.
  • cysteine residues comprised in epitopes of the polyepitope construct may be "reversibly or irreversibly blocked".
  • An “irreversibly blocked cysteine” is a cysteine of which the cysteine thiol-group is irreversibly protected by chemical means.
  • “irreversible protection” or “irreversible blocking” by chemical means refers to alkylation, preferably alkylation of a cysteine in a protein by means of alkylating agents, such as, for example, active halogens, ethylenimine or N-(iodoethyl)trifluoro-acetamide.
  • Alkylation can be performed by any method known in the art, such as, for example, active halogens X(CH 2 ) n R in which X is a halogen such as I, Br, Cl or F.
  • active halogens are methyliodide, iodoacetic acid, iodoacetamide, and 2- bromoethylamine.
  • a “reversibly blocked cysteine” is a cysteine of which the cysteine thiol-groups is reversibly protected.
  • the term “reversible protection” or “reversible blocking” as used herein contemplates covalently binding of modification agents to the cysteine thiol-groups, as well as manipulating the environment of the protein such, that the redox state of the cysteine thiol-groups remains (shielding). Reversible protection of the cysteine thiol-groups can be carried out chemically or enzymatically.
  • reversible protection by enzymatical means contemplates reversible protection mediated by enzymes, such as for example acyl-transferases, e.g. acyl-transferases that are involved in catalysing thio- esterif ⁇ cation, such as palmitoyl acyltransferase.
  • acyl-transferases e.g. acyl-transferases that are involved in catalysing thio- esterif ⁇ cation, such as palmitoyl acyltransferase.
  • reversible protection by chemical means contemplates reversible protection, using conditions or agents well known to the person skilled in the art.
  • the removal of the reversibly protection state of the cysteine residues can chemically or enzymatically be accomplished by e.g.: - a reductant, in particular DTT, DTE, 2-mercaptoethanol, dithionite, SnCl 2 , sodium boro hydride, hydroxylamine, TCEP, in particular in a concentration of 1-200 mM, more preferentially in a concentration of 50-200 mM; removal of the thiol stabilising conditions or agents by e.g.
  • cysteine residues can be carried out in vitro or in vivo, e.g. in a cell or in an individual.
  • one cysteine residue, or 2 or more cysteine residues comprised in the HCV epitopes as described herein may be mutated to a natural amino acid, preferentially to methionine, glutamic acid, glutamine or lysine.
  • compositions and vaccines are provided.
  • compositions comprising a polynucleotide, a polypeptide or a vector comprising the HCV polyepitope construct as described herein, or a combination thereof.
  • the composition furthermore comprises at least one of a pharmaceutically acceptable excipient, i.e. a carrier, adjuvant or vehicle.
  • a pharmaceutically acceptable excipient i.e. a carrier, adjuvant or vehicle.
  • composition can be used interchangeably.
  • said immunogenic composition is a vaccine composition.
  • said vaccine composition is a prophylactic vaccine composition.
  • the prophylactic vaccine composition refers to a vaccine aimed for preventing HCV infection and to be administered to healthy persons who are not yet infected with HCV.
  • said vaccine composition may also be a therapeutic vaccine composition.
  • the therapeutic vaccine composition refers to a vaccine aimed for treatment of HCV infection and to be administered to patients being (chronically) infected with HCV.
  • Hepatitis C is a blood- bourne infectious disease that is caused by the hepatitis C virus (HCV) infecting the liver.
  • HCV hepatitis C virus
  • the infection can cause liver inflammation (hepatitis) that is often asymptomatic, but ensuing chronic hepatitis can result later in cirrhosis (f ⁇ brotic scarring of the liver) and liver cancer.
  • a vaccine or vaccine composition is an immunogenic composition capable of eliciting an immune response sufficiently broad and vigorous to provoke at least one or both of: a stabilizing effect on the multiplication of a pathogen already present in a host and against which the vaccine composition is targeted.
  • a vaccine composition may also induce an immune response in a host already infected with the pathogen against which the immune response leading to stabilization, regression or resolving of the disease; and an increase of the rate at which a pathogen newly introduced in a host, after immunization with a vaccine composition targeted against said pathogen, is resolved from said host.
  • the vaccine composition of the invention is a HCV vaccine composition.
  • the vaccine or vaccine composition comprises an effective amount of the peptides, polypeptide, nucleic acids or polynucleotide of the present invention.
  • said vaccine composition comprises a vector, a plasmid, a recombinant virus and/or host cell comprising the polyepitope construct of the present invention.
  • Said vaccine composition may additionally comprise one or more further active substances and/or at least one of a pharmaceutically acceptable excipient, being a carrier, adjuvant or vehicle.
  • an "effective amount" of a polypeptide or polynucleotide in a vaccine or vaccine composition is referred to as an amount required and sufficient to elicit an immune response. It will be clear to the skilled artisan that the immune response sufficiently broad and vigorous to provoke the effects envisaged by the vaccine composition may require successive (in time) immunizations with the vaccine composition as part of a vaccination scheme or vaccination schedule.
  • the "effective amount” may vary depending on the health and physical condition of the individual to be treated, the age of the individual to be treated (e.g. dosing for infants may be lower than for adults), the taxonomic group of the individual to be treated (e.g.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the vaccine may be administered in conjunction with other immunoregulatory agents.
  • the dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • the "subject or “individual” as mentioned in the present invention may be non-human, e.g. a mammal, or human.
  • the subject or individual is a primate. Even more preferably, the subject or individual is a human.
  • the present invention furthermore relates to a method of inducing an immune response against HCV in an individual comprising administering the polynucleotide, the vector, the polypeptide, the host cell or the composition of the present invention.
  • polypeptides and polynucleotides encoding them can be delivered in a pharmaceutically acceptable carrier or as colloidal suspensions, or as powders, with or without diluents. They can be "naked” or associated with delivery vehicles and delivered using delivery systems known in the art.
  • a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable adjuvant” is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection.
  • a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen.
  • a “pharmaceutically acceptable vehicle” includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc.
  • vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles.
  • a composition or vaccine is prepared as an injectable, either as a liquid solution or suspension.
  • Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal, intraepidermal, or by "gene gun".
  • Other types of administration comprise electroporation, implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization or nasal spray or drops.
  • Solid forms, suitable for dissolving in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect.
  • a liquid formulation may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred.
  • Another drug delivery system for increasing circulatory half-life is the liposome.
  • the peptides and nucleic acids of the invention may also be administered via liposomes, which serve to target a particular tissue, such as lymphoid tissue, or to target selectively infected cells, as well as to increase the half-life of the peptide and nucleic acids composition.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility.
  • Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art.
  • the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients.
  • the composition is preferably administered to subjects using those methods that are known to those skilled in the art.
  • naked DNA is currently being used for intramuscular (IM) administration in clinical trials.
  • IM intramuscular
  • an alternative method for formulating purified plasmid DNA may be desirable.
  • a variety of methods have been described, and new techniques may become available.
  • Cationic lipids can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould- Fogerite 1988; U.S. Pat No. 5279833; WO 91/06309; and Feigner et al, 1987).
  • glyco lipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • DNA-based delivery technologies include facilitated (bupivicaine, polymers, peptide- mediated) delivery, cationic lipid complexes, particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US 5 922 687), DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, emulsified DNA, DNA included in a viral vector, DNA formulated with a transfection- facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with calcium precipitating agents, DNA coupled to an inert carrier molecule, and DNA formulated with an adjuvant.
  • facilitated e.g., polymers, peptide- mediated
  • cationic lipid complexes e.g., cationic lipid complexes
  • particle-mediated e.g., a particle-mediated (“gene gun”) or pressure-mediated delivery
  • DNA formulated with charged or uncharged lipids DNA included in a viral vector
  • Recombinant virus or live carrier vectors may also be directly used as live vaccines in humans. Accordingly the present invention also relates to a recombinant virus, a bacterial vector, a yeast vector or a plasmid, and a host cell comprising the polynucleotide as described herein.
  • the polynucleotide is introduced in the form of a vector wherein expression is under control of a promoter. Therefore, further embodiments of the present invention are an expression vector which comprises a polynucleotide encoding at least the polyepitope construct as described herein, and which is capable of expressing the respective peptides, a host cell comprising the expression vector and a method of producing and purifying the herein described peptides, pharmaceutical compositions comprising the herein described peptides and a pharmaceutically acceptable carrier and/or adjuvants.
  • nucleic acid vaccines examples include attenuated viral hosts, such as a pox virus.
  • attenuated viral hosts such as a pox virus.
  • vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention.
  • the recombinant vaccinia virus Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response.
  • Vaccinia vectors for example Modified Vaccinia Ankara (MVA), and methods useful in immunization protocols are described in, e.g., US 4 722 848.
  • Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al, 1991.
  • Preferable yeast vectors are Sacharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha.
  • Alphaviruses Semliki Forest Virus, Sindbis Vrius, Venezuelan Equine Encephalitis Virus (VEE)
  • Herpes simplex Virus HSV
  • replication- deficient strains of Adenovirus human or simian
  • SV40 vectors CMV vectors
  • papillomavirus vectors derived from Epstein Barr virus.
  • retroviral vectors Salmonella typhi vectors
  • detoxified anthrax toxin vectors and the like
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the polynucleotide construct.
  • mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing polynucleotide expression.
  • immunostimulatory sequences appear to play a role in the immunogenicity of nucleic acid vaccines. These sequences may be included in the vector, outside the polynucleotide coding sequence, if desired to enhance immunogenicity.
  • a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used.
  • proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL- 12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), and costimulatory molecules.
  • Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes.
  • immunosuppressive molecules e.g. TGF-P
  • TGF-P immunosuppressive molecules
  • polyepitope constructs are described in, e.g., US 6 534 482 (Pharmexa Inc.); An and Whitton, 1997; Thomson et al., 1996; Whitton et al., 1993; Hanke et al., 1998.
  • a polyepitope DNA plasmid encoding supermotif- and/or motif-bearing HCV epitopes derived from multiple regions of the HCV polyprotein sequence, the PADRE ® universal helper T cell epitope (or multiple HTL epitopes from HCV), and an endoplasmic reticulum-translo eating signal sequence can be engineered.
  • the present invention also relates to the polynucleotide, the vector, the host cell, the polypeptide or the composition of the present invention for use as a medicament.
  • said medicament is a vaccine.
  • the present invention relates to the use of the polyepitope construct comprising the epitopes of the present invention, or the nucleic acid sequence encoding said epitopes, for the manufacture of a medicament for preventing and/or treating an HCV infection.
  • the invention also relates to a vector, a plasmid, a recombinant virus or host cell comprising the polynucleotide as described herein for the manufacture of a medicament for preventing and/or treating an HCV infection or hepatitis C.
  • the invention includes the polynucleotide, the polypeptide, the vector or the composition as described herein for use as a medicament, and more particular, for use in treating and/or preventing hepatitis C.
  • the present invention relates to the use of the polynucleotide, the vector, the host cell, the polypeptide or the composition for inducing an immune response against HCV in an individual.
  • Said use can be characterized in that said polynucleotide, vector, host cell, polypeptide or composition is used as part of a series of time and compounds.
  • a series of time and compounds refers to administering with time intervals to an individual the compounds used for eliciting an immune response.
  • the latter compounds may comprise any of the following components: polynucleotide, vector, host cell, polypeptide or composition of the present invention.
  • the immune response comprises or consists of a T cell response. More particular, the T cell response is a CTL response and/or a HTL response. Even more specific, the CTL response is a CD8+ T cell response and the HTL response is a CD4+ T cell response.
  • a polypeptide comprising the polyepitope construct as given herein is especially suited for use as a priming agent in a heterologous prime boost treatment regimen.
  • heterologous refers to a different presentation format, i.e. protein versus vector, of the epitopes in the priming versus the boosting agent.
  • the boosting composition or boosting agent may be provided in a variety of different forms. Specifically, the boosting agent is a vector.
  • the present invention relates to the use of a polypeptide comprising the polyepitope construct of the invention, or a composition comprising it, for the manufacture of a medicament for inducing a T cell response against HCV in a prime boost treatment regimen, comprising the steps of: a. administering the polypeptide, or a composition comprising it, as a priming agent; and b. administering a boosting agent comprising a vector encoding at least one CTL epitope which is the same as a CTL epitope of the priming composition.
  • the present invention relates to a polypeptide comprising the polyepitope construct of the invention, or a composition comprising it, for use in treating and/or preventing hepatitis C in a prime boost treatment regimen, comprising the steps of: a. administering the polypeptide, or a composition comprising it, as a priming agent; and b. administering a boosting agent comprising a vector encoding at least one CTL epitope which is the same as a CTL epitope of the priming composition.
  • the vector is a plasmid, a bacterial, a viral vector or a yeast vector.
  • the epitopes encoded by the vector of the boosting agent are the same as the epitopes of the polyepitope construct of the priming polypeptide.
  • the present invention thus also relates to a vector comprising a polynucleotide encoding a polyepitope construct.
  • the polyepitope construct of this invention can be provided in kit form together with instructions for vaccine administration.
  • the kit would include desired polypeptide compositions in a container, preferably in unit dosage form and instructions for administration.
  • An alternative kit would include a polynucleotide construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration.
  • Lymphokines such as IL-2 or IL- 12 may also be included in the kit.
  • kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.
  • HLA-typed chronic HCV patients infected with genotype Ib were used for direct sequencing of the HCV viral genome.
  • Patient samples were selected based on the presence of at least one of the following HLA class I molecules: HLA AOl, A02, A03, Al 1, A24, B07, B08, B35, B44, CwO4, CwO6, and CwO7.
  • HLA class I typing of the samples was performed using the INNO-LiPA HLA-A Multiplex kit and INNO-LiPA HLA-A Update kit, INNO-LiPA HLA-B Multiplex Plus kit and INNO- LiPA HLA-B Update Plus kit, INNO-LiPA HLA-C Multiplex kit and INNO-LiPA HLA-C Update kit (Innogenetics, Belgium).
  • oligonucleotide primers were designed and combined in 21 suitable primer sets (table 2).
  • Viral RNA was extracted from a 1 mL plasma sample using the QIAamp ® Viral RNA Mini Kit (Qiagen) according to the manufacturer's protocol. Viral RNA was eluted in 60 ⁇ L RNase-free water. Negative extraction controls were included. Nine microliter of the isolated RNA was transcribed to cDNA using one of the primer sets described in table 2. This was done essentially as described in the ThermoScriptTM RT-PCR System Protocol (InvitrogenTM). No-template negative controls were included. PCRs were performed with the AccuPrimeTM Taq DNA Polymerase System (Invitrogen).
  • Thermal cycling was performed as outlined in the protocol with an initial denaturation step of 2 min at 95°C, followed by 40 cycles (30 s at 95°C, 30 s at the annealing temperature varying between 50-60 0 C, 30 s at 68°C) and a final extension step of 10 min at 68°C.
  • a sample of the reaction was evaluated for specificity and correct length of the PCR fragment by agarose gel electrophoresis. Prior to DNA sequencing, single specific PCR fragments were digested with ExoSAP-IT (United States Biochemicals; US). Samples with multiple (specific and aspecif ⁇ c) bands were separated on a preparative agarose gel.
  • DNA sequence analysis was performed by AGOWA (Germany) and BaseClear (The Netherlands) according to standard methods (ABI Prism BigDye Terminator Cycle Sequencing v3.1 on an ABI 3730 XL 96 Capillary DNA sequencer). The result of DNA sequence analysis was evaluated by alignment with the HCV genotype Ib consensus sequence (see Figure 12) after translation of the appropriate open reading frame to amino acid sequence.
  • the HCV amino acid sequencing data from the 63 patients were compared with the amino acid sequence of the immunogenic epitopes.
  • Each designed DNA construct contains HLA-restricted epitopes which bind to at least one HLA molecule with an affinity ⁇ 500 nM. These epitopes were demonstrated to be immunogenic in the respective HLA transgenic mice when administered as a pool of peptides emulsified in IFA (results shown in Table 13 of WO 05/118626 GENimmune N.V. et al.; incorporated herein by reference).
  • DNA constructs were generated using a selection of these epitopes. Several epitopes are included in more than 1 construct. The epitope order and amino acid spacers were designed to avoid generation of junctional epitopes and to maximize proteosomal processing. The amino acid sequences were back-translated using a mammalian codon usage table and the online back-translation tool both provided by Entelechon (Germany). The DNA sequences were inserted into pMB75.6 vector using Pstl and BamHI restriction sites (Figure 2). A Kozak sequence, a mouse Igk signal sequence (MGMQ VQIQSLFLLLLWVPGSRG, SEQ ID NO 114), and a stop codon were also included.
  • HLA transgenic mice are generated on a C57BL/6 background.
  • the immunogenicity of HCV HLA class I-restricted epitopes encoded in the DNA constructs was tested in the relevant HLA transgenic mice.
  • the immunogenicity of HLA-A*0201-, HLA-A* 1101-, and HLA-B *0702-restricted epitopes is tested in resp.
  • Fl HLA-A*0201/Kb.C57BL/6xBalb/c Fl HLA-A* 1101/Kb.C57BL/6xBalb/c
  • mice Both male and female mice were used, and their age ranged between 8 and 14 weeks. Each experimental group consisted of 3 mice and the na ⁇ ve group (non-immunized HLA transgenic mice) consisted of 4 mice. Each DNA vaccine was tested in two or more independent experiments. A typical immunization and testing scheme is shown below.
  • HLA transgenic mice were pretreated by injecting 50 ⁇ l 10 ⁇ M cardiotoxin bilaterally into each tibialis anterior muscle; 3-5 days later, the same muscles were injected with a total of 100 ⁇ g plasmid DNA diluted in PBS.
  • mice Na ⁇ ve animals (non- immunized HLA transgenic mice) were included in each experiment as the background control group. Eleven to 14 days after immunization, the mice were euthanized, and the spleens were removed. The splenocytes were used as the source of lymphocytes to measure CTL responses.
  • the cells were removed from the column in culture medium consisting of RPMI- 1640 medium with HEPES (Gibco Life Technologies) supplemented with 10% FBS, 2 mM L- glutamine, 50 ⁇ M 2-ME, 0.5 mM sodium pyruvate, lOO ⁇ g/ml streptomycin and 100 U/ml penicillin. (RPMI-10 medium), washed, and counted again.
  • the responses to CTL epitopes were evaluated using an IFN- ⁇ ELISPOT assay. Briefly, IP membrane-based 96-well plates (Millipore, Bedford MA) were pretreated with 70% MeOH, washed 3x with sterile water, and coated overnight at 4 0 C with anti-mouse IFN- ⁇ monoclonal antibody (Mabtech MabAN18) at a concentration of lO ⁇ g/ml in PBS. After washing 3 times with PBS, RPMI-10 medium was added to each well, and the plates were incubated at 37 0 C for 1 hour to block the plates. The purified CD8+ cells were applied to the wells of the blocked membrane plates at a cell concentration of 4x10 5 cells/well.
  • the immunogenicity of all of the epitopes in the DNA polyepitope construct was tested.
  • the peptides were dissolved in RPMI-10 medium (final peptide concentration lO ⁇ g/ml), and mixed with target cells (10 5 HLA- A2. I/Kb transfected Jurkat cells/well and 10 5 HLA-Al 101/Kb transfected Jurkat cells/well for resp. HLA- A02- and HLA-AI l -restricted peptides). Controls of irrelevant peptide and ConA (lO ⁇ g/ml) were also utilized.
  • the target cell/peptide mixture was layered over the effector CD8+ cells in the wells of the membrane plates, which were incubated for 20 hours at 37 0 C in 5% CO 2 . Media and cells were then washed off the ELISPOT plates with PBS + 0.05% Tween-20, and the plates were incubated with filtered biotinylated anti-mouse IFN- ⁇ antibody (Mabtech MabR4-6A2-Biotin) at a final concentration of 1 ⁇ g/ml for 4 hours at 37 0 C. After washing, the plates were incubated with Avidin-Peroxidase Complex (Vectastain), prepared according to the manufacturer's instructions, and incubated at room temperature for 1 hour.
  • Avidin-Peroxidase Complex Vectastain
  • the raw data for the irrelevant peptide control were averaged for each group (both na ⁇ ve and immunized). Net spots were calculated by subtracting the average media control for each group from the raw data values within the group. The average and standard error were then calculated for each peptide, and the average and standard error were normalized to 10 6 cells (by multiplying by a factor of 2.5). Finally, a type 1, one-tailed T test was performed to compare the data from immunized groups to that from na ⁇ ve controls. The data was reported as the number of peptide-specif ⁇ c IFN ⁇ -producing cells, termed Spot-Forming Cells (SFCyiO 6 CD8 + cells. Data was considered positive if significantly different than the na ⁇ ve controls (p ⁇ 0.05) and if result is > 30 SFC/10 6 cells.
  • SFCyiO 6 CD8 + cells Spot-Forming Cells
  • HLA transgenic SFC/10e6 10e6 genie SEQ mouse model sequence cells ⁇ S.E. T test cells + S.E. ID NO
  • VLVGGVLAAL 11 ,7 ⁇ 5,7 0,02 0,8 + 9,3 - 83 A0201/Kb.C57BL/6 x Balb/c HMWNFISGI 10,4 ⁇ 6,9 0,22 4,6 ⁇ 8,7 - 30
  • KLQDCTMLV 5 8 ⁇ 5,3 0,12 -1 ,7 ⁇ 6,2 - 36
  • HLA transgenic SFC/10e6 10e6 genie SEQ mouse model sequence cells ⁇ S.E. T test cells ⁇ S.E. ID NO
  • VLVGGVLAAL 10,0 ⁇ 5,8 0,15 0,8 ⁇ 9,3 - 83 A0201/Kb.C57BL/6 x Balb/c HMWNFISGI 357,5 ⁇ 133,3 0,02 4,6 + 8,7 + 30
  • KLQDCTMLV 25,4 ⁇ 10,5 0,00 -1 ,7 ⁇ 6,2 - 36
  • Example 4 Immunogenicity of HCV-derived HLA-class I-restricted epitopes encoded in a HCV DNA polyepitope construct: alternative protocol
  • the immunogenicity can also be tested using alternative protocols.
  • the evaluation of the immunogenicity of HCV-derived HLA-A01-, HLA- A02-, HLA-AI l- and HLA-A24-restricted epitopes encoded in different DNA constructs was done using an adapted protocol.
  • HLA transgenic mice (Fl HLA-A02/K b xBalb/c, HLA- A02/ K b , HLA-AOl/ K b , HLA-Al 1/K b , and HLA-A24/K b transgenic mice) were immunised with one of the selected DNA constructs. To ensure an equal distribution of mice between different groups, a randomisation procedure based on body weight was performed. Female and male mice (age between 8 and 14 weeks) were ranked by body weight, extreme light or heavy animals were excluded. The remaining animals were grouped by sequentially assigning animals for the 2 experiments.
  • mice were pre-treated with cardiotoxin (Sigma, C9759) on day -5 by bilateral intramuscular injection of 50 ⁇ l (for mice ⁇ 20g) to lOO ⁇ l (for mice >20g) of a lO ⁇ M cardiotoxin solution.
  • mice Five days later, all mice were immunised with lOO ⁇ g HCV-DNA plasmid by bilateral injection of 50 ⁇ g in the m. tibialis anterior. Mice were euthanised between 13 and 15 days after the DNA immunisation. For all mice, the spleen was removed and placed into a well of a 6-well plate containing 3ml RPMI-5 medium (RPMI1640 medium + 5% iFCS).
  • the spleens were pooled per 2 or 3 mice. By circular motion, the spleens were pressed against the bottom of the well with the plunger of a 10ml- syringe until mostly fibrous tissue remains. The suspension was transferred into a centrifuge tube through a 70 ⁇ M-nylon cell strainer and centrifuged for 10 minutes at 1100 rpm. Before counting the spleen cells, the red blood cells were lysed by resuspending the pellet in 2 ml AKC lysing buffer and incubating for 5 minutes at room temperature, followed by an additional washing step. The pellet was resuspended in a suitable volume of RPMI-5 medium. Viability was assessed using trypan blue exclusion.
  • IFN ⁇ ELISPOT A 96-well ELISPOT plate (Millipore, transparent non-sterile MAIP HTS plates) was coated overnight at 4°C with an anti-IFN ⁇ antibody (Mabtech, clone ANl 8). The ELISPOT plate was then washed twice with PBS and blocked for at least 2 hours at room temperature (RT) with assay medium. In triplicate, purified CD8+ T cells (between 5x10 4 and 2x10 5 cells/well, depending on availability) and antigen-presenting cells (loaded or not-loaded with peptide) were added.
  • antigen-presenting cells were either LCL 721.221 cells transfected with an HLA-A24/K b hybrid construct (10 4 cells/well) or syngeneic spleen cells from non- immunised HLA-A24/K b transgenic mice (2x10 5 cells/well).
  • APC antigen-presenting cells
  • syngeneic spleen cells from non- immunised HLA-A01/K b transgenic mice (2x10 5 cells/well) were used.
  • HLA- A02- and HLA-Al 1 -restricted epitopes respectively Jurkat cells transfected with an HLA-A02/K b hybrid construct (2x10 4 cells/well) and LCL 721.221 cells transfected with an HLA-Al 1/K b hybrid construct (10 4 cells/well) were used.
  • As a positive control cells were stimulated with the polyclonal stimulus PHA (2 ⁇ g/ml). Plates were then incubated and left undisturbed for 20 hours. After incubation, cells were removed, plates were washed several times and incubated with biotinylated anti-IFN ⁇ antibody (Mabtech, clone R46A2) for 2 hours at RT.
  • Peptides eliciting a specific delta CTL response of > 30 specific spots/10 6 CD8 cells and a response ratio > 2 in at least one pool are categorised as immunogenic. A minimum of 2 pools is to be tested.
  • TYSTYGKFL 33 0,4 1 ,8 81 YLNTPGLPV 0 0,9 0,8 88
  • the data show that only some of the epitopes are immunogenic when embedded in a pDNA construct.
  • Some epitopes although formerly shown to induce specific immunogenic responses upon peptide immunization in HLA transgenic mice, loose their immunogenic potential when included in a DNA construct. Others retain their immunogenic potential when present in certain DNA constructs, but loose their immunogenic potential when encoded by other DNA constructs.
  • a few epitopes show immunogenicity independent of the construct used. Remarkably, six epitopes, i.e.
  • the epitopes represented by SEQ ID NO 9, SEQ ID NO 21, SEQ ID NO 53, SEQ ID NO 61, SEQ ID NO 75, and SEQ ID NO 78 induce immunogenic responses in the HLA-matched transgenic mice irrespective of the DNA construct containing these epitopes, irrespective of the protocol used for the analysis (either the protocol as described in example 3 or the protocol of example 4) and with a minimum of 4 constructs tested.
  • these epitopes proved to be highly conserved. It can thus be concluded that these epitopes are particularly useful to include into a HCV polyepitope construct for use in the prevention or treatment of HCV infection.
  • HHHMFHHHWWHHHMWHHH SEQ ID NO 117
  • NAA three amino acid linker sequence
  • Other tags such as e.g. the hexahistidine tag, or another linker can also be used.
  • the complete HCV polyepitope coding regions ( Figures 14 and 15 - SEQ ID NO 118 and SEQ ID NO 119 respectively) were subcloned into E. coli vectors for expression using the temperature-inducible bacteriophage Lambda pR-based expression system known in the art.
  • the final expression plasmids were transformed by a standard heat-shock method into competent E. coli host strains BL21 (Novagen, USA) and SG4044 (Gottesman et al, 1981) already transformed with resp.
  • the HCV polyepitope protein was produced from a (pre)culture in medium consisting of 20 g/1 of yeast extract (Becton Dickinson, ref. 212750 500G), 10 g/L of tryptone (Becton Dickinson, ref. 211705 500G), 5 g/L of NaCl and 10 mg/L of tetracycline.
  • Preculture medium 500 mL in 2L baffled shake flasks
  • Precultures were incubated at 28°C and 200-250 rpm for 22 to 24h.
  • Baffled shake flasks (2L) were filled with 500 mL of culture medium and inoculated 1/20
  • Ni 2+ -IMAC capture and intermediate purification performance was evaluated for the polyepitope construct encoded by SEQ ID NO 119, under denaturing conditions, after cell disruption by Gu.HCl-solubilization and disulphide bridge disruption, reversible cystein blocking and clarification.
  • cell pellet obtained from 2.7 L culture was resuspended in 10 volumes (10 mL buffer/gram wet weight cell pellet) of lysis buffer (6M Gu.HCl, 50 mM Na 2 HP0 4 .2H 2 0, pH 7.2) and sodium sulfite, sodium tetrathionate and L-cystein were added to final concentrations of respectively 320 mM, 65 mM and 0.2 mM. After subsequent pH adjustment to pH 7.2, solution was stirred overnight at room temperature in contact with air and shielded from the light. The cell lysate obtained was clarified by centrifugation (18.500 g for 60 minutes at 4°C).
  • n-dodecyl-N,N-dimethylglycine also known as lauryldimethylbetaine or Empigen BB ® , Albright & Wilson
  • imidazole were added to the protein solution to a final concentration of 3% (w/v) and 20 mM respectively and the pH was adjusted to pH 7.2.
  • the sample was filtrated through a 0.22 ⁇ m pore size bottle top filter with prefilter (Millipore).
  • the protein sample was loaded on the column.
  • the column was washed sequentially with IMAC-E buffer containing 3 % Empigen BB ® and IMAC-E buffer without 3 % Empigen BB ® till the absorbance at 280 nm reached the baseline level.
  • Further washing and elution of the fusion product was performed by the sequential application of IMAC-F buffer (20 mM Tris, 8 M urea, pH 7.2) supplemented with 20 mM imidazole, 50 mM imidazole, 200 mM imidazole and 700 mM imidazole respectively till the absorbance at 280 nm reached the baseline level.
  • Protein concentration in the 200 mM and 700 mM imidazole IMAC elution pools was determined by measuring absorbance at 280 nm and subtraction of the absorbance at 320 nm, assuming that a protein solution of 1 mg/mL in a cuvette with 1 cm optical pathlength yields an absorbance at 280 nm of 1.5. Results:
  • the polyepitope protein comprising the polyepitope construct represented by the amino acid sequence SEQ ID NO 147, was mainly recovered in the 700 mM imidazole fraction ( Figure 20b) with > 90% purity ( Figure 20a).
  • the intact N-terminus was confirmed by sequencing and the intact C-terminus was confirmed by Western Blot with a monoclonal antibody against the C-terminus. 93% of the polyepitope protein sequence was covered by peptide mass finger printing.
  • Example 7 Use of the HCV polyepitope protein in a prime boost treatment regimen
  • the purified protein as obtained in example 6 was diluted in desalting buffer (7M Urea, 2OmM Tris, 10% sucrose, pH 8) towards a concentration of 1 mg/ml and lOO ⁇ l (100 ⁇ g) was injected subcutaneously at the base of the tail using a BD MicrofineTM plus 1.0 cc insulin syringe in HLA transgenic mice.
  • the pMB75.6 vector comprising the nucleotide sequence represented by SEQ ID NO 103 ( Figure 7), generated as described in example 2 was diluted with PBS towards a concentration of lmg/ml and lOO ⁇ g was administered by bilateral injection of 50 ⁇ l in both m.tibialis anterior (after anaesthesia).
  • All injections with the polyepitope protein were administered subcutaneously at the base of the tail at a 100 ⁇ g dose.
  • DNA injections were given intramuscularly in the m.tibialis anterior at a 100 ⁇ g dose.
  • mice were euthanized 11 days after the last injection.
  • ELISPOT analyses for CTL responses (using methods as described in example 4) were performed on pooled spleen cells from 3 mice within the same immunization group.
  • ELISPOT analyses for ThI (IFN ⁇ ) were performed on pooled spleen cells from all 18 mice.
  • irradiated syngeneic spleen cells (2xlOE5 cells/well) were used as APC in vitro. These APC were loaded for 2 to 4 hours at a density of 5xlO 6 cells/ml with 10 ⁇ g/ml of peptide (Table 2). Cells were ⁇ -irradiated at 10 Gy, washed and pipetted through a cell strainer before addition to the wells.
  • CD4+ cells were purified by magnetic separation using anti-CD4 antibody- coated magnetic MACS beads (Miltenyi), according to the manufacturer's instructions.
  • a 96-well ELISPOT plate (MAIP HTS plates, Millipore) was coated overnight with an anti- IL-5 antibody (clone TRFK5, Mabtech) or an anti-IFNg antibody (clone ANl 8, MabTech) and blocked for 2 hours at room temperature with RPMI- 1640 medium supplemented with 5% Fetal Bovine serum.
  • CD4+ spleen cells (2x10E5 cells/well) together with peptide-loaded antigen presenting cells were added.
  • HCV-specif ⁇ c HLA-A24-restricted and HLA-Al 1 -restricted CTL responses were high when a protein prime immunization was boosted with DNA (figure 21 A and 21B). Positive responses towards both HLA-A24-restricted and to HLA-Al 1 -restricted epitopes were detected. These responses were higher than the responses following DNA only immunizations (i.e. a single injection of DNA) as shown for the HLA A24-restricted epitopes. Also high T helper 1 responses could be detected upon protein priming, followed by DNA boosting ( Figure 22).
  • HCV polyepitope protein is especially useful as a priming agent in a heterologuous prime/boost treatment regimen.

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Abstract

La présente invention porte sur un produit de construction de polyépitope HCV et sur son utilisation pour la prévention et/ou le traitement d'une infection par HCV.
PCT/EP2008/052517 2007-03-02 2008-02-29 Produit de construction de polyépitope hcv et ses utilisations WO2008107400A1 (fr)

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JP2012507280A (ja) * 2008-10-29 2012-03-29 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 改良型ｈｃｖワクチンおよびその使用方法
US20130337002A1 (en) * 2010-12-02 2013-12-19 Bionor Immuno As Peptide scaffold design
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JP2014502156A (ja) * 2010-12-02 2014-01-30 ビオノール イミュノ エーエス ペプチド骨格設計
EP2646459A4 (fr) * 2010-12-02 2014-12-03 Bionor Immuno As Conception d'échafaudage peptidique
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