WO2008107400A1

WO2008107400A1 - Hcv polyepitope construct and uses thereof

Info

Publication number: WO2008107400A1
Application number: PCT/EP2008/052517
Authority: WO
Inventors: Marie-Ange Buyse; Denise Baker; Gert Verheyden
Original assignee: Genimmune N.V.; Pharmexa Inc.
Priority date: 2007-03-02
Filing date: 2008-02-29
Publication date: 2008-09-12

Abstract

The present invention is directed to a HCV polyepitope construct and the use thereof for the prevention and/or treatment of HCV infection.

Description

HCV polyepitope construct and uses thereof

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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).

Virus-specific, human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes

(CTL) and HLA class Il-restricted helper T lymphocytes (HTL) are known to play a major role in the prevention of chronic infection and viral clearance in vivo (Houssaint et al, 2001; Gruters et al., 2002; Tsai et al., 1997; Murray et al., 1992; Tigges et al., 1992; Bowen and Walker, 2005).

Major Histocompatibility Complex (MHC) molecules are classified as either class I or class II. MHC class I molecules are expressed on virtually all nucleated cells. Peptide fragments presented in the context of MHC class I molecules are recognized by CD8+ T lymphocytes (cytotoxic T lymphocytes or CTLs). CD 8+ T lymphocytes frequently mature into cytotoxic effector cells which can lyse cells bearing the stimulating antigen. CTLs are particularly effective in eliminating tumor- and virus-affected cells.

MHC class II molecules are expressed primarily on activated antigen-presenting cells and lymphocytes. CD4+ T lymphocytes (helper T lymphocytes or HTLs) are activated upon recognition of a unique peptide fragment presented by a MHC class II molecule, usually found on an antigen presenting cell like a macrophage or dendritic cell. 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).

Of the many thousand possible peptides that are encoded by a complex foreign pathogen, only a small fraction ends up in a peptide form capable of binding to MHC class I or class II antigens and can thus be recognized by T cells if containing a matching T-cell receptor. This phenomenon is known as 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.

In view of the heterogeneous immune response observed after HCV infection, induction of a multi-specific cellular immune response directed simultaneously against multiple HCV epitopes is important for the development of an efficacious vaccine against HCV. The technology relevant to polyepitope vaccines is developing and a number of different approaches are available which allow simultaneous delivery of multiple epitopes. Several independent studies have established that induction of simultaneous immune responses against multiple and individual peptides can be achieved (Doolan et al (1997), Bertoni and colleagues (1997)). In terms of immunization with polyepitope nucleic acid vaccines, several examples have been reported where multiple T cell responses were induced. For example, minigene vaccines composed of approximately ten MHC Class I epitopes in which a plurality of epitopes were immunogenic and/or antigenic have been reported. Specifically, minigene vaccines composed of 9 EBV (Thomson et al, Proc, 1995), 7 HIV (Woodbeπy et al, 1999), 10 murine (Thomson et al, (1998) and 10 tumor-derived (Mateo et al., 1999) epitopes have been shown to be active. It has also been shown that a polyepitope DNA plasmid encoding nine different HLA- A2.1 and - Al 1 -restricted epitopes derived from HBV and HIV, induced CTL against all epitopes (Ishioka et al., 1999). WO04/031210 and WO05/089164 (Innogenetics et al.) disclose respectively HBV and HPV polyepitope constructs comprising more than 20 CTL epitopes with optimized immunogenicity.

Also in the field of HCV, 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. As an example, the expression of 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.

Hence, 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. However, 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. Not all epitopes remain immunogenic when administered within the context of a polyepitope construct compared to peptide immunization in the same species (Street et al., 2002). Moreover, not all immunogenic epitopes retain their antigenicity in the context of different polyepitope constructs. To address these factors, epitope selection should include identification of epitopes retaining their immunogenic properties in different microenvironments.

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). Therapeutic vaccination of 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. However, CTL escape mutations are constrained by a fitness cost to replication that varies between epitopes (Sόderholm 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).

There is a need to identify strategies to optimize polyepitope constructs for therapeutic and/or prophylactic use. Such optimization is important in terms of induction of potent immune responses and ultimately, for clinical efficacy. Accordingly, 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.

SUMMARY OF THE INVENTION

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. In one embodiment, 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. In a further embodiment, the polyepitope construct of the invention further comprises at least one CTL and/or HTL epitope. Preferably, 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.

In another embodiment, 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 125.

Optionally, the epitopes in the polyepitope construct are linked to each other by one or more spacer amino acids. Preferably, 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.

In a further embodiment, the CTL and/or HTL epitopes comprised in the polyepitope construct are sorted to minimize the number of CTL and/or HTL junctional epitopes.

In a specific embodiment, 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. In a further embodiment, 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.

Moreover, the current invention also relates to a polynucleotide encoding the polypeptide as described herein.

Optionally, the polynucleotide of the present invention further comprises one or more regulatory sequences. Preferably, said regulatory sequence is an internal ribosome binding site (IRES).

In a specific embodiment, the polynucleotide of the present invention further comprises one or more promoters. Preferably, the promoter is a CMV promoter.

In another embodiment, the polynucleotide of the invention comprises one or more MHC class I and/or MHC class II-targeting sequences. Preferably, 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.

Specifically, 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. In a further embodiment, 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.

Furthermore, the invention encompasses a vector comprising the polynucleotide as described herein. Preferably, the vector is an expression vector. More preferably, the vector is a plasmid, a viral or bacterial vector. In a further embodiment, the viral vector is a pox virus. Preferably, 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. Preferably, the composition further comprises a pharmaceutical acceptable excipient. In a specific embodiment, the composition is a vaccine.

In another embodiment, 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

Specifically, 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.

In another embodiment, 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.

In a specific embodiment, 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.

Moreover, 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.

More particular, the T cell response comprises a Cytotoxic T Lymphocyte (CTL) response and/or a T Helper (HTL) response. More specific, the CTL response is a CD8+ T cell response and the HTL response is a CD4+ T cell response. Even more particular, the epitopes encoded by the vector are the same as the epitopes comprised in the polypeptide. In a specific embodiment, the vector for use as a boosting agent is a plasmid, a viral vector, a bacterial vector or a yeast vector.

Furthermore, 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.

Moreover, the present invention includes a cell comprising the polypeptide, the polynucleotide, or the vector as described herein.

In a further embodiment, 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. In a particular embodiment, 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.

Furthermore, the invention covers a method of making the polypeptide, the polynucleotide, the vector, the composition, or the cell as described herein.

It is generally noted that the wording "use of the composition, the polynucleotide, the vector or the polypeptide for the preparation of a medicament for treating and/or preventing HCV" in the present invention can alternatively be construed as "the composition, the polynucleotide, the vector or the polypeptide for use in treating and/or preventing HCV ". FIGURE LEGENDS

Figure 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;

"..J "_: 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;

"... x — " _: more than 1 AA has been identified in the respective sample at position 4 of the epitope instead of the AA found in the epitope as shown in the top of the table;

"????????": no sequencing results available of this HCV region for this particular sample.

Figure 2: Organization of polyepitope-encoding insert. Figure 3: Construct ICCG5754: A. Amino acid sequence of the signal sequence and the poly epitope,

B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded,

C. Amino acid sequence of the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 4 : Construct ICCG5755 :

A. Amino acid sequence of the signal sequence and the polyepitope,

C. Amino acid sequence of the polyepitope, D. DNA sequence encoding the polyepitope.

Figure 5: Construct ICCG5756:

A. Amino acid sequence of the signal sequence and the polyepitope,

B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded, C. Amino acid sequence of the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 6: Construct ICCG5796:

A. Amino acid sequence of the signal sequence and the polyepitope, B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded,

C. Amino acid sequence of the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 7: Construct ICCG5959:

A. Amino acid sequence of the signal sequence and the polyepitope,

Figure 8: Construct ICCG5946:

A. Amino acid sequence of the signal sequence and the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 9: Construct ICCG5947:

A. Amino acid sequence of the signal sequence and the polyepitope,

C. Amino acid sequence of the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 10: Construct ICCG5768:

C. Amino acid sequence of the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 11: Construct ICCG5741 : A. Amino acid sequence of the signal sequence and the polyepitope,

C. Amino acid sequence of the polyepitope,

D. DNA sequence encoding the polyepitope. Figure 12: HCV Ib consensus sequence (SEQ ID NO 112).

Figure 13: A. Amino acid sequence of the HCV poly epitope protein (CTL-HTL) with N-terminal translation initiator Met (M),

B. Amino acid sequence of the HCV polyepitope protein (HTL-CTL) with N-terminal translation initiator Met (M),

C. Amino acid sequence of the HCV polyepitope protein (CTL-HTL),

D. Amino acid sequence of the HCV polyepitope protein (HTL-CTL).

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

(HTL-CTL), linker and tag,

B. Nucleic acid sequence encoding the HCV polyepitope protein (HTL-CTL). 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. coli SG40440 (pcI857) 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. DETAILED DESCRIPTION OF THE INVENTION

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. More specifically, 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.

Identification of the epitopes

Based on the hundreds of known HCV genotypes and subtypes (at least 3000 amino acids per sequence), thousands of theoretical CTL and/or HTL epitopes can be predicted. The HLA class I binding epitopes of the present invention have been identified by the method as described in WO05/118626 -GENimmune NV et al. (incorporated herein by reference). 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. To this, 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). At the end of the culture period, T cell activity was detected using assays such as ⁵¹Cr 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. To this, a single immunization with CTL peptide pools together with a common HTL epitope emulsified in IFA was performed and up to 14 days later, CD8+ spleen cells were isolated and evaluated for epitope specificity using a direct ex vivo IFNγ ELISPOT assay. A large set of immunogenic epitopes was identified.

Design of the polyepitope construct

Several 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.

Identification of conserved and efficiently processed epitopes For the design of the most efficacious construct we evaluated the conservancy of the selected candidate therapeutic CTL epitopes within HLA-matched chronic HCV patients, as is shown in Example 1. A combined approach of prediction and selection of epitopes within highly conserved regions and evaluation of HLA-restricted viral mutations of these epitope sequences has lead to the identification of a subset of HCV-derived T-cell epitopes highly suitable to be included in an HCV vaccine. Epitopes were considered not stable and prone to HLA-driven viral adaptation when there was a statistical difference (p<0,05 using two-tailed Fisher's exact test) in the number of mutated epitopes in HLA-matched patients versus HLA-unmatched patients. Certain epitopes were found to be clearly prone to HLA-related viral escape, though several of the epitopes were highly conserved within the set of samples tested. Consequently, including a large set of highly conserved epitopes (within the context of a matching HLA) in a therapeutic polyepitope T cell vaccine for HCV minimizes the chance that the virus escapes immune pressure through HLA-related viral mutations.

In addition, efficient processing of the individual epitopes was assessed as described in Examples 2 to 4. The data indicate that not all epitopes remain immunogenic when administered within the context of polyepitope plasmid DNA compared to peptide immunization, which is probably due to less efficient T-cell priming. Moreover, not all immunogenic epitopes retain their antigenicity in the context of different polyepitope constructs, pointing to the sensitivity of the molecular environment for these epitopes. However, the present invention has identified for the first time epitopes that are immunogenic irrespective of their location and micro-environment in the constructs tested.

Accordingly, 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. In a preferred embodiment, 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. Moreover, as shown in Example 1, said epitopes are not prone to viral escape. Accordingly, 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. Furthermore, 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. Preferably, the epitopes of the poly epitope construct are directly or indirectly linked to one another in the same reading frame.

The term "construct" as used herein generally denotes a composition that does not occur in nature. As such, 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". The term "isolated" refers to material that is substantially free from components that normally accompany it as found in its naturally occurring environment. However, it should be clear that the 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. As such, 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. It is thus to be understood that 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.

With regard to a particular amino acid sequence, 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.

With regard to a particular nucleic acid sequence, a "nucleic acid epitope" is a set of nucleic acids that encode for a particular amino acid sequence that forms an epitope. In an immune system setting, in vivo or in vitro, 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.

The term "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.

The term "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, and 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. 8, 9, 10, or 11), and most preferably 9 or 10 amino acids in length. 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. In a preferred embodiment, 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). Accordingly, 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. Preferably, the at least one CTL and/or HTL epitope is derived from HCV. Even more preferably, 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. As shown in Examples 2-4, 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 at least one of the constructs tested.

Multiple HLA class I or class II epitopes present in a polyepitope construct can be derived from the same antigen, or from different antigens. For example, 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. In a preferred embodiment, 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. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more amino acids. 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.

In a preferred embodiment, the polyepitope construct of the present invention further comprises the universal T cell epitope called PADRE^® (Pharmexa A/G, Hørsholm); 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). 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. Alternatively 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. Hence, 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. Furthermore, 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.

The term "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:

1) Evaluation of primary 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 ⁵¹Cr-release assay involving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth et al., 1996; Wentworth et al., 1996a; Alexander et al., 1997) or surrogate mice. In this method, peptides (e.g. formulated in incomplete Freund's adjuvant) are administered subcutaneously to HLA transgenic mice or surrogate mice. Eleven to 14 days following immunization, 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 ⁵¹Cr-release assay involving peptide- sensitized target cells and/or target cells expressing endogenously generated antigen.

Alternatively, 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.

3) Demonstration of recall T cell responses from immune individuals who have effectively been vaccinated, recovered from infection, and/or from chronically infected patients (see, e.g., Rehermann et al., 1995; Doolan et al., 1997; Bertoni et al., 1997; Threlkeld et al., 1997; Diepolder et al., 1997). In applying this strategy, recall 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. At the end of the culture period, T cell activity is detected using assays including ⁵¹Cr release involving peptide-sensitized target cells, T cell proliferation, or cytokine release. 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.

Based on the data derived from the different constructs, a further selection of candidate epitopes is made according to their immunogenicity profile in the different settings. Immunogenicity for the epitopes of the invention is indicated in Tables 4 and 5. A "+" indicates T cell reactivity in at least one sample.

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.

Table A

Sequence name in Los Alamos lb.274933RU AF176573 lb.AB016785 AB016785 lb.Conl AJ238799 lb.D89815 D89815 lb.HC-C2 D10934 lb.HCRβ AY045702 lb.HCU16362 U16362 lb.HCU89019 U89019 lb.HCV-AD78 AJ132996 lb.HCV-A AJ000009 lb.HCV-BK M58335 lb.HCV-CGIB AF333324 lb.HCV-JS D85516 lb.HCV-J D90208 lb.HCV-Kl-Rl D50480 lb.HCV-Kl-R2 D50481 lb.HCV-Kl-R3 D50482 lb.HCV-L2 U01214 lb.HCV-N AF 139594 lb.HCV-N D63857 lb.HCV-Sl AF356827 lb.HCV-S AY460204 lb.HCV-TRl AF483269 lb.HCVT050 AB049087 lb.HCVT094 AB049088 lb.HCVT109 AB049089 lb.HCVT140 AB049090 lb.HCVT142 AB049091 lb.HCVT145 AB049092 lb.HCVT150 AB049093 lb.HCVTlβl AB049094 lb.HCVT169 AB049095 lb.HCVT191 AB049096 lb.HCVT197 AB049097 lb.HCVT209 AB049098 lb.HCVT212 AB049099 lb.HCVT217 AB049100 lb.HCVT221 AB049101 lb.HD-1 U45476 lb.HEBEI L02836 lb.HPCGENANTI M84754 lb.HPCHCPO D45172 lb.HPCPP D30613 lb.HPCUNKCDS M96362 lb.J33 D14484

IbJKl-Ml X61596 lb.JP1993130874-A/l_E05027 lb.JT Dl ] 1168

Ib .MILE AB080299

Ib .MDl-I AF 165045

Ib .MD 10-l AF 165063

Ib .MDI l AF207752

Ib .MD 12 AF207753

Ib .MD 13 AF207754

Ib .MD14_ AF207755

Ib .MD 15 AF207756

Ib .MD 16 AF207757

Ib .MD 17 AF207758

Ib .MD 18 AF207759

Ib .MD 19 AF207760

Ib .MD2-1 AF 165047

Ib .MD20 AF207761

Ib .MD21 AF207762

Ib .MD22 AF207763

Ib .MD23 AF207764

Ib .MD24 AF207765

Ib .MD25 AF207766

Ib .MD26 AF207767

Ib .MD27 AF207768

Ib .MD28 AF207769

Ib .MD29 AF207770

Ib .MD3-1 AF 165049

Ib .MD30 AF207771

Ib .MD31 AF207772

Ib .MD32 AF207773

Ib .MD33 AF207774

Ib .MD34 AF208024

Ib .MD4-1 AF 165051

Ib .MD5-1 AF 165053 lb.MD6-l AF165055 lb.MD7-l AF165057 lb.MD8-l AF165059 lb.MD9-l AF165061 lb.NCl AJ238800 lb.pCV-J4L6S_AF054247 lb.Source AF313916 lb.TMORF D89872

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

(direct of via a linker), including, without limitation, recombinant fusion, covalent binding, non- covalent binding, disulfide binding, ionic binding, hydrogen binding, polymerization, cyclization, electrostatic binding and connecting through a central linker or carrier. Polymerization can be accomplished for example by reaction between glutaraldehyde and the - NH2 groups of the lysine residues using routine methodology.

In a specific embodiment, 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.

To develop polyepitope constructs using the epitopes of the present invention, 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. As described herein, 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, ⁵¹Cr 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). According to a specific embodiment, 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. It is preferred that 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. For example, 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). For separating class I epitopes, or separating a class I and a class II epitope, 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. In a preferred embodiment, 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.

In some polyepitope constructs, it is sufficient that 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.

The only outer limit on the total length and nature of each spacer sequence derives from considerations of ease of synthesis, proteolytic processing, and manipulation of the polypeptide.

The (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. Thus, "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.

Moreover, 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. In order to carry out the expression of the polyepitopic polypeptide in bacteria, in eukaryotic cells (including yeast) or in cultured vertebrate hosts such as Chinese Hamster Ovary (CHO), Vero cells, RK13, COSl, BHK, and MDCK cells, or invertebrate hosts such as insect cells, the following steps are carried out: 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, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence, culture of said transformed cellular host under conditions enabling the expression of said insert. As such, the present invention also relates to a cell or host cell comprising a polypeptide, a polynucleotide or a vector containing a polyepitope construct.

The polyepitopic polypeptide can be purified by methods well known to the person skilled in the art.

For therapeutic or prophylactic immunization purposes, 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.

In addition to a promoter sequence, 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.

In a particular embodiment, the polynucleotide of the present invention further comprises one or more regulatory sequences. By "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, for example, 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.

In a further embodiment, 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".

The use of a 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

WO04/031210 (Pharmexa Inc. et al.). In a specific embodiment, 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.

The phrase "operably linked" or "operatively 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). For example, 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 (such as a protein encoding sequence and a promoter region sequence linked to the 5' end of the encoding sequence) 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. Thus, a promoter region would be operably linked to a nucleotide sequence if the promoter were capable of effecting transcription of that nucleotide sequence.

One or more 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. In particular, "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. In this respect, it is to be understood that alkylation of cysteine thiol-groups refers to the replacement of the thiol-hydrogen by (CH₂)_nR, in which n is 0, 1, 2, 3 or 4 and R= H, COOH, NH₂, CONH₂ , phenyl, or any derivative thereof. Alkylation can be performed by any method known in the art, such as, for example, active halogens X(CH₂)_nR in which X is a halogen such as I, Br, Cl or F. Examples of 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. In particular, 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. The term "reversible protection by enzymatical means" as used herein contemplates reversible protection mediated by enzymes, such as for example acyl-transferases, e.g. acyl-transferases that are involved in catalysing thio- esterifϊcation, such as palmitoyl acyltransferase. The term "reversible protection by chemical means" as used herein 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, SnCl₂, 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. pH increase; enzymes, in particular thioesterases, glutaredoxine, thioredoxine, in particular in a concentration of 0,01-5 μM, even more particular in a concentration range of 0,1-5 μM.; combinations of the above described chemical and/or enzymatical conditions. The removal of the reversibly protection state of the cysteine residues can be carried out in vitro or in vivo, e.g. in a cell or in an individual. Alternatively, 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

The current invention furthermore relates to compositions comprising a polynucleotide, a polypeptide or a vector comprising the HCV polyepitope construct as described herein, or a combination thereof. In a specific embodiment, the composition furthermore comprises at least one of a pharmaceutically acceptable excipient, i.e. a carrier, adjuvant or vehicle. The terms "composition", "immunogenic composition" and "pharmaceutical composition" can be used interchangeably. More particularly, said immunogenic composition is a vaccine composition. Even more particularly, 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. Alternatively, 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. The infection can cause liver inflammation (hepatitis) that is often asymptomatic, but ensuing chronic hepatitis can result later in cirrhosis (fϊbrotic 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. In particular the vaccine composition of the invention is a HCV vaccine composition. In particular, the vaccine or vaccine composition comprises an effective amount of the peptides, polypeptide, nucleic acids or polynucleotide of the present invention. In a specific embodiment, 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. human, non-human primate, primate, etc.), the capacity of the individual's immune system to mount an effective immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment, the strain of the infecting pathogen and other relevant factors. 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. Preferably, the subject or individual is a primate. Even more preferably, the subject or individual is a human.

Carriers, adjuvants and vehicles - delivery

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.

Various art-recognized delivery systems may be used to deliver a polyepitope construct into appropriate cells. The 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. Preferably, 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. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles.

Typically, 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. After 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. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art.

The approach known as "naked DNA" is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of DNA vaccines, 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). In addition, 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.

Further examples of 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. In this context it is noted that practically all considerations pertaining to the use of adjuvants in traditional vaccine formulation apply to the formulation of DNA vaccines.

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.

In a preferred embodiment of the invention, 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.

Detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, e.g. by Donnelly J.J. et al, 1997 and 1997 'a. Examples of expression vectors include attenuated viral hosts, such as a pox virus. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention. 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. Further examples are: 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, and vectors derived from Epstein Barr virus. A wide variety of other vectors useful for therapeutic administration or immunization of the epitopes of the invention, e.g. retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Additional vector modifications may be desired to optimize polynucleotide expression and immunogenicity. In some cases, 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. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing polynucleotide expression. In addition, immunostimulatory sequences (ISSs or CpGs) 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. In some embodiments, 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. Examples of 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. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-P) may be beneficial in certain diseases.

The use of polyepitope constructs is 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. For example, 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.

All disclosures herein which relate to use of adjuvants in the context of protein or (polypeptide based pharmaceutical compositions apply mutatis mutandis to their use in nucleic acid and vector vaccination technology. The same holds true for other considerations relating to formulation and mode and route of administration and, hence, also these considerations discussed herein in connection with a traditional pharmaceutical composition apply mutatis mutandis to their use in nucleic acid and vector vaccination technology.

Medical use

In a further embodiment, 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. Preferably, said medicament is a vaccine. More specifically, 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. In a specific embodiment 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. Furthermore, 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.

In a further embodiment, 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. In this regard, it is to be understood that the term "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. In a particular embodiment, 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.

It has also been demonstrated in the present invention that the use of 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. The term "heterologous" as used herein 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. In a particular embodiment, 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. In a further embodiment, 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.

In a particular embodiment, the vector is a plasmid, a bacterial, a viral vector or a yeast vector. More preferably, the epitopes encoded by the vector of the boosting agent are the same as the epitopes of the polyepitope construct of the priming polypeptide. Accordingly, 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. Typically 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. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.

Other arrangements of the methods and tools embodying the invention will be obvious for those skilled in the art.

It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for the methods and tools according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.

The present invention is illustrated by the following Examples, which should not be understood to limit the scope of the invention to the specific embodiments therein.

EXAMPLES

Example 1: Evaluation of epitope conservancy within HLA-matched HCV patients

Frozen plasma samples from 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).

- : not available

Primer design

Based on a HCV genotype Ib consensus sequence derived from the Los Alamos database or GenBank Accession number D90208, 30 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 μL 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 ThermoScript™ RT-PCR System Protocol (Invitrogen™). No-template negative controls were included. PCRs were performed with the AccuPrime™ 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 50-60⁰C, 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 aspecifϊc) bands were separated on a preparative agarose gel. In this case, the correct fragment was isolated, extracted and purified according to the MiniElute^® or QiaExII^® protocol (Qiagen). 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.

Results

The HCV amino acid sequencing data from the 63 patients were compared with the amino acid sequence of the immunogenic epitopes.

As an example, detailed sequencing results for a selection of immunogenic epitopes are shown in Figure 1 and table 3. Table 3

Seven of these epitopes are highly conserved (>80% conservancy) in this sample set. However, for one of these epitopes (GPRLGVRAT), there is an increased frequency of mutation in HLA-matched patient samples. Four of the eleven HLA-matched patients show mutations within the epitope. However, no mutations are found in patients who have no matching HLA. This indicates that this epitope is prone to HLA-related viral escape. A second example concerns a less conserved epitope (LIRLKPTLH). For this epitope, there is no indication that HLA-related viral escape is the driver for the mutations within the epitope.

Example 2: Poly epitope construct design

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. The respective amino acid and DNA sequences of the constructs ICCG 5754, ICCG 5755, ICCG 5756, ICCG 5796, ICCG 5959, ICCG 5946, ICCG 5947, ICCG 5768 and ICCG5741 are given in Figures 3-11.

Example 3: Immunogenicity of HCV-derived HLA-class I- restricted epitopes encoded in HCV DNA constructs

In the following examples, all 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. As an example, 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 and Fl HLA- B*0702/Kb.C57BL/6xBalb/c trangenic 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 naϊve 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. For the DNA immunizations, (HLA transgenic) mice were pretreated by injecting 50 μl 10 μM cardiotoxin bilaterally into each tibialis anterior muscle; 3-5 days later, the same muscles were injected with a total of 100 μg plasmid DNA diluted in PBS.

Each group was tested in two independent experiments. Naϊve 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.

Immunization and testing schedule for peptide immunogenicity experiments

In vivo In vitro r 3-5 days \ f - 14 days 2 days I

Group Week 3 Week 2 Week I

1 Cardiotoxsn DMA If

2 - Naive < Spleens were disrupted with a 15-ml tissue grinder and the resulting single cell suspensions were treated with DNAse solution (10 μl/spleen of 30 mg/ml DNAse in PBS), washed in RPMI- 1640 medium with 2% FCS, and counted. Splenocytes were then incubated at 4° C for 15-20 minutes in 300μl MACS^® buffer (PBS with 0.5% BSA and 2mM EDTA) with 35μl of MACS^® CD8a(Ly-2) Microbeads/10⁸ cells according to the manufacturer's specifications. The cells were then applied to a MACS^® column (Miltenyi Biotech) and washed four times. 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 μM 2-ME, 0.5 mM sodium pyruvate, lOOμg/ml streptomycin and 100 U/ml penicillin. (RPMI-10 medium), washed, and counted again.

IFNγ ELISPOT

The responses to CTL epitopes were evaluated using an IFN-γ 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 4⁰C with anti-mouse IFN-γ monoclonal antibody (Mabtech MabAN18) at a concentration of lOμg/ml in PBS. After washing 3 times with PBS, RPMI-10 medium was added to each well, and the plates were incubated at 37⁰C 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 4x10⁵ 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 lOμg/ml), and mixed with target cells (10⁵ HLA- A2. I/Kb transfected Jurkat cells/well and 10⁵ HLA-Al 101/Kb transfected Jurkat cells/well for resp. HLA- A02- and HLA-AI l -restricted peptides). Controls of irrelevant peptide and ConA (lOμg/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 37⁰C in 5% CO₂. 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-γ antibody (Mabtech MabR4-6A2-Biotin) at a final concentration of 1 μg/ml for 4 hours at 37⁰C. 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. Finally, the plates were developed with AEC (1 tablet 3-Amino-9- ethylcarbazole dissolved in 2.5 ml dimethylformamide, and adjusted to 50 ml with acetate buffer; finally, 25μl of 30% H₂O₂ was added to the AEC solution), washed, and dried. Spots were counted using an AID plate reader. Each peptide was tested for recognition in both the immunized group and the naϊve group. Data was collected in triplicate for each experimental condition.

Data analysis

The raw data for the irrelevant peptide control were averaged for each group (both naϊve 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 10⁶ 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 naϊve controls. The data was reported as the number of peptide-specifϊc IFNγ-producing cells, termed Spot-Forming Cells (SFCyiO⁶ CD8⁺ cells. Data was considered positive if significantly different than the naϊve controls (p<0.05) and if result is > 30 SFC/10⁶ cells.

Results

The results for the different constructs are shown in Table 4.

Table 4

ICCG 5754 Naϊve

SFC/ immuno

HLA transgenic SFC/10e6 10e6 genie SEQ mouse model Sequence cells + S.E. Ttest cells + S.E. (+/-) ID NO

QIVGGVYLL -1 ,3 + 2,9 0,15 -0,4 + 2,6 57

YLLPRRGPRL 338,3 ± 17,5 0,00 2,5 ± 2,1 + 87

SMVGNWAKV 2,1 + 3,5 0,13 2,9 + 1 ,8 73

CLVDYPYRL 1095,0 ± 72,2 0,00 2,5 ± 2,1 + 12

YLVTRHADV 2,9 ± 1 ,8 0,32 2,5 ± 1 ,8 90

GMFDSSVLC 5,4 + 1 ,2 0,09 1 ,3 + 1 ,9 23

F1 HLA- SVFTGLTHI 262,5 ± 30,1 0,00 2,1 ± 1 ,6 + 76

A0201/Kb.C57BL/6 YLVAYQATV 35,8 ± 12,5 0,25 5,8 ± 1 ,7 89 x Balb/c QMWKCLIRL 890,0 + 40,4 0,00 0,0 + 2,8 + 59

RLGAVQNEV 1 ,3 ± 2,2 0,11 2,9 ± 2,8 62

HMWNFISGI 431 ,3 ± 43,6 0,01 -0,4 ± 2,0 + 30

LLFNILGGWV 17,1 + 15,1 0,11 -0,8 + 0,8 42

KLQDCTMLV 1628,3 ± 59,8 0,00 0,8 ± 2,1 + 36

TLWARMILM 1308,3 + 47,7 0,00 5,8 + 2,0 + 78

YLFNWAVRT 0,0 + 2,1 0,02 2,1 + 2,2 86

ICCG 5755 Naϊve SFC/ immuno

HLA transgenic SFC/10e6 10e6 genie SEQ mouse model Sequence cells ± S.E. Ttest cells + S.E. ID NO

YLLPRRGPRL 255,0 ± 37,5 0,00 -6,3 ± 1 ,1 + 87

DLMGYIPLV 34,6 ± 6,4 0,00 -1 ,7 + 1 ,6 + 13

YIPLVGAPL 3,3 ± 2,8 0,04 -3,8 + 1 ,8 - 85

FLLALLSCL 0,8 ± 2,6 0,09 -5,8 ± 2,4 - 17

FLLLADARV -2,5 ± 1 ,9 0,23 -5,8 + 2,7 - 18

GLLGCIITSL -2,9 ± 1 ,2 0,25 -5,0 ± 2,4 - 22

F1 HLA- KVLVLNPSV -0,8 ± 2,3 0,24 -3,8 ± 2,7 - 40

A0201/Kb.C57BL/6 YLNTPGLPV -0,4 ± 1 ,2 0,04 -6,3 + 2,4 - 88 x Balb/c YQATVCARA -4,6 ± 1 ,4 0,50 -4,6 ± 1 ,9 - 91

TLHGPTPLL -3,8 ± 1 ,2 0,42 -2,9 ± 2,8 - 77

VLVGGVLAAL -2,1 ± 2,3 0,09 -7,9 + 2,2 - 83

HMWNFISGI 232,9 ± 20,8 0,00 -4,2 ± 2,3 + 30

ILAGYGAGV -2,5 ± 1 ,1 0,00 -8,3 + 1 ,2 - 33

ALYDWSTL 650,8 ± 224,4 0,02 -4,6 + 1 ,8 + 3

NIIMYAPTL 3,8 ± 2,7 0,15 -1 ,7 ± 2,5 - 54

ICCG 5946 Naϊve

SFC/ immuno

HLA transgenic SFC/10e6 10e6 genie SEQ mouse model sequence cells ± S.E. T test cells + S.E. ID NO

YLLPRRGPRL 16,3 ± 3,7 0,16 5,0 + 10,3 87

GLLGCIITSL 15,0 ± 5,8 0,05 3,3 ± 7,6 - 22

F1 HLA-

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

NIIMYAPTL 16,3 ± 6,2 0,07 7,5 + 7,3 - 54

TLWARMILM 314,2 ± 83,2 0,01 17,5 ± 7,3 + 78

ICCG 5947 Naϊve

SFC/ immuno

HLA transgenic SFC/10e6 10e6 genie SEQ mouse model sequence cells ± S.E. T test cells ± S.E. ID NO

YLLPRRGPRL 105,4 ± 40,3 0,01 5,0 ± 10,3 + 87

GLLGCIITSL -0,4 ± 2,2 0,28 3,3 + 7,6 - 22

F1 HLA-

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

NIIMYAPTL 3,3 ± 3,4 0,25 7,5 ± 7,3 - 54

TLWARMILM 1043,8 ± 52,3 0,00 17,5 + 7,3 + 78

ICCG 5756 Naϊve

SFC/ immuno

HLA transgenic SFC/10e6 10e6 genie SEQ mouse model Sequence cells ± S.E. T test cells ± S .E. (+/-) ID NC

F1 HLA- A1101/Kb.C57BL/6 STNPKPQRK 1569,2 ± 26,1 0,00 -4,6 ± 0,8 75 x Balb/c RLGVRATRK -7,9 0,7 0 ,00 -2, ,9 ± 0,7 63

KTSERSQPR 103,8 12,5 0 ,02 2. ,9 ± 2,1 38

QLFTFSPRR 3,3 1 ,2 0 ,36 0_; A ± 2,1 58

RLLAPITAY 6,3 1 ,9 0 ,25 0: A ± 1 ,9 64

AVCTRGVAK 979,6 81 ,3 0 ,00 3_; ,8 ± 1 ,5 9

HLHAPTGSGK 2,1 0,8 0 ,39 0_; A ± 1 ,5 28

AAYAAQGYK 11 ,7 3,1 0 ,07 -6_; ,7 ± 1 ,3 2

HLIFCHSKK 1 ,3 1 ,3 0 , 19 -3_; ,3 ± 1 ,2 29

LIFCHSKKK 1 ,3 1 ,0 0 , 10 -3_; ,3 ± 1 ,0 41

KVLVDILAGY 3,3 1 ,9 0 ,05 -8_; ,3 ± 0,8 39

GWCAAILRR 28,8 1 ,9 0 ,00 -3_; ,3 ± 1 ,2 27

GWCAAILR 5,8 1 ,3 0 , 16 -1 ,7 ± 1 ,6 26

RVCEKMALY 8,3 2,5 0 ,06 -3_; ,8 ± 1 ,2 68

LVNAWKSKK -0,4 0,5 0 , 13 -4_; ,6 ± 0,9 51

ASAACRAAK 4,2 1 ,2 0 , 19 -0: ,8 ± 1 ,1 8

RVFTEAMTR 123,3 8,2 0 ,00 -1. ,3 ± 1 ,0 70

ICCG 5768 Naϊve

SFC immuno

HLA transgenic SFC /10⁶ /10⁶ genie SEQ mouse model Sequence cells + S.E. T test cells + S.E. (+/-) ID NO

LPRRGPRLGV 64,2 + 22,2 0 ,03 3,3 + 4,4 + 48

LPRRGPRLG 18,3 + 13,8 0 ,28 6,3 + 5,8 - 49

QPRGRRQPI 5,8 ± 29,4 0 ,49 5,0 ± 8,9 60

SPRGSRPSW 12,1 + 26,2 0 ,33 -1 ,3 + 2,5 - 74

IPLVGAPL -3,3 ± 25,7 0 ,38 7,1 ± 7,8 34

LPGCSFSIF -4,6 + 23,7 0 ,40 2,5 + 3,5 - 46

HPNIEEVAL -25,0 ± 17,4 0 ,14 0,0 ± 3,9 31

F1 HLA- AAKLSALGL -2,9 + 25,4 0 ,44 2,1 + 5,8 - 1

B0702/Kb.C57BL/6 IPTSGDVVV -2,1 ± 28,8 0 ,45 2,9 ± 7,6 - 35 x Balb/c LPVCQDHLEF -15,8 ± 23,2 0 ,28 1 ,3 ± 5,1 50

FPYLVAYQA -14,6 + 22,9 0 ,34 -2,5 + 4,6 - 19

NPAIASLMAF -8,3 ± 24,5 0 ,32 6,7 ± 7,5 - 55

EPEPDVAVL -5,4 + 27,1 0 ,47 -2,9 + 4,4 - 16

EPDVAVLTSM -11 ,3 ± 21 ,4 0 ,35 -1 ,7 ± 3,3 15

LPINALSNSL 152,1 + 33,2 0 ,00 11 ,7 + 10,8 + 47

APTLWARMIL 996,3 ± 278,0 0 ,01 0,4 ± 6,6 + 6

APTLWARMI 626,7 ± 192,1 0 ,01 22,5 ± 9,3 + 7

ICCG 5741 Naϊve

SFC immuno

HLA transgenic SFC /10⁶ /10⁶ genie SEQ mouse model Sequence cells ± S.E. T test cells ± S.E. (+/-) ID NO

F1 HLA- LPRRGPRLGV 422,9 + 111 ,9 0,01 3,3 4,4 + 48

B0702/Kb.C57BL/6 LPRRGPRLG 357,1 ± 102,0 0,01 6,3 5,8 + 49 x Balb/c GPRLGVRAT 2,5 + 12,0 0,27 -4,6 1 ,3 24

DPRRRSRNL 15,0 ± 21 ,7 0,28 4,2 4,8 14

APLGGAARAL 7,5 ± 19,0 0,30 -0,8 5,2 4

LPALSTGLI 3,3 + 18,9 0,44 5,8 5,7 45

YAAQGYKVL -5,8 ± 15,0 0,28 0,0 6,2 84

TPGERPSGM 7,9 ± 22,5 0,31 -3,8 ± 3,2 79

RPSGMFDSSV -5,8 ± 13,1 0,38 -10,0 ± 2,1 67 APPPSWDQM 10,0 ± 15,2 0,17 -3_; ,8 ± 3,4 5

KPTLHGPTPL -2,9 ± 13,3 0,45 -4_; ,6 ± 2,9 37

GPTPLLYRL 1 ,7 + 18,2 0,40 -3_; ,3 + 2,2 - 25

LPAILSPGAL 4,6 ± 19,5 0,28 -6_; ,7 ± 2,7 - 44

RPDYNPPLL 5,8 + 21 ,0 0,28 -7. ,1 + 1 ,5 - 66

PPVVHGCPL -1 ,7 ± 17,3 0,41 -5 ,0 ± 4,7 56

SAACRAAKL 7,5 + 23,7 0,28 -7. ,1 + 1 ,4 - 71

APTLWARMIL 178,3 ± 53,8 0,01 o. A ± 6,6 + 6

APTLWARMI 104,2 ± 32,9 0,01 22 _; ,5 ± 9,3 + 7

S. E. = standard error

The results show that not all 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.

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. As an example, 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^bxBalb/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 50μl (for mice <20g) to lOOμl (for mice >20g) of a lOμM cardiotoxin solution. Five days later, all mice were immunised with lOOμg HCV-DNA plasmid by bilateral injection of 50μg 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 70μM-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.

CD8+ cells were purified from pooled spleen cells by magnetic separation using anti-CD8 antibody-coated magnetic beads (MACS^®, Miltenyi). Isolation was performed according to the manufacturer's instructions. Finally, CD8+ cells were resuspended in complete RPMI-10 medium (RPMIi64o medium + 10% iFCS, 50μM β-mercapto-ethanol, Ix MEM non-essential amino acids, 1 mM MEM sodium pyruvate and 50μg/ml gentamycin (= assay medium)) and counted. Purity of the CD8+ cells was determined by FACS staining using PE-labelled anti- CD8 antibodies (Pharmingen).

IFNγ ELISPOT A 96-well ELISPOT plate (Millipore, transparent non-sterile MAIP HTS plates) was coated overnight at 4°C with an anti-IFNγ 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 5x10⁴ and 2x10⁵ cells/well, depending on availability) and antigen-presenting cells (loaded or not-loaded with peptide) were added. For HLA-A24-restricted epitopes, antigen-presenting cells (APC) were either LCL 721.221 cells transfected with an HLA-A24/K^b hybrid construct (10⁴ cells/well) or syngeneic spleen cells from non- immunised HLA-A24/K^b transgenic mice (2x10⁵ cells/well). For HLA-AOl -restricted epitopes syngeneic spleen cells from non- immunised HLA-A01/K^b transgenic mice (2x10⁵ cells/well) were used. For HLA- A02- and HLA-Al 1 -restricted epitopes, respectively Jurkat cells transfected with an HLA-A02/K^b hybrid construct (2x10⁴ cells/well) and LCL 721.221 cells transfected with an HLA-Al 1/K^b hybrid construct (10⁴ cells/well) were used. As a positive control, cells were stimulated with the polyclonal stimulus PHA (2μg/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-IFNγ antibody (Mabtech, clone R46A2) for 2 hours at RT. After washing, plates were incubated with streptavidin-HRP (Becton Dickinson) for 1 hour at RT. After this incubation period, spots were visualised using AEC as a substrate. Rinsing the plates with tap water stopped the colour reaction. Plates were then dried and read using an AELVIS ELISPOT reader. Since each spot represents a cytokine-producing cell, the number of spots per well was determined.

Data analysis

Peptides eliciting a specific delta CTL response of > 30 specific spots/10⁶ 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.

Results

The results for the different HCV constructs are shown in table 5.

Table 5: Immunogenicity data for HLA- restricted peptides encoded in different HCV constructs

TYSTYGKFL 33 0,4 1 ,8 81 YLNTPGLPV 0 0,9 0,8 88

ICCG5946

ICCG5946

ICCG5946

ICCG5947

ICCG5947

ICCG5947

ICCG5959

ICCG5959

ICCG5959

ICCG 5959

Delta Ratio

SFC/10⁶ SFC/10⁶ SEQ

HLA transgenic cells cells ImmunoID mouse model Sequence (MIN) (MAX) (MIN) (MAX) genic NO

VATDALMTGY 410 1439 42 214 + 122

FTDNSSPPAV 1 3 0,3 1 ,3 - 123

HLA-A01/K^b LVDILAGYGA 1 7 0,7 1 ,7 - 124

6 pools of 2 ATDALMTGY 579 1389 59 197,5 + 125 mice FTDNSSPPA 1 10 0,8 2 - 126

ATDALMTGF 13 383 2,3 39,3 + 127

ICCG 5959

Comparable to the results described in example 3, 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. In addition, and as shown in Example 1 , 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.

Example 5: Generation of the HCV polyepitope protein

Generation of recombinant E. coli strains

Based on the amino acid sequence of the HCV polyepitope protein (Figure 13A - SEQ ID NO 115; or Figure 13B - SEQ ID NO 116 ) an optimized coding sequence was designed and synthesized by GeneArt (Regensburg, Germany) using their GeneOptimizer sequence optimization software. During design, appropriate endonuclease restriction sites were introduced in the 5 ' and 3 ' flanking regions to simplify subcloning into the expression vectors, and an affinity tag (consisting of the amino acid sequence

HHHMFHHHWWHHHMWHHH, SEQ ID NO 117) was added, optionally preceded by a three amino acid (NAA) 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 plasmid pAcI (SEQ ID NO 120; Figures 16-17) or plasmid pcI857 (SEQ ID NO 121; Figures 18-19) ensuring the expression of the temperature-sensitive mutant of the bacteriophage Lambda cl repressor. All subcloning was performed using standard recombinant DNA technology mainly based on the use of restriction enzymes and PCR techniques known in the art.

After transformation, individual colonies were transferred into culture 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, grown at 28°C and induced by a temperature shift to 37°C and/or 42°C. At several time intervals up to 4 hour post induction, samples (total cell lysates) of non- induced, induced, and wild-type cells were analyzed by western blot analysis with polyclonal rabbit antisera against the HCV poly epitope protein.

Production of a HCV polyepitope protein in E. coli (Fermentation)

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) was inoculated with 500 μL from a cell bank glycerol slant. 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

(v/v) with preculture broth. The culture was allowed to grow for 4h at 28°C and was induced for 3h at 37°C. Cells were recovered from the culture broth by centrifugation in a Beckman JLA10.500 rotor at 9000 rpm at 4°C for 25 min. Cell pellets were stored at -70⁰C.

Example 6: IMAC-purifϊcation of the HCV polyepitope construct

Materials and methods: Ni²⁺-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.

In brief, 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 Na₂HP0₄.2H₂0, 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). Pellet was discarded and the supernatant, containing the soluble fusion protein fraction, was recovered. Then, n-dodecyl-N,N-dimethylglycine (also known as lauryldimethylbetaine or Empigen BB^®, Albright & Wilson) and 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 μm pore size bottle top filter with prefilter (Millipore).

All further chromatographic steps were executed on an Akta Explorer 100 workstation (GE healthcare Bio-Sciences). A XK 16/20 column (GE healthcare Bio-Sciences) was packed with 20 mL of Ni²⁺-charged Chelating Sepharose FF resin (GE healthcare Bio-Sciences) and equilibrated with 50 mM phosphate, 6 M Gu.HCl, 20 mM imidazole , pH 7.2 (IMAC-E buffer) supplemented with 3 % Empigen BB^®.

Next, 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.

All protein fractions obtained were analyzed by SDS-PAGE under non-reducing conditions (+ subsequent silver staining) and western-blotting using polyclonal rabbit antisera directed against the HCV fusion protein that were pre-incubated with E. coli lysate (MC 1061 (pAcI) + BL21 (pAcI)).

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.

No host cell protein bands (also not around ~25 kDa) were observed on SDS-PAGE gel in the 700 mM imidazole elution pool. Removal of histidine-rich host contaminants (e.g. SIyD) was accomplished in the 200 mM imidazole washing.

Example 7: Use of the HCV polyepitope protein in a prime boost treatment regimen

Preparation of immunogens 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 lOOμl (100 μg) was injected subcutaneously at the base of the tail using a BD Microfine™ 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 lOOμg was administered by bilateral injection of 50 μl in both m.tibialis anterior (after anaesthesia).

Immunization schedule Two groups of 18 homozygous HLA-A24/K^b and one group of 18 homozygous HLA-Al 1/K^b transgenic mice were included. One group of each received a double protein prime and DNA as boost. As a control, the second group of HLA-A24/K^b transgenic mice received only a single DNA injection (without prior cardiotoxin pre-treatment).

All injections with the polyepitope protein were administered subcutaneously at the base of the tail at a 100 μg dose. DNA injections were given intramuscularly in the m.tibialis anterior at a 100 μg 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 (IFNγ) were performed on pooled spleen cells from all 18 mice.

For the evaluation of HLA Class II epitopes, 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 5xlO⁶ cells/ml with 10 μg/ml of peptide (Table 2). Cells were γ-irradiated at 10 Gy, washed and pipetted through a cell strainer before addition to the wells.

From each individual mouse, the spleen was removed and spleen cells were isolated in pools of three mice. 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. In triplicate, CD4+ spleen cells (2x10E5 cells/well) together with peptide-loaded antigen presenting cells were added. Plates were then left undisturbed overnight. After incubation, cells were removed, plates were washed several times and incubated with biotinylated anti-IL-5 antibody (clone TRFK4, Mabtech) or anti-IFNg antibody (clone R46A2, MabTech) for 2 hours at room temperature. After washing, plates were incubated with streptavidin-HRP (BD Biosciences) for 1 hour at room temperature. Then, spots were visualized using AEC (BD Biosciences) as substrate. Rinsing the plates with tap water stops the color reaction. Plates are then dried and analyzed using an automated ELISPOT reader (AELVIS).

Responses were reported as spot forming cells (SFC)/ 10⁶ cells and the criteria for determining the immunogenicity for individual epitopes were established using background responses measured with unloaded antigen presenting cells as negative control condition. Any delta response > 30 SFC/10⁶ cells with a response ratio > 2 were classified as positive.

Results HCV-specifϊc 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).

This demonstrates that the HCV polyepitope protein is especially useful as a priming agent in a heterologuous prime/boost treatment regimen.

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Claims

1. 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.

2. The polypeptide of claim 1, wherein the polyepitope construct further comprises at least one CTL and/or HTL epitope.

3. The polypeptide of claim 2, wherein the HTL epitope is a PADRE® epitope.

4. The polypeptide of claim 2, wherein the CTL and/or HTL epitope is derived from HCV.

5. The polypeptide of claim 4, wherein the at least one CTL epitope is 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.

6. The polypeptide of any of claims 1 to 5, wherein the polyepitope construct further comprises one or more spacer amino acids.

7. The polypeptide of claim 6, wherein the one or more spacer amino acids are selected from the group consisting of: K, R, N, Q, G, A, S, C, G, P and T.

8. The polypeptide of any of claims 1 to 7, wherein the CTL epitopes are sorted to minimize the number of CTL junctional epitopes .

9. The polypeptide of any of claims 1 to 8, wherein the HTL epitopes are sorted to minimize the number of HTL junctional epitopes.

10. 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.

11. A polynucleotide encoding the polypeptide of claims 1 to 10.

12. The polynucleotide of claim 11, further comprising one or more regulatory sequences.

13. The polynucleotide of claim 12, wherein the regulatory sequence is an internal ribosome binding site (IRES).

14. The polynucleotide of any of claims 11 to 13, further comprising one or more promoters.

15. The polynucleotide of claim 14, wherein the promoter is a CMV promoter.

16. The polynucleotide of any of claims 11 to 15, comprising one or more MHC Class I and/or MHC Class II targeting sequences.

17. The polynucleotide of claim 16, wherein 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.

18. 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.

19. A vector comprising the polynucleotide of any of claims 11 to 18.

20. The vector of claim 19, which is an expression vector.

21. The vector of claim 20, which is a plasmid, a viral or bacterial vector.

22. The vector of claim 21 , wherein the viral vector is a pox virus.

23. The vector of claim 22, which is a vaccinia virus.

24. A composition comprising the polypeptide of any of claims 1 to 10, or the polynucleotide of any of claims 11 to 18, or the vector of any of claims 19 to 23, or any combination thereof.

25. A composition according to claim 24, further comprising a pharmaceutical acceptable excipient.

26. A composition according to claim 25, which is a vaccine.

27. The composition, polynucleotide, vector or polypeptide of claims 1 to 26 for use as a medicament.

28. Use of the composition, polynucleotide, vector or polypeptide of any of claims 1 to 26 for the preparation of a medicament for treating and/or preventing HCV infection.

29. The composition, polynucleotide, vector or polypeptide of any of claims 1 to 26 for use in the treatment and/or prevention of hepatitis C.

30. A cell comprising the polypeptide of any of claims 1 to 10, or the polynucleotide of any of claims 11 to 18, or the vector of any of claims 19 to 23.

31. A method of inducing an immune response against HCV in an individual, comprising administering the polypeptide of any of claims 1 to 10, the polynucleotide of any of claims 11 to 18, the vector of any of claims 19 to 23, the composition of any of claims 24 to 26, or the cell of claim 29 to said individual.

32. A method of making the polypeptide of any of claims 1 to 10, the polynucleotide of any of claims 11 to 18, the vector of any of claims 19 to 23, the composition of any of claims

24 to 26, or the cell of claim 29.