WO2007110098A1 - HBx PEPTIDES, CAPABLE OF ELICITING A PROMISCUOUS IMMUNODOMINANT CD4+ RESPONSE DIRECTED AGAINST HBV - Google Patents

Abstract

The present invention relates to HBx peptides, and analogues and sub-fragments thereof, which are useful for eliciting a CD4+ response against HBV, and more a CD4+ promiscuous immunodominant response. The present invention also relates to mutant peptides, which have lost their capacity of eliciting a CD4+ response, and which are useful for prognosis a predisposition to develop a HCC.

Description

TITLE

HBx peptides, capable of eliciting a promiscuous immunodominant CD4+ response directed against HBV

TECHNICAL FIELD OF THE INVENTION

The present invention relates to peptides, analogues and sub-fragments thereof, which are, or comprise, epitopes that are recognized by T cells during HBV infection, and more particularly during chronic HBV infection. The peptides of the invention are fragments of the HBx protein of the Hepatitis B

Virus (HBV).

The HBx fragments, analogues and sub-fragments of the invention are capable of eliciting a CD4+ response directed against HBV.

As a very advantageous feature of the invention, there are provided HBx peptides, analogues and sub-fragments thereof, which are, or comprise, promiscuous and immunodominant CD4+ epitopes.

The present invention also relates to products deriving from the peptides, analogues and sub-fragments of the invention.

The present invention further describes mutant peptides, which have lost their capacity of eliciting a CD4+ response, and which are useful hepatocellular carcinoma (HCC) prognosis tools.

The present invention further relates to the medical and/or biotechnological applications of the peptides, analogues, sub-fragments and derived products of the invention.

BACKGROUND

Hepadna viruses including Hepatitis B virus (HBV) are small hepatotrophic DNA viruses that replicate by reverse transcription and establish persistent liver infection in humans and other animals.

Despite intensive vaccination programs, HBV infection remains an important health problem world-wide, with over 350 million chronic carriers. Patients with chronic hepatitis B have a high risk of developing liver cirrhosis, which is associated with a high mortality rate due to the development of hepatocellular carcinoma (HCC) or non-carcinomatous complications of cirrhosis (portal hypertension and liver failure).

The small 3.2-kb DNA genome of HBV codes for capsid proteins (HBc, HBe), for polymerase (Pol), for envelop proteins (HBs), for the HBx protein, which consists of 154 amino acids and is encoded by gene X. These proteins are translated from the pregenomic 3.5kb RNA and from 2.5, 2.1 and 0.8 kb mRNA. An additional protein HBSP (hepatitis B spliced protein) is translated from a spliced RNA.

HBx is well conserved among the mammalian hepadnaviruses, and is produced very early during infection. HBx is a multifunctional protein with a number of reported activities.

HBx has been reported to be a promiscuous transactivator, which activates a variety of viral and cellular promoters and enhancers.

HBx has also been reported to either induce or block apoptosis.

A number of reports have also shown that HBx contains several HLA class I- restricted epitopes, such as the H LA-A0201 -restricted epitopes described in

Malmassari et al. 2005 (Microbes and Infection 7:626-634 "In vivo hierarchy of immunodominant and subdominant HLA-A^*0201 -restricted T-cell epitopes of HBx antigen of hepatitis B virus").

For example, Chung et al. 1999 (Journal of Immunotherapy 22(4):279-287 "Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in hepatitis B virus X antigen") disclose five CTL epitope peptides, which binds to HLA-A2.1 molecules and elicit a CTL response.

The HLA class l-restricted peptides of Chung et al. are referred to as peptide D4

(positions 91-100 of HBx), peptide D5 (positions 92-100 of HBx), peptide D6 (positions 99-108 of HBx), peptide D8 (positions 115-123 of HBx) and peptide E3

(positions 133-141 of HBx).

It has also been reported that HBx may induce a CD4+ response. Jung et al. 1991 (Hepatology 13:637-643 "Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus") disclose an HBx peptide, which induces a CD4^÷ CD8^" response. This peptide is referred to as p12 (positions 111-126 of HBx). To the best of the inventors' knowledge, little progress has been made since 1991 in the knowledge of the HBx potential to induce a CD4+ response.

As a matter of fact, research of T cell epitopes contained in HBV proteins, such as HBx, mainly focuses on CD8⁺ CD4^" T-cell epitopes, with a view to elicit a CTL response.

However, HBx has up to now not led to any efficient vaccine composition.

Indeed, HBx is a complex molecule, in the sense that it has numerous functions, which are not all identified and/or completely understood. Its T cell- activation/induction properties are only one of many other reported properties (such as, e.g., promiscuous transactivator, apoptosis regulator, etc.).

Another obstacle is that the restriction of an HBx peptide to a defined MHC molecule often limits the spectrum of patients, to which the peptide may be usefully administered. Furthermore, it is generally believed that CTL induction is the best approach to obtain an efficient anti-HBV therapeutic vaccine capable of controlling viral infection. Under such circumstances, research on HBx nowadays mainly focuses on CTL induction, in the context of the elucidation of its pro- or anti-apoptotic effects, and of its transactivator potential. Due to its functions HBx cannot be included in vaccine composition as a complete protein.

There however still remains a need for effective anti-HBV tools, and more particularly anti-HBV therapeutic tools, as well as for effective HCC prognostic tools.

SUMMARY OF THE INVENTION

Despite the fact that today trends in HBV research focus on CTL induction, the present inventors chose to conduct a comprehensive analysis of HBx-specific CD4+ T cell responses, and produced a range of HBx peptides covering the entire length of the molecule. They screened this range of HBx peptides with PBMC coiiected from a set of human patients affected with chronic hepatitis, having less than 100,000 HBV copies/mL (this set of patients also had normal transaminase levels). This specific selection of chronic hepatitis patients, which has been made by the inventors (i.e., chronic HBV patients, in which replication is controlled to less than 100,000 HBV copies/ml_, and preferably normal transaminase levels), enabled the inventors to identify HBx peptides, which are capable of inducing a specific CD4+ response. The present invention thus relates to HBx epitope peptides, which are capable of eliciting a CD4+ response. Illustrative peptides of the invention, and nucleic acids coding therefor, are shown in Table 1 below, and in figures 7 (underlined, and underlined and bold characters), and in figures 8, 9, 10.

Hence, whereas research on T cell response induced by HBV has up to now mainly focused on CTL induction, the present inventors provide peptides, which induce a CD4+ response, i.e., an immune response that is an important component of the generation of memory T and/or B cells, and that, in turn, is able to activate and/or stimulate a CD8+ and/or antibody response.

As a very advantageous feature of the invention, there are provided HBx peptides, which are, or comprise, CD4+ epitopes that are promiscuous: they induce a significant CD4+ response in almost all the patients tested, whereas these patients have very different HLA types. The invention thereby provides peptides, which induce a significant CD4+ response for such a broad spectrum of different patients' HLA types, that these peptides can be considered to be not limited by the particular patient HLA type, and that a vaccine composition comprising at least one of these peptides can be considered to be somehow "HLA-independent".

As another very advantageous feature of the invention, there are provided HBx peptides, which are, or comprise, CD4+ epitopes that are immunodominant: the CD4+ response they elicit has an intensity that is so much higher than the one induced by each of the other or prior art peptides, that these HBx peptides can be considered to be immunodominant. As another very advantageous feature of the invention, there are provided HBx peptides, which are, or comprise, CD4+ epitopes that are both immunodominant and promiscuous.

To the best of the inventors' knowledge, the present invention is the first description of promiscuous immunodominant HBx peptides.

The fact that an HBx peptide is promiscuous and immunodominant has direct implications in terms of vaccine composition: it allows for the first time the provision of an anti-HBV vaccine composition, which can be efficiently and/or usefully administered to a very broad spectrum of patients.

The present invention relates to the HBx peptides as such, and to the analogues and sub-fragments thereof, as well as to products deriving therefrom, such as multimers and/or MHC complexes deriving therefrom, nucleic acids, vectors and cells. The present invention also relates to the biological, biotechnological, medical, clinical, preventive, palliative, therapeutic, diagnostic, prognostic applications thereof. It more particularly relates to a pharmaceutical composition, a diagnostic/prognostic composition, an immunizing composition, and a vaccine composition, comprising at least one of the products of the invention.

The present invention also relates to mutant peptides, which have lost their capacity of eliciting a CD4+ response, and which are useful hepatocellular carcinoma (HCC) prognosis tools.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Percentage of HBx-specific IFN-gamma-secreting T-cell responses in 49 chronic HBV carriers. PBMC were in vitro stimulated with HBx-derived 15-mer peptides divided in three pools (A, B and C) for 10-14 days. IFN-gamma-secreting T cells were determined by ELISPOT using the same peptide pools. The response was considered positive if the median number of spot-forming cells in triplicate wells was at least twice that in control wells in the absence of peptides and if at least 62 spots were detected per 1 X 10⁶ PBMC after background subtraction. Total and diversity of pool-specific IFN-gamma-secreting T-cell responses obtained with HBx pools A, B and C are shown.

Figure 2. Number of IFN-gamma-secreting T cells after in vitro stimulation with pool C determined by ELISPOT and expressed as the number of specific spot- forming cells (SFC)/10⁶ PBMC (left panel). Each peptide from the pool C was tested separately in order to map T-cell responses to single peptides. Results showed that the pool C-specific T-cell response is targeted against one immunodominant epitope designated x26 (middle panel). T-cells responses activated to single pool C peptides other than x26 peptide are also shown (right panel). ND: not done.

Figure 3. Phenotyping of IFN-gamma-producing T cells expanded from PBMC for 10 days with x26 peptide. T cells were cultured overnight with (left panels) or without (right panels) x26 peptide in the presence of brefeldin A and then stained with anti-CD4-PE (upper panels) and anti-CD8-PerCP (lower panels) and anti- IFN-gamma-FITC monoclonal antibodies.

Figure 4. x26 epitope HLA class ll-restriction. In vitro expanded PBMC were pre- incubated with anti-class Il HLA antibodies: anti-HLA-DR (L243), anti-HLA-DQ (SPVL3) and anti-HLA-DP (B7-21) or with an irrelevant antibody (anti-HLA-A2). PBMC were then tested in Elispot assays as described in Materials and Methods. Results obtained with 3 chronic carriers (FRAAN, PREXA and HUEAL) are shown.

Figure 5. Number of IFN-gamma- and IL-10-secreting T cells after in vitro stimulation with peptide pool C or individual x26 peptide determined by Elispot assays. Results are expressed as the number of specific spot-forming cells (SFC)/10⁶ PBMC. Number of IFN-gamma- or IL-10-SFC are indicated on each column (grey and black columns respectively). A) specific T cells from 7 patients secreting only IFN-gamma; B) specific T cells from 6 patients secreting IFN- gamma and IL-10. Figure 6. Cross-recognition of viral mutants by x26-specifιc T cells: PBMC from 7 patients with x26-specific T-cell response were activated in vitro with wild-type x26 peptide or with variant peptides V2 and V3 to assess cross-reactivity in IFN- gamma Elispot tests as described before.

Figure 7. Nucleic and consensus amino acid sequences of the HBx protein (SEQ ID NO:1 and NO:2). Bold characters = peptide "x22-x27"; bold and underlined characters = peptide "x26".

Figures 8, 9 and 10. Nucleic acid and amino acid sequences of illustrative HBx peptides of the invention: Table 1 :

Start and stop positions are included within the peptide sequence.

DETAILED DESCRIPTION

To define new T-cell epitopes other than HLA-A2-restricted epitopes, 15-mer peptides, corresponding to the HBx consensus sequence and overlapping by 10 amino-acids, were produced by the inventors, and used to stimulate PBMC from chronically infected patients with less than 100,000 HBV copies/mL and normal transaminase levels.

The results of the inventors show: i) a sporadic detection of HLA-A2-restricted HBx-specific CD8+ T cells secreting

IFN-gamma, with weak cytotoxic capacities;

H) more than 60% of studied patients (24/36) presented IFN-gamma-secreting and/or IL-10-secreting T cells specific for epitopes located in the carboxy-terminal part of HBx;

Hi) promiscuous and immunodominant CD4+ T-cell epitope peptides which stimulate specific T cells were identified by the inventors, and were widely detected during the chronic phase of HBV infection; /Vj the mutation of certain residues within the CD4+ T-cell epitopes of HBx results in the loss the CD4+ activation function, and that it is a prognosis indicator reflecting the fact that the patient is at risk of developing a HCC.

Overall, these results show a peripheral low frequency of CD8+ T cells and a predominance of CD4+ T cells specific for HBx.

Hence, although CD8+ cytotoxic T lymphocytes are generally thought to be the most important effector cells for the elimination of virally infected cells, the present inventors show that, the cells that play a central role in the antiviral immune response in the context of HBV, and more particularly of the HBx protein of HBV, are CD4+ T cells. CD4+ T cells induce and maintain cytotoxic activity, and secrete antiviral cytokines. Therefore, the definition and characterisation of epitopes within HBx have important implications for the production of preventive and/or palliative and/or therapeutic vaccine for HBV infected patients, and more particularly for HBV chronically infected patients.

HBx peptides of the invention, analogues and sub-fragments thereof; nucleic acids coding therefore, vectors and cells:

The CD4+ epitope peptides of the invention are peptides, which are capable of eliciting a CD4+ response directed against HBV. They consist of:

- a HBx fragment of SEQ ID NO:4 (peptide "x24-x27"), SEQ ID NO:6 (peptide "x24-x26"), SEQ ID NO:8 (peptide "x24-x25"), SEQ ID NO:10 (peptide "x25-x26"), SEQ ID NO:12 ("peptide x25-x27"), SEQ ID NO:14 (peptide "x26-x27"), SEQ ID NO:16 (peptide "x24"), SEQ ID NO:18 (peptide "x25"), SEQ ID NO:20 (peptide "x26"), SEQ ID NO:22 (peptide "x27"), SEQ ID NO:26 (peptide "x22"), SEQ ID NO:28 (peptide "x22-x23"), SEQ ID NO:30 (peptide "x22-x24"), SEQ ID NO:32 (peptide "x22-x25"), SEQ ID NO:34 (peptide "x22-x26"), SEQ ID NO.36 (peptide "x22-x27"), SEQ ID NO:38 (peptide "x23-x24"), SEQ ID NO:40 (peptide "x23- x25"), SEQ ID NO:42 (peptide "x23-x26"), or SEQ ID NO:44 (peptide "x23-x27"), [see table 1 above, as well as figure 7 -bold characters, bold and underlined characters- and figures 8, 9, 10], or of

- a MHC class ll-restricted analogue thereof, which derives from said HBx fragment by substitution and/or deletion of one or several amino acid(s), and which has retained a capacity of eliciting a CD4+ response directed against HBV,

- a MHC class ll-restricted sub-fragment of said fragment or of said analogue, which has retained a capacity of eliciting a CD4+ response directed against HBV.

According to a preferred embodiment of the present invention, said CD4+ epitope peptides consist of:

- a HBx fragment of SEQ ID NO:20 (peptide "x26"), or of

- a MHC class ll-restricted analogue thereof, which derives from said HBx fragment by substitution and/or deletion of one or several amino acid(s), and which has retained a capacity of eliciting a CD4+ response directed against HBV, - a MHC class ll-restricted sub-fragment of said fragment or of said analogue, which has retained a capacity of eliciting a CD4+ response directed against HBV.

According to a particular embodiment of the present invention, an analogue and/or sub-fragment of the present invention does not bind to any HLA class I molecule, most preferably to any MHC class I molecule, i.e., it has no significant affinity with any HLA class I molecule, most preferably to any MHC class I molecule.

To determine whether a given analogue or sub-fragment does not bind to any MHC class I molecule, any means that the skilled person may find appropriate may be used. For example, computer programs for the prediction of peptide binding to MHC class-l alleles are available to the skilled person, e.g., the online available ProPredi software

(http://www.imtech.res.in/raghava/propred1/page2.html), which has also been described in Singh, H. and Raghava, G. P. S. (2003) "ProPredi : Prediction of promiscuous MHC class-l binding sites" Bioinformatics 19(8): 1009-1014.

The present inventors demonstrate that, compared to other and to prior art HBx peptides, the "x26" peptide of the present invention (SEQ ID NO:20) has the advantage of being a promiscuous and immunodominant epitope.

Example 1 below gives an illustration of these features: see e.g., figure 2, which shows that:

- said "x26" peptide is a promiscuous epitope: it elicits a CD4+ response in 22 of 25 chronic HBV patients, whereas these patients have very different HLA types, and that

- said "x26" peptide is an immunodominant epitope: the intensity of the CD4+ response obtained with a pool of nine HBx fragments ("pool C"), which are 15 mers overlapping by 10 residues from position 116 to position 154 of the HBx consensus sequence of SEQ ID NO:2 is roughly similar to the one obtained with said "x26" peptide alone (positions 126-140 of the

HBx consensus sequence of SEQ ID NO:2).

Hence, the inventors demonstrate that peptide "x26" induce a strong CD4+ response for such a broad spectrum of different patients' HLA types, that it can be considered to be not limited by the particular patient HLA type, and that a vaccine composition comprising it can be considered to be somehow "HLA-independent".

Therefore, the "x26" peptide of the present invention (SEQ ID NO: 20), as well as any peptide comprising it (e.g., SEQ ID NO: 4, 6, 10, 12, 14), is very advantageous compared to other and prior art HBx peptides, for the generation of an efficient preventive and/or palliative and/or therapeutic anti-HBV composition.

By "CD4+ response", it is herein intended any CD4+ response that the person of ordinary skill in the art would contemplate. A CD4+ response may e.g., be the induction and/or stimulation of the activation of a CD4+ T-cell, such as e.g., the induction and/or stimulation of the production and/or secretion of one or several cytokine(s) (e.g., IFN-gamma and/or IL-10).

Example 1 below gives an illustration of means enabling the detection of CD4+ responses elicited by HBx peptides.

For the sake of conciseness, the term "peptide" encompasses small peptides (e.g., a peptide of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids), as well as (poly)peptides, such as e.g., a polypeptide of 21 amino acids or higher, up to about 45 amino acids, preferably up to 40 amino acids, such as e.g., a polypeptide of 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids.

Peptides of the invention can be natural peptides, or fragments of natural polypeptides or proteins, or recombinant peptides, or synthetic peptides.

Any method of peptide synthesis that is known to the skilled person can be used. Examples of synthesis methods, such as the Merrifield solid phase synthesis, can e.g., be found in « Solid Phase Peptide Synthesis » (J. M. Steward & J. D. Young, 1969, Ed. W.H. Freeman Co., San Francisco), or in « Peptide synthesis » (M. Bodansky et al. 1976, John Wiley & Sons, 2nd Edition).

The present invention also encompasses the nucleic acids coding for the peptides, analogues and sub-fragments of the invention in accordance with the genetic code, taking into account the degeneracy of this code, as well as the vectors comprising at least one of such nucleic acids, and the cells, which have been transfected, infected or transformed by at least one of such nucleic acids or vectors.

Illustrative nucleic sequences coding for peptides x24-x27, x24-x26, x24-x25, x25- x26, x25-x27, x26-x27, x24, x25, x26, x27, x22, x22-x23, x22-x24, x22-x25, x22- x26, x22-x27, x23-x24, x23-x25, x23-x25, x23-x26, x23-x27 of the invention are shown in figures 8, 9 and 10: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 , SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 , SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43 (see Figure 7 -bold characters, bold and underlined characters-, see Figures 8, 9 and 10, see also table 1). Any vector or cell that the skilled person finds appropriate may be used. Appropriate vectors may e.g., be integrative viral vectors (retrovirus, adeno- associated virus, lentivirus, etc.) or non integrative viral vectors (adenovirus, alphavirus, Herpes simplex virus, etc.), plasmids, phages, YAC, and more generally any expression vectors. An advantageous expression vector has such a structure and comprises such regulatory and modulatory elements, that it enables the production of the coded peptide in vitro and/or in vivo, preferably in a human being.

In addition to the sequence coding for at least one peptide, analogue or sub- fragment of the invention, the vector can thus comprise one or several elements among: transcription regulation regions (such as promoters, enhancers, ribosome binding sites, polyA signals), termination signals, prokaryote or eukaryote replication origins, genes coding for selection markers, genes coding for a tag such as Myc, His, Tag or Flag.

The present invention also encompasses: α) any HBx-specific T cell or cell line (e.g., an HBx-specific rodent, rat, murine, or human T cell or cell line), preferably an HBx-specific human T cell or cell line, which is obtainable by stimulation of a T cell or cell line with a peptide or analogue or sub-fragment of the invention, as well as β) any monoclonal HBx-specific T cell hybridoma obtainable by fusion of an HBx- specific T cell or cell line of α), with a tumor cell, preferably a thymoma (e.g., the BW5147 thymoma).

Plurality of fused, or coupled or otherwise associated, peptides of the invention; nucleic acids coding for them, vectors and cells (= combination products of the invention):

The peptides of the invention can be used in as such, or in the form of a polymer, or of a complex, or of a conjugate, or otherwise physically and/or functionally associated.

A first peptide of the invention (such as the peptide of SEQ ID NO:20), or a MHC class ll-restricted analogue or sub-fragment thereof, can be fused, or coupled, either directly or indirectly via a linker, or otherwise associated, to at least one second peptide of the invention, or MHC class ll-restricted analogue or sub- fragment thereof, resulting in what can be referred to as a combination product of the invention. Said first peptide of the invention, or MHC class ll-restricted analogue or sub-fragment thereof, can be identical or different from said at least one second peptide of the invention, or MHC class ll-restricted analogue or sub- fragment thereof.

The present invention thus relates to a peptide combination product, which comprises at least two elements which, independently of each other, are chosen among the peptides of the invention, and the MHC class ll-restricted analogues and sub-fragments thereof, and wherein said at least two elements are fused, or coupled or otherwise associated together.

Said at least one second peptide of the invention may be not directly contiguous to the HBx fragment, to which the first peptide of the invention corresponds (such as, for example, the "x24" and/or the "x22" peptide, which are not contiguous to the "x26" peptide of the invention).

Such a combination product of the invention is useful to elicit an anti-HBV preventive and/or palliative and/or therapeutic immune response in an organism in need thereof, such as a person having, or susceptible of having, a HBV infection, more particularly a chronic HBV infection, and/or a cirrhosis and/or a hepatocellular carcinoma (HCC).

The present invention also encompasses the nucleic acids coding for the combination products of the invention in accordance with the genetic code, taking into account the degeneracy of this code, as well as the vectors comprising at least one of such nucleic acids, and the cells, which have been transfected, infected or transformed by at least one of such nucleic acids or vectors, as well as α) any HBx-specific T cell or cell line (e.g., an HBx-specific rodent, rat, murine, or human T cell or cell line), preferably an HBx-specific human T cell or cell line, which is obtainable by stimulation of a T cell or cell line with a peptide combination product of the invention, as well as β) any monoclonal HBx-specific T cell hybridoma obtainable by fusion of an HBx- specific T cell or cell line of α), with a tumor cell, preferably a thymoma (e.g., the BW5147 thymoma).

Peptide(s) of the invention fused, or coupled or otherwise associated, to at least one other molecule; nucleic acids coding for them, vectors, and cells (= hybrid combination products of the invention):

Alternatively or additionally, a peptide of the invention (such as the peptide of SEQ ID NO: 20), or a MHC class ll-restricted analogue or sub-fragment thereof, can advantageously be fused, or coupled, either directly or indirectly via a linker, or otherwise associated, to at least one other molecule, which is not a peptide or a MHC class ll-restricted analogue or sub-fragment of the invention, thereby resulting in what will be referred to as a hybrid combination product of the invention.

Combination of peptide(s), analogue(s) or sub-fragment(s) of the invention with HBV protein(s) or peptide(s) (HBx fragment, which is not a peptide or analogue or sub-fragment of the invention; HBV protein, or HBV protein fragment other than HBx fragment): Said at least one other molecule, which is not a peptide or a MHC class II- restricted analogue or sub-fragment of the invention, can for example be an HBx fragment, which is not a peptide or analogue or sub-fragment of the invention. According to a particular embodiment of the invention, said HBx fragment is non- contiguous, i.e., it is an HBx fragment, which, in the HBx protein, is not directly contiguous to the HBx fragment, to which the peptide of the invention corresponds.

For example, a peptide of the invention, or a MHC class ll-restricted analogue or sub-fragment thereof, can be directly fused, or coupled through a linker that is not an HBV fragment, or otherwise associated, to at least one non-contiguous HBx fragment, which is not a peptide of the invention. Said at least one non-contiguous HBx fragment preferably comprises at least 5 amino acids, more preferably at least 6 amino acids. Said at least one non-contiguous HBx fragment advantageously is a peptide comprising at least one CD4+ epitope, and/or at least one CD8+ epitope, and/or at least one B-cell epitope.

Such a fused, or otherwise coupled or associated, peptide or MHC class ll- restricted analogue or sub-fragment of the invention is useful to elicit an anti-HBV preventive and/or palliative and/or therapeutic immune response in an organism in need thereof, such as a person having, or susceptible to have, a HBV infection, more particularly a chronic HBV infection, and/or a cirrhosis and/or a HCC.

Said at least one other molecule, which is not a peptide or a MHC class ll- restricted analogue or sub-fragment of the invention, can for example be a molecule, which is not an HBx fragment. A peptide of the invention, or a MHC class ll-restricted analogue or sub-fragment thereof, can thus be fused, or coupled, either directly or indirectly via a linker (i.e., a HBV linker or non-HBV linker), or otherwise associated, to at least one other molecule, which is not an HBx fragment.

Said at least one other molecule, which is not an HBx fragment, can be an HBV molecule, or may derive from HBV, i.e., it can be an HBV protein, or HBV protein fragment that is not an HBx fragment, such as, advantageously, at least one HBV envelop protein or HBV envelop fragment (e.g., at least one HBs protein or HBs fragment), and/or at least one HBV capsid protein or HBV capsid fragment (e.g., at least one HBc protein or HBc fragment, and/or at least one HBe protein or HBe fragment), and/or at least one HBV polymerase or HBV polymerase fragment, and/or at least one HBSP protein or HBSP fragment.

Said HBV protein fragment that is not an HBx fragment preferably comprises at least 5 amino acids, more preferably at least 6 amino acids.

HBV protein fragment that is not an HBx fragment advantageously comprises at least one CD4+ epitope, and/or at least one CD8+ epitope, and/or at least one B- cell epitope.

Combination of peptide(s), analogue(s) or sub-fragment(s) of the invention with MHC molecule(s):

Alternatively or additionally, said at least one other molecule, which is not an HBx fragment, can be a molecule, which is not, or does not derive from HBV. For example, a peptide of the invention, or a MHC class ll-restricted analogue or sub-fragment thereof, can be fused, or coupled, either directly or indirectly, or otherwise associated, to at least one lipid fraction.

Advantageously, a peptide of the invention, or a MHC class ll-restricted analogue or sub-fragment thereof, can be fused, or coupled, either directly or indirectly, or otherwise associated, to at least one MHC molecule, preferably to at least two, more preferably at least three, most preferably at least four MHC molecules. Said MHC molecule advantageously is a HLA molecule, preferably a HLA class Il molecule, more preferably a HLA-D molecule (for example a HLA-DR₁ HLA-DP, HLA-DQ, HLA-DM, HLA-DO, HLA-DS, or HLA-DZ molecule), most preferably a HLA-DR molecule (for example, a HLA-DR1 , HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR52 or HLA-DR53 molecule).

Said at least two, three or four MHC molecules can be chosen independently from each other: they can be all identical, or all different from each other, or there can be at least two identical MHC molecules, and at least one different MHC molecule. For example, a peptide, analogue or sub-fragment of the invention can be fused, or coupled or otherwise associated, to four HLA-DR molecules. According to a preferred embodiment of the present invention, a peptide of the invention, or a MHC class ll-restricted analogue or sub-fragment thereof, is fused, or coupled or otherwise associated, to four CMH class Il molecules, preferably to four HLA class Il molecules, thereby forming tetramers having a high TCR-CD4+ affinity.

Such MHC-fused or -coupled peptides are particularly useful for binding to CD4+ TCR+ cells, and more particularly for sorting CD4+ TCR+ cells from a cell population, e.g., by flow cytometry, preferably by fluorescence activated cell sorting, whereby CD4+ T cells, which are capable of binding to the peptide(s) contained in said MHC-fused or -coupled peptide(s), are isolated. Advantageously, said sorted CD4+ T cells are activated CD4+ T cells, more preferably CD4+ T cells, which are specifically activated against the peptide(s) contained in said MHC-fused or -coupled peptide(s).

Such sorted CD4+ T cells are useful for eliciting an anti-HBV preventive and/or palliative and/or therapeutic immune response in an organism in need thereof, such as a person having, or susceptible of having, a HBV infection, more particularly a chronic HBV infection, and/or a cirrhosis and/or a HCC.

Such MHC-fused or -coupled peptides are also useful T cell activating agents, which may used in a pharmaceutical composition, preferably an immunising composition, more preferably a vaccine composition, intended for the prevention and/or palliation and/or therapy of an HBV infection, more particularly a HBV chronic infection and/or a cirrhosis and/or a HCC.

The herein described hybrid combination products of the invention can be further fused, or coupled, or otherwise associated, together, and/or further fused, or coupled, or otherwise associated, to at least one additional peptide or analogue or sub-fragment of the invention. For example,

- at least one peptide or analogue or sub-fragment of the invention, and - at least one MHC molecule, such as at least one HLA class Il molecule, and

- at least one of the following HBV elements: HBV proteins, HBV protein fragments other than an HBx fragment, HBx fragments other than a fragment of the invention, can be fused, and/or coupled, and/or otherwise associated together. The present invention also encompasses the nucleic acids coding for the hybrid combination products of the invention in accordance with the genetic code, taking into account the degeneracy of this code, as well as the vectors comprising at least one of such nucleic acids, and the cells, which have been transfected, infected or transformed by at least one of such nucleic acids or vectors, as well as α) any HBx-specific T cell or cell line (e.g., an HBx-specific rodent, rat, murine, or human T cell or cell line), preferably an HBx-specific human T cell or cell line, which is obtainable by stimulation of a T cell or cell line with a hybrid combination product of the invention, as well as β) any monoclonal HBx-specific T cell hybridoma obtainable by fusion of an HBx- specific T cell or cell line of α), with a tumor cell, preferably a thymoma (e.g., the BW5147 thymoma).

Anti-HBV compositions and treatments:

The present invention also relates to compositions, more particularly to pharmaceutical compositions, comprising at least one of the products of the invention.

A composition of the invention thus comprises at least one of the products described in the present application, and more particularly at least one of the following elements, still more particularly at least two of the following elements: - the peptides of the invention [(SEQ ID NO:4 (peptide "x24-x27"), SEQ ID NO:6 (peptide "x24-x26"), SEQ ID NO:8 (peptide "x24-x25"), SEQ ID NO: 10 (peptide "x25-x26"), SEQ ID NO: 12 ("peptide x25-x27"), SEQ ID NO: 14 (peptide "x26- x27"), SEQ ID NO:16 (peptide "x24"), SEQ ID NO:18 (peptide "x25"), SEQ ID NO:20 (peptide "x26"), SEQ ID NO:22 (peptide "x27"), SEQ ID NO:26 (peptide "x22"), SEQ ID NO:28 (peptide "x22-x23"), SEQ ID NO:30 (peptide "x22-x24"), SEQ ID NO:32 (peptide "x22-x25"), SEQ ID NO:34 (peptide "x22-x26"), SEQ ID NO:36 (peptide "x22-x27"), SEQ ID NO:38 (peptide "x23-x24"), SEQ ID NO:40 (peptide "x23-x25"), SEQ ID NO:42 (peptide "x23-x26"), or SEQ ID NO:44 (peptide "x23-x27")], the analogues and sub-fragments of the invention, as well as the nucleic acids coding therefor, the vector comprising at least one of such nucleic acids, and the cells transfected, infected or transformed by at least one of such nucleic acids or vectors,

- the combination products of the invention (see above, the paragraphs relating to the combination products of the invention),

- the hybrid combination products of the invention (see above, the paragraphs relating to the hybrid combination products of the invention).

The present invention also relates to a method of administering an anti-HBV treatment in a person in need thereof, which comprises administering at least one of such compositions.

Nucleic acids or vectors can be administered to a patient, e.g. intramuscularly (Loirat et al. 2000 J Immunol 765, 4748-4755; Malmassari et al. 2005 Microbes Infect 7, 626-634; Mancini-Bourgine et al. hepatogy 2004, 40: 874-882). Such a DNA or vector can be fused, or coupled, or otherwise associated, to at least one cell or tissue targeting molecule and/or at least one molecule, which stimulates the immune system, and/or to at least one element which facilitates nucleic acid transfection, such as liposome, conjugate, polymer.

A composition of the invention advantageously comprises at least one of the following elements: a) a HBx fragment of SEQ ID NO: 20 (EIRLKVFVLGGCRHK; peptide "x26"), or b) the MHC class ll-restricted analogues thereof, which derive from said

SEQ ID NO:20 HBx fragment by substitution and/or deletion of one or several amino acid(s), and which have retained a capacity of eliciting a CD4+ response directed against HBV, c) the MHC class ll-restricted sub-fragments of said SEQ ID NO: 20 HBx fragment or of said analogues of b), wherein said sub-fragments have retained a capacity of eliciting a CD4+ response directed against HBV, d) the nucleic acids, coding for the peptide of SEQ ID NO: 20, for said analogues of b) and for said sub-fragments of c), in accordance with the universal genetic code, taking into account the degeneracy of this code, e) the vectors comprising at least one nucleic acid of d), f) the cells, which have been transfected, infected or transformed by at least one nucleic acid of d), and/or at least one vector of e), g) any HBx-specific T cell or cell line (e.g., a HBx-specific rodent, rat, murine, or human T cell or cell line), preferably a HBx-specific human T cell or cell line, which is obtainable by stimulation of a T cell or cell line with a peptide combination product of the invention, h) any monoclonal HBx-specific T cell hybridoma obtainable by fusion of a HBx-specific T cell or cell line of g), with a tumor cell, preferably a thymoma (e.g., the BW5147 thymoma).

According to a particular embodiment of the present invention, said analogue and/or sub-fragment does not bind to any HLA class I molecule. As above described (see the paragraphs relating to the combination products and to the hybrid combination products of the invention), the fragment of SEQ ID NO: 20 can be fused, or coupled, or otherwise associated, to at least one other HBx fragment, which is not a contiguous fragment in the HBx protein, and/or to at least one HBV protein or fragment other than a HBx fragment, and/or to at least one non-HBV molecule, such as at least one MHC molecule, preferably at least one HLA class Il molecule. Such a "x26-containing" composition may further comprise at least one of the following elements: i) the peptides of SEQ ID NO:4 (peptide "x24-x27"), SEQ ID NO:6 (peptide "x24-x26"), SEQ ID NO:8 (peptide "x24-x25"), SEQ ID NO: 10 (peptide "x25-x26"), SEQ ID NO:12 ("peptide x25-x27"), SEQ ID NO:14 (peptide "x26-x27"), SEQ ID NO: 16 (peptide "x24"), SEQ ID NO: 18 (peptide "x25"), SEQ ID NO:20 (peptide "x26"), SEQ ID NO:22 (peptide "x27"), SEQ ID NO:26 (peptide "x22"), SEQ ID NO:28 (peptide "x22-x23"), SEQ ID NO:30 (peptide "x22-x24"), SEQ ID NO:32 (peptide "x22-x25"), SEQ ID NO:34 (peptide "x22-x26"), SEQ ID NO:36 (peptide "x22-x27"), SEQ ID NO:38 (peptide "x23-x24"), SEQ ID NO:40 (peptide "x23- x25"), SEQ ID NO:42 (peptide "x23-x26"), and SEQ ID NO:44 (peptide "x23-x27"), ii) the MHC class ll-restricted analogues thereof, which derive from the peptides of i) by substitution and/or deletion of one or several amino acid(s), and which have retained a capacity of eliciting a CD4+ response directed against HBV, iii) the MHC class ll-restricted sub-fragments of said peptide of i) or of said analogue of ii), which have retained a capacity of eliciting a CD4+ response directed against HBV, iv) the nucleic acids coding for the peptides of i), the analogues of ii) and the sub-fragments of iii), in accordance with the universal genetic code, taking into account the degeneracy of this code, v) the vectors, comprising at least one nucleic acid of iv), vi) the cells, which have been transfected, infected or transformed by at least one nucleic acid of iv), and/or at least one vector of v), vii) any HBx-specific T cell or cell line (e.g., an HBx-specific rodent, rat, murine, or human T cell or cell line), preferably an HBx-specific human T cell or cell line, which is obtainable by stimulation of a T cell or cell line with a peptide combination product of the invention viii) any monoclonal HBx-specific T cell hybridoma obtainable by fusion of an HBx-specific T cell or cell line of vii), with a tumor cell, preferably a thymoma (e.g., the BW5147 thymoma).

The compositions of the invention may further comprise at least one agent, which is not an anti-HBV agent, for example at least one anti-HCV agent.

The compositions of the present invention may further comprise at least one pharmaceutically and/or physiologically acceptable vehicle (diluent, excipient, additive, pH adjuster, emulsifier or dispersing agent, preservative, surfactant, gelling agent, as well as buffering and other stabilizing and solubilizing agent, etc.).

Appropriate pharmaceutically acceptable vehicles and formulations include all known pharmaceutically acceptable vehicles and formulations, such as those described in "Remington: The Science and Practice of Pharmacy", 20^th edition, Mack Publishing Co.; and "Pharmaceutical Dosage Forms and Drug Delivery Systems", Ansel, Popovich and Allen Jr., Lippincott Williams and Wilkins. In general, the nature of the vehicle will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise, in addition to the one or more contrast agents, injectable fluids that include pharmaceutically and physiologically acceptable fluids, including water, physiological saline, balanced salt solutions, buffers, aqueous dextrose, glycerol, ethanol, sesame oil, combinations thereof, or the like as a vehicle. The medium also may contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. The carrier and composition can be sterile, and the formulation suits the mode of administration.

For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional nontoxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, sodium saccharine, cellulose, magnesium carbonate, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated with traditional binders and carriers, such as triglycerides.

The present invention more particularly relates to immunogenic compositions, immunogenic drugs, and vaccines.

Such compositions can be used in therapy and/or prophylaxis. The term vaccine thus herein encompasses therapeutic, as well as prophylactic vaccine.

The immunogenic compositions, immunogenic drugs, and vaccines of the present invention, can be intended for the treatment and/or prevention and/or palliation of a disease related to, and/or involving, HBV, more particularly a chronic HBV infection and/or a cirrhosis and/or a HCC.

In addition to at least one product of the invention, such compositions advantageously comprise at least one carrier molecule and/or at least one adjuvant. The term "carrier molecule" means an immunogenic molecule containing antigenic determinants recognized by T cells. A carrier molecule can be a protein or can be a lipid. A carrier protein is conjugated to a polypeptide to render the polypeptide immunogenic. Carrier proteins include keyhole limpet hemocyanin, horseshoe crab hemocyanin, and bovine serum albumin.

The term "adjuvant" means a substance that nonspecifically enhances the immune response to an antigen. Adjuvants include Freund's adjuvant, either complete or incomplete; Titermax gold adjuvant; alum; and bacterial LPS.

The present invention also encompasses a method for activating CD4+ T cells, which comprises in vivo or in vitro contacting at least one CD4+ T cell with:

- at least one peptide, analogue or sub-fragment of the invention, or with at least one nucleic acid coding therefor, or with at least one vector comprising at least one of such nucleic acids, or at least one cell transfected, transformed, or infected by at least one nucleic acid or vector of the invention, or

- at least one combination product of the invention (peptide, nucleic acid, vector, cell),

- at least one hybrid combination product of the invention (peptide, nucleic acid, vector, cell). Said contacting thereby induces and/or stimulate the activation of said CD4+ T cell, and the HBx-specific activation of said CD4+ T cell.

The present invention further encompasses any CD4+ T cell, obtainable by this method of the invention, and more particularly any HBx-specific CD4+ T cell obtainable by said contacting.

The present invention also encompasses a method for inducing and/or stimulating the maturation of a dendritic cell, which comprises in vivo or in vitro contacting at least one dendritic cell with:

- at least one peptide, analogue or sub-fragment of the invention, or with at least one nucleic acid coding therefor, or with at least one vector comprising at least one of such nucleic acids, or at least one cell transfected, transformed, or infected by at least one nucleic acid or vector of the invention, or

- at least one combination product of the invention (peptide, nucleic acid, vector, cell), - at least one hybrid combination product of the invention (peptide, nucleic acid, vector, cell).

The present invention further encompasses any dendritic cell, obtainable by this method of the invention.

Mutant products:

The present applications also relates to mutant products, i.e., to mutant peptides, which have lost their capacity of eliciting a CD4+ response, as well as to nucleic acids coding for them, and vectors and cells comprising such nucleic acids or vector.

Indeed, the present inventors describe mutants of the CD4+ epitope peptides of the present invention, which have lost their capacity of inducing a CD4+ response. These mutants may derive from the parent epitope peptides by mutation of at least one amino acid, e.g., of one, two, or three amino acid(s), e.g., mutation of amino acid I in position 127 and/or K in position 130 and/or V in position 131 (amino acids positions are given by reference to the consensus sequence of the complete HBX - SEQ ID NO: 2). Such positions may advantageously be the ones of MHC class Il anchor residues or aminoacids interacting with TCR. Appropriate mutation(s) notably include/s amino acid substitution(s) and/or deletion(s). Illustrative mutant peptides comprise the peptides of SEQ ID NO.23 (EIRLMIFVLGGCRHK) and SEQ ID NO:24 (ETRLMIFVLGGCRHK). The present inventors thereby show that, if the HBx protein, or the CD4+ epitope peptides deriving therefrom, is mutated in at least one these positions, then the patient is (or has become) unable to rise a CD4+ response against HBx. The inventors further made the link with the fact that these mutations can be observed in hepatocellular (HCC) tissues, i.e., once HBV has integrated the host's genome. The work of the present inventors demonstrates that a detection of said mutations in said HBx protein, or epitope peptides thereof, is indicative of a predisposition to develop an HCC.

Whereas prior art only suggests the detection of such mutations in integrated HBV, i.e., when HCC has already developed, the present invention allows to proceed to the detection of mutated amino acid(s) at an early stage, long before

HBV has integrated the tissues, i.e., at a stage where prognostic can still be very beneficial to the patient's health.

The present invention also relates to the nucleic acids coding for a mutant peptide of the invention, to the vector comprising at least one of such nucleic acids, and to cells, such as APC, comprising at least one of such nucleic acids or vectors.

The present invention thus relates to prognostic applications, and more particularly to prognostic applications of the mutant products of the present invention. Such a mutant product notably is a useful hepatocellular carcinoma (HCC) prognosis tool, more particularly a useful target for HCC prognosis, still more particularly a useful HCC prognosis indicator (the presence of which is indicative of a predisposition to develop an HCC).

Diagnosis / prognosis methods:

The present invention also encompasses the diagnosis and/or prognosis applications of:

- the CD4+ epitope-containing products of the invention (i.e., the peptides, analogues or sub-fragments of the invention, and the nucleic acids coding therefore, as well as the combination and hybrid combination products of the invention), and of

- the mutant products of the invention, which have lost their capacity of eliciting a CD4+ response.

The present invention thus encompasses a method for the diagnosis of the HBV infection stage, for example in a representative sample collected from a HBV- infected patient, which comprises determining the absence or presence of at least one mutant product of the invention, wherein the absence of a mutant product of the invention is indicative of a replicative HBV infection stage or of a chronic infection stage, and wherein the presence of a mutant peptide product of the invention is indicative of a risk to develop HCC. Illustrative mutant peptides comprise the peptides of SEQ ID NO:23 (EIRLMIFVLGGCRHK) and SEQ ID NO:24 (ETRLMIFVLGGCRHK). The present invention also encompasses a method, which enables to assess the reactivity, or the potential of reactivity, that a patient has, or can have, against

HBV. The present invention indeed encompasses a method for prognosing that a patient, who is chronically infected by HBV, but in which HBV is not genomically integrated yet, has, or has acquired, a predisposition to develop a hepatocarcinoma, which comprises: detecting in a HBV-containing sample collected from said patient, wherein said HBV is not genomically integrated yet, the presence of at least one mutation of

HBx in amino acid I in position 127 and/or in amino acid K in position 130 and/or in amino acid V in position 131 , said positions being computed with respect to the complete HBx sequence of SEQ ID NO:2.

Said detection of the presence of at least one mutation can be achieved by detecting the presence in said sample of at least one mutant peptide of the invention, or of a cell expressing or containing such a mutant peptide.

To detect such mutant peptides, antibody directed against them can be used, preferably antibody which binds to at least one peptide of the invention, without binding to a non-mutant HBx peptide. Such antibodies, and specific antibodies, are encompassed by the present invention.

The diagnosis and prognosis methods of the invention can be implemented on any sample, which is representative of the HBV infection occurring, or suspected of occurring, in the patient.

Advantageously, the teaching of the present invention enables to implement them of a sample, which is a body fluid, which contains or is susceptible to contain, circulating hematopoietic cells, such as blood, preferably serum.

Other definitions:

In the present application, all terms are used and meant in their ordinary meaning, i.e., the meaning that the person of ordinary skill in the art would give to them. Amino acids:

In the context of the present invention, 'amino acid residue¹ means any amino acid residue known to those skilled in the art (see e.g.: Sewald et a/., 2002; IUPAC nomenclature under http://www.chem.qmul.ac.uk/iupac/AminoAcid/). This encompasses naturally occurring amino acids (including for instance, using the three-letter code, Ala, bAla, Arg, Asn, Asp, Cys, GIn, GIu, GIy, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, VaI), as well as rare and/or synthetic amino acids and derivatives thereof (including for instance Aad, Abu, Acp, Ahe, Aib, Apm, Dbu, Des, Dpm, HyI, MeLys, MeVaI, Nva, HAO, NCap, Abu, Aib, MeXaa and the like (see e.g.: (Mϋller et a/., 1993; Aurora et a/., 1998; Obrecht et a/., 1999; Maison et al., 2001 ; Formaggio et al., 2003; Nowick et al., 2003. Said amino acid residue or derivative thereof can be any isomer thereof, especially any chiral isomer, e.g., the L- or D- isoform. By amino acid derivative, we hereby mean any amino acid derivative as known in the art (see e.g.: Sewald et al., 2002; IUPAC nomenclature under http://www.chem.qmul.ac. uk/iupac/AminoAcid/).

For instance, amino acid derivatives include residues derivable from natural amino acids bearing additional side chains, e.g. alkyl side chains, and/or heteroatom substitutions. Further examples of amino acid derivatives comprise amino acid bearing chemical modifications such the one fund in mimetic peptides or peptidomimetics, which are compounds containing non-peptidic structural elements that are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic usually does no longer have classical peptide characteristics such as enzymatically scissille peptidic bonds. Preferably, said amino acid belongs to the group of the non-essential amino acids. Preferred non-essential amino acids are glycine, alanine, proline, serine, cysteine, tyrosine, asparagines, glutamine, aspartic acid, glutamic acid, arginine, histidine. Appropriate amino acids may be accurately selected by selecting those amino acids which are in lower amounts in the patient into which the drug is to be administered. Dosage and administration regimen can be determined as a function of the patient's level in said amino acid. Preferred dosage and administration regimen are those which intend to increase the patient's amino acid level up to the normal standard level. Comprising / consisting:

The term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term "consisting of is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited. The term "essentially consisting of is a partially open term, which does not exclude additional, unrecited element(s), step(s), or ingredient(s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the invention. The term "comprising" (or "comprise(s)") hence includes the term "consisting of ("consist(s) of), as well as the term "essentially consisting of ("essentially consist(s) of). Accordingly, the term "comprising" (or "comprise(s)") is, in the present application, meant as more particularly encompassing the term "consisting of ("consist(s) of), and the term "essentially consisting of ("essentially consist(s) of).

Significant:

The term "significantly" is herein used in its usual meaning in the field of statistics

(e.g., t test, z test, chi squared value, or F ratio, etc.), i.e., for comparing a value to another one, and determining whether these values differ from each other. The term "significantly" hence encompasses the fact that the skilled person may take into account the standard deviation (if any), which measures the amount of spread of data in a frequency distribution. The desired p value is usually set at an alpha level of 5%, or at the more stringent alpha level of 1 %.

Each of the relevant disclosures of all references cited herein is specifically incorporated by reference. The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES:

Despite the availability of an effective vaccine against hepatitis B virus (HBV) infection for more than two decades, more than 5% of the world population, i.e., 400 million people, are currently infected with HBV, and >1 million people die each year from HBV-related liver cirrhosis and hepatocellular carcinoma. HBx protein of HBV is a critical component of the infection process in vivo and has been implicated in the development of hepatocellular carcinoma. We were interested in the detection and characterisation of CD8+ and CD4+ peripheral HBx-specific T cells. For this purpose, chronically infected patients with less than 100,000 HBV copies/ml are being enrolled. At present, 49 out of 60 patients were studied. To analyze the HBx-specific T cells, we used Elispot assays or intracellular cytokine staining to measure IFN-gamma and IL-10 secretion. A total of 29 fifteen-mer peptides corresponding to the HBx consensus sequence were used for in vitro stimulation of PBMC from all patients. This study allowed us to define an immunodominant promiscuous T-cell epitope located at the carboxy-terminal part of HBx and recognized by CD4+ T cells from a vast majority of the patients. The pathogenesis of HBV infection is modulated through the selection and expression of a number of common viral mutants. As the HBV genome encodes its essential genes with overlapping ORFs, a mutation in the HBV genome can alter the expression of multiple proteins. The common double mutation in the HBV pre-core promoter region A1762T/G1764A, which has been largely found in tumors, corresponds to the double mutation in codons 130 and 131 of the HBV X gene. Importantly, these and other HBx mutations described during the evolution of chronic HBV infection are located at the immunodominant CD4+ T-cell epitope. Therefore, we evaluated the cross-recognition by T cells specific for the promiscuous HBx epitope of viral sequence variants occurring during the natural history of HBV infection. In this case, fifteen-mer peptides corresponding to the HBx wild-type promiscuous epitope or viral mutant sequences were used for in vitro stimulation of PBMC.

In summary, we described here a novel immunodominant promiscuous T-cell epitope within HBx protein largely recognized during the chronic phase of HBV

Materials and Methods

Patient population

Sixty chronically HBV-infected subjects with less than 100,000 HBV copies/ml, HBsAg+, HBeAg+, anti-HBe antibodies+ and with normal transaminases level were enrolled. In one group patients were inactive carriers of HBsAg (n=30) and in another group patients had controlled viral replication either spontaneously or due to effective antiviral treatment (n=30). All patients were 18 to 60 years-old, had no immunosuppression or infections associated with human immunodeficiency virus (HIV), hepatitis C virus (HCV) or hepatitis D virus (HDV), or liver diseases other than HBV infection and had an alcohol consumption inferior than 40 g/d. HLA-A2 and HLA-B7 phenotyping was performed using PE-coupled anti-HLA-A2 (BB7-2) and FITC-coupled anti-HLA-B7 monoclonal antibodies (Serotec, Oxford, UK). HLA-DR genotyping was performed using Olerup SSP™ Genovision kit (Saltsjόbaden, Sweeden). Two blood samples from each patient were collected. At present, 49 out of 60 patients were analysed. This study was approved by the ethics committee of Necker Hospital, and all study participants gave informed, ritten consent for participation in line with French ethical guidelines.

The HLA haplotypes of the patients have been analysed. Herein below are shown the DR haplotypes (table 2).

Table 2: DR haplotypes

DR

Cone.

Cells Positive lanes SSP genotypes DRBl DRB3 DRB4 DRB5 T-response

(ng/μL) Haplotype

Patients

FRAAN PBMC 44 6, 13, 15, 16, 17, 19, 21 DRB1*11, DRB1*13, V X26 positive DR52

MOUIB PBMC 63 6,13,15,16,17, 21 DRB1*11, DRB1*13, V X26 positive DR52

PHYME PBMC 38 13,14,15,16,21 DRB1*11, DRB1*12, V X26 positive DR52

YEJIA PBMC 48 13.14.15.16.21 DRB1*11, DRB1*12, V X26 positive DR52

BONMO PBMC 42 8,9,22 DRB 1*04, DRB 1*07, V X26 negative DR1,DR53

KONMO PBMC 81 1,5,17,21 DRB 1*01,DRB 1*03, V X26 positive DR1,DR52

DELMI EBV-B 101 5,6,15,16,17,21 DRB1*O3, DRB1*13, V X26 positive DR52

HACVI EBV-B 148 5,6,15,16,17,21 DRB 1*03, DRB 1*13, V X26 positive DR52

NGUNQ EBV-B 147 5,6,15,16,17,21 DRBl *03, DRBl *13, V X26 positive DR52

GUIDI PBMC 41 3,14,16,21,23 DRB1*12, DRB1*15, V V X26 negative DR51,DR52

TUREL PBMC 10 5.6.9.17.21.22 DRBl *03, DRB 1*07, V V X26 negative DR52, DR53

DELJE PBMC 23 3, 10, 16, 23 DRB1*O8, DRB1*15, X26 negative DR51

LAIJE PBMC 13 1.8.22 DRBl *01, DRB 1*04, V? X26 negative DR1,DR51

DUTJE PBMC 13 1.8.23 DRB1*O1, DRB1*O4, X26 positive DR1,DR53

ROTJE PBMC 16 13,14,15,16,21 DRB1*11, DRB1*12, V X26 positive DR52

GIMJA PBMC 43 8,22 DRB1*O4, (DRB1*1367), V X26 negative DR53

DOUNA EBV-B 122 5, 6, 17, 21, 22, DRB 1*03, DRB3, V X26 negative DR53

BONLI EBV-B 83 6, 8?, 15, 16, 17, 21, 22, DRB1*O4, DRB1*13, V V X26 negative DR52, DR53

BOCSO EBV-B 134 5,6,15,16,17,21, DRB1*O3, DRB1*13, V NOT DONE DR52

LOBLE EBV-B 119 5,9,17,21,22 DRB 1*03, DRB 1*07, V V NOT DONE DR52, DR53

DIACO EBV-B 65 5, 6, 13, 15, 16, 17, 21 DRB1*O3, DRB1*11, ■J NOT DONE DR52 X26

DRAFA EBV-B 82 5,17,21 DRB 1*03, (DRB 1*1367), V DR52 positive**

JURRO EBV-B 53 6,8? ,15,16 ,17 ,21,22, DRB1*O4,DRB1*13, ■J V X26 negative DR52, DR53 DUFJC EBV-B 60 8, 18 , 19, 2071 ,22 DRB 1*04, DRBl* 14, V V X26 negative DR52, DR53

HUEAL PBMC 16 8,13 ,15,16, 22 DRB 1*04, DRB 1*11, V X26 positive DR53 PERCU PBMC 73 9, 13 , 15,16, ?1 22 DRBl *07, DRB 1*11, V V X26 positive DR52, DR53

WADPH PBMC 34 1,3, 23 DRBl *01, DRBl* 15, V X26 positive DR1,DR51 ANDCO PBMC 67 9,11 ,19,22 DRB 1*07, DRB 1*09, V X26 negative DR53 PREXA PBMC 32 3,6, 13, 15, 16 21,23 DRB1*11, DRB1*15, V X26 positive DR52, DR51

GESLI PBMC 54 5,6, 13, 15, 16 17,21 DRB 1*03, DRB 1*11, V X26 positive DR52

MEKMO PBMC 42 8, 13 , 15,16 71 ,22 DRB 1*04, DRB 1*11, V ^■J X26 positive DR52, DR53

NGUER PBMC 45 5,6, 11, 17, 19 21,22 DRB1*O3, DRB1*9, V -J NOT DONE DR52, DR53

but main reactive peptide is x25

CO

Synthetic peptides

The synthetic peptides were purchased from NeoMPS (Strasbourg, France). The consensus sequence of the HBx protein (MAARLCCQLDPARDVLCLRPVGAESRGRPLSGPLGTLSSPSPSAVPTDHGAHL SLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQILPKVLHKRTLGLSAMSTTDL EAYFKDCLFKDWEELGEEIRLKVFVLGGCRHKLVCAPAPCNFFTSA; SEQ ID NO: 2) was covered by 29 peptides of 15 mers overlapping by 10 residues. These peptides were divided in 3 pools: - pool A (peptides x1 to x10),

- pool B (peptides x11 to x20) and

- pool C (peptides x21 to x29).

The peptides were re-suspended at 1 mg/ml and stored at -20 ⁰C until used. These peptides were used as peptide pools or as individual peptides for in vitro stimulation of PBMC and in the Elispot assay. The promiscuous CD4+ T-cell epitope described in this study was identified by activating PBMC of HBV chronic carriers with the HBx-derived peptide number 26 (EIRLKVFVLGGCRHK; SEQ ID NO:20), located at the amino-acids 126 to 140 within HBx protein and called hereafter x26 peptide. Two other peptides corresponding to variants sequences of the wild-type x26 peptide were used: EIRLMIFVLGGCRHK (SEQ ID NO: 23) and ETRLMIFVLGGCRHK (SEQ ID NO: 24), called hereafter V2 and V3 respectively.

In vitro expansion of PBMC

PBMC were re-suspended at 3 x 10⁶ cells per ml in complete medium (RPMI 1640 medium, Life Technologies, Gaithersburg, MD) supplemented with 2 mM L- glutamine, 1 mM sodium pyruvate, non-essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% human AB serum (Institut Jacques Boy, Reims, France) plus 20 ng/ml IL-7 (Roche, Meylan, France) and 100 pg/ml IL=12 (R&D Systems Inc., MN, USA) in 24-well plates. Cells were stimulated by incubation with peptide pools A, B and C at 1 microg/ml of each peptide or with individual peptides at 10 microg/ml. Half the medium was replaced every 3-4 days with complete medium supplemented with recombinant IL-2 (50 Ill/ml) (Roche, Meylan, France). After 10-14 days of culture, IFN-gamma- and IL-10-producing cells were specifically quantified by Elispot assays. Elispot assay

Sterile nitrocellulose HA 96-well plates (Millipore, Bedford, MA) were coated overnight at 4°C with 15 microg/ml anti-IFN-gamma mAb (clone 1-DIK; Mabtech, Stockholm, Sweden) in 50 microL 0.1 M bicarbonate buffer (pH 9.6) or with 10 microg/ml anti-IL-10 mAb (clone B-N10, Diaclone, Besancon, France) in 50 microL PBS (pH 7.0). Wells were blocked with 200 microL of 5% human AB serum in PBS at room temperature for 2h and then washed three times with serum-free medium. The coated wells were filled in triplicate with in vitro- stimulated cells (1 to 2 x 10⁵/well) in complete medium and the appropriate peptides (1 microg/ml) or with medium alone as negative control. After 20h of incubation at 37°C, plates were washed with PBS-Tween and incubated with 50 microL of 1 microg/ml biotinylated anti-IFN-gamma mAb (clone 7B6-1 ; Mabtech) or with 50 microL of 20 microg/ml biotinylated anti-IL-10 mAb (clone B-T10; Diaclone) for 2h at room temperature. Plates were then washed as described above and an alkaline phosphatase-conjugated streptavidin (Mabtech) was added for 1.5 h. After washing three times with PBS-Tween and two times with PBS, spots were developed by adding the substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Promega, Madison, Wl). After appearance of spots, the reaction was stopped with tap water and air-dried. A Zeiss Elispot automated counter was used to score the number of spots. The response was considered positive if the median number of spot-forming cells (SFC) in triplicate wells was at least twice that in control wells containing no peptide and at least 5 spots were detected per 10⁵ PBMC after background subtraction. The specificity and cut-off of Elispot assays was determined with PBMC from healthy individuals (n=9) and with PBMC from hemochromatosis patients (n=2). These PBMC were subjected to in vitro expansion with HBx-derived peptides and tested in Elispot assays in experimental conditions identical to PBMC from HBV chronic carriers. The cut-off was fixed at 62 IFN-gamma- and 40 IL-10-SFC and was calculated as (X + 2 sd) of SFC per million of PBMC from HBV-negative subjects.

Inhibition of Elispot assays In order to determine class Il HLA-restriction, aliquots of in vitro expanded PBMC were pre-incubated at 37°C for 90 min with 10 microg/ml of anti-class Il HLA antibodies: anti-HLA-DR (L243), anti-HLA-DQ (SPVL3) and anti-HLA-DP (B7-21). Anti-class I HLA-A2 antibodies (BB7-2) were used as negative control. Pre- incubated PBMC were tested in Elispot assays as described above.

Intracellular staining

In vitro expanded PBMC were incubated either with Staphylococcal Enterotoxin B at 500 ng/ml (Sigma, St. Louis, MO, USA) as positive control, with medium alone as negative control or with individual HBx-derived peptides at 1 microg/ml in a total volume of 200 microL complete medium. Brefeldin A (Sigma) was added at a final concentration of 2 microg/ml, and cells were then incubated overnight at 37°C. Cells were washed twice with fluorescence-activated cell sorter (FACS) buffer (PBS, bovine serum albumin 1%, sodium azide 0.1%) and stained with 2 microL of CD4-PE (clone RPA-T4; BD Pharmingen) and 2 microL of CD8-PerCP (clone SK1 ; BD Biosciences) in 50 microL of FACS buffer for 30 min at 4°C. After two washes with FACS buffer, the cells were fixed with 2% formaldehyde-FACS buffer for 15 min at 4°C. The cells were then washed twice in FACS buffer and once with permeabilization buffer (0.1 % saponin in FACS buffer). Cells were then stained with 1 microL of anti-human IFN-gamma-FITC antibody (clone 4S.B3; BD Biosciences) in 50 microL of permeabilization buffer for 30 min at 4°C. The cells were washed twice with permeabilization buffer, once with FACS buffer and then fixed with 1% formaldehyde-FACS buffer. Samples were stored in the dark at 4°C. At least 50,000 lymphocyte-gated events were acquired using FACSCalibur flow cytometer (BD Biosciences) and analysed with the Cellquest software (BD Biosciences). Background staining was assessed using a control isotype- matching monoclonal antibody and subtracted from all values.

Results

Presence of HBx-specific IFN-gamma-secreting T cells in chronic HBV carriers

Among 49 patients studied, HBx-specific IFN-gamma-secreting T cells were detected in 35/49 (71.4%) chronic carriers. Total and diversity of HBx-specific IFN-gamma-secreting T-cell responses obtained with pools A, B and C are shown in Figure 1. Fifty-one per cent (25/49) of patients had T cells specific for epitopes located in the carboxy-terminal part of the protein. T-cell responses were mainly focused on pool C only and less than 10% of patients had responses specific for the 3 peptide pools.

Mapping of the IFN-gamma-secreting T-cell response to individual pool C peptides

The quantification of IFN-gamma-secreting T cells upon stimulation with peptide pool C and mapping of the T-cell response to single peptides is shown in Figure 2.

IFN-gamma-secreting T cells recognizing the carboxy-terminal domain of HBx targeted a single immunodominant epitope x26 in 24/29 patients (82.8%) (Figure

2, middle panel). For some patients, weak T-cell reactivities to other pool C peptides were detected {Figure 2, right panel). T cells specific for peptides HBx24 and HBx 25 were found in 2 and 3 patients respectively. For another patient

(WADPH) INF-gamma ELISPOT assay was positive (>1 ,000 spots/ million PBMC) using peptide pool C for stimulation. Mapping of T cell response to single peptides revealed that HBx22 (183 spots), HBx25 (112 spots), HBx26 (1750 spots) and

HBx27 (150 spots) peptides were reactive. Background level was 45 spots/million PBMC.

All together, these results showed that T-cell response is focussed on x26 peptide or clustered in the region that overlaps x26 peptide.

Characterisation of the x26 promiscuous epitope The phenotyping of IFN-gamma-producing x26-specific T cells of one representative out of 10 patients tested is shown in Figure 3. The promiscuous x26 epitope specifically stimulated IFN-gamma-secreting CD4+ T cells (see upper right panel). Pre-incubation of PBMC with anti-HLA-DR antibodies resulted in at least 80% of inhibition of IFN-gamma-secretion upon stimulation with x26 peptide. This effect was neither observed after pre-incubation of PBMC with anti-HLA-DP or anti-HLA- DQ antibodies, nor with the irrelevant control anti-HLA-A2 antibodies (Figure 4). These results indicated that the promiscuous x26 epitope is presented by HLA-DR molecules. Presence of HBx-specific IFN-gamma- and IL-10-secreting T cells^' in chronic HBV carriers

Among 25 chronic carriers studied, 12 patients presented neither IFN-gamma- nor IL-10-producing specific T cells. T cells secreting IFN-gamma only were observed in 7 patients (Figure 5A). Finally, 6 patients had T cells secreting IFN-gamma or

IL-10 (Figure 5B). Except for one patient, the frequency of IFN-gamma-secreting

T cells was always higher than for IL-10-secreting T cells (Figure 5B). All together, peptide pool C or individual x26 peptide activated efficiently IFN-gamma-secreting T cells, but IL-10 secretion was comparatively weak or even undetectable.

Cross-recognition of viral mutants by x26-specific T cells

PBMC from 7 patients with x26-specific T-cell response were activated in vitro with variant peptides V2 and V3 to assess cross-reactivity (Figure 6). In 4 patients x26-specific T cells were not activated by the variant peptides. In 3 patients the number of cells activated by the variant peptides was lower than with the wild type x26 peptide. In conclusion, viral mutant sequences were less potent or even unable to activate T cells specific of the promiscuous wild-type x26 epitope.

Conclusions

• although CD8+ cytotoxic T lymphocytes are generally thought to be the most important effector cells for the elimination of virally infected cells, CD4+ T cells play a central role in the antiviral immune response, not only by inducing or maintaining cytotoxic activity but also by secreting antiviral cytokines

• the characterisation of the immunodominant T-cell response directed against

HBx in HBV-infected individuals could have important implications for clinical follow-up and/or therapeutic vaccine design for chronically infected patients • the degenerate HLA-DR binding of the x26 peptide make this epitope a valuable component of a vaccine that is designed to cover a large and ethnically diverse patient population • production of IFN-gamma by activated T cells may have important implication for the non cytolytic clearance of HBV from hepatocytes

• mutations in HBx sequences that are frequently found in patients with pre- core mutation, fulminant hepatitis or hepatocellular carcinoma can affect T- cell recognition of the HBx x26 promiscuous epitope. This makes the x26 peptide a valuable tool to monitor HBV sequence evolution involved in disease progression.

Claims

1. A peptide, which is capable of eliciting a promiscuous immunodominant CD4+ response directed against HBx protein of HBV, characterized in that it consists of:

- an HBx fragment of SEQ ID NO:20 (EIRLKVFVLGGCRHK; peptide "x26"), or of

- a MHC class ll-restricted analogue thereof, which derives from said HBx fragment by substitution and/or deletion of one or several amino acid(s), and which has retained a capacity of eliciting a CD4+ response directed against HBV, but which does not bind to any HLA class I molecule,

- a MHC class ll-restricted sub-fragment of said HBx fragment or of said analogue, wherein said sub-fragment has retained a capacity of eliciting a CD4+ response directed against HBV, but does not bind to any HLA class I molecule.

2. A nucleic acid, which codes for the peptide of claim 1 , in accordance with the universal genetic code, taking into account the degeneracy of this code.

3. A vector, which comprises at least one nucleic acid of claim 2.

4. A cell, which has been transfected, infected or transformed by at least one nucleic acid of claim 2, and/or by at least one vector of claim 3.

5. The cell of claim 4, which is an antigen presenting cell.

6. A peptide of claim 1 , which is fused or coupled to at least one protein other than HBx, or to a peptide fragment other than an HBx peptide fragment.

7. The fused or coupled peptide of claim 6, wherein said at least one other protein is an HBV protein other than HBx, or said at least one other peptide fragment is a HBV peptide fragment other than an HBx peptide fragment.

8. The fused or coupled peptide of claim 7, wherein said at least one other HBV protein is an HBV envelop protein, or an HBV capsid protein, or an HBV polymerase protein, or an HBSP protein, or wherein said at least one other HBV peptide fragment is an HBV envelop peptide fragment, or an HBV capsid peptide fragment, or an HBV polymerase fragment, or an HBSP peptide fragment.

9. The fused or coupled peptide of claim 7 or 8, wherein said at least one other HBV peptide fragment is, or comprises, at least one CD8+ and/or at least one B cell HBV epitope.

10. The peptide of claim 6, wherein said at least one other protein or peptide is a protein or peptide other than a HBV protein or peptide.

11. The fused or coupled peptide of claim 10, wherein said at least one non-HBV protein is a MHC molecule.

12. The fused or coupled peptide of claim 11 , wherein said at least one MHC molecule is a HLA class Il molecule.

13. The fused or coupled peptide of any one of claims 10-12, which is fused or coupled to four HLA class Il molecules.

14. A nucleic acid, which codes for the fused or coupled peptide of any one of claims 6-13, in accordance with the universal genetic code, taking into account the degeneracy of this code.

15. A vector, which comprises at least one nucleic acid of claim 14.

16. A cell, which has been transfected, infected or transformed by at least one nucleic acid of claim 14, and/or at least one vector of claim 15.

17. The cell of claim 16, which is an antigen presenting cell.

18. A pharmaceutical composition, which is intended for eliciting an immune response in an organism in need thereof, which comprises: - at least one peptide of claim 1 , or at least one nucleic acid of claim 2, or at least one vector of claim 3, or at least one cell of any one of claims 4-5, or

- at least one fused or coupled peptide of any one of claims 6-13, or at least one nucleic acid of claim 14, or at least one vector of claim 15, or at least one cell of any one of claims 16-17.

19. The pharmaceutical composition of claim 18, which further comprises at least one carrier molecule and/or at least one adjuvant.

20. The pharmaceutical composition of claim 18 or 19, which is intended for the prevention and/or palliation and/or therapy of a disease or condition involving an HBV infection.

21. The pharmaceutical composition of any one of claims 18-20, wherein said disease or condition is hepatitis, or a chronic HBV infection, or a hepatocellularcarcinoma, or a cirrhosis.

22. The peptide of claim 1 , for use a CD4+ antigen.

23. A method for activating CD4+ T cells, which comprises in vitro contacting at least one CD4+ T cell with:

- at least one peptide of claim 1 , or at least one nucleic acid of claim 2, or at least one vector of claim 3, or at least one cell of any one of claims 4-5, or - at least one fused or coupled peptide of any one of claims 6-13, or at least one nucleic acid of claim 14, or at least one vector of claim 15, or at least one cell of any one of claims 16-17.

24. A method for inducing and/or stimulating the maturation of a dendritic cell, which comprises in vitro contacting at least one dendritic cell with:

- at least one peptide of claim 1, or at least one nucleic acid of claim 2, or at least one vector of claim 3, or at least one cell of any one of claims 4-5, or - at least one fused or coupled peptide of any one of claims 6-13, or at least one nucleic acid of claim 14, or at least one vector of claim 15, or at least one cell of any one of claims 16-17.

25. The fused or coupled peptide of any one of claims 11-13, for use in the isolation of HBV-activated CD4+ T cells.

26. A mutant peptide, which derives from at least one peptide of claim 1 by at least one amino acid substitution, but which has lost the capacity of eliciting a CD4+ response directed against HBV.

27. The mutant peptide of claim 26, which is mutated in amino acid I in position 127, and/or in amino acid K in position 130, and/or in amino acid V in position 131 , said positions being computed with respect to the complete HBx sequence of SEQ ID NO:2.

28. The mutant peptide of claim 26 or 27, which consists of SEQ ID NO: 23 or of SEQ ID NO: 24.

29. The mutant peptide of any one of claims 26-28, for use as a hepatocarcinoma predisposition indicator.