Treatment of hepatitis b virus associated diseases

Abstract

The present invention provides novel immunogenic peptides derived from the X protein and the polymerase protein of the hepatitis B virus (HBV). These peptides contain epitopes that are very conserved across multiple HBV variants and are derived from protein regions that are essential for viral replication. Furthermore, novel HBV antigens have been identified that bind to multiple HLA types and epitopes that elicit IFNγ responses in PBMCs from HBV resolvers.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080084855.0, entitled “Treatment of Hepatitis B Virus-Related Diseases”, filed on December 4, 2020. Technical Field

[0002] This invention relates to the field of infectious diseases. In particular, this invention relates to immunogenic peptides, polynucleotides, immunogenic compositions, and methods for treating hepatitis B virus (HBV)-related diseases. Background of the Invention Chronic hepatitis B virus (HBV) infection is a major global health problem. HBV is a prototypical member of the hepatotropic DNA virus family and has a strong preference for infecting hepatocytes (Ganem et al., 2004 N Engl J Med [New England Journal of Medicine] 350:1118).

[0003] Despite the availability of effective preventative vaccines against hepatitis B for 30 years, an estimated 2 billion people are infected with HBV, and more than 240 million are currently infected with chronic (long-term) hepatitis B, primarily outside of Western Europe and North America (World Health Organization, July 2013).

[0004] Human-to-human transmission of the virus occurs through direct blood-to-blood contact or via the semen or vaginal fluid of an infected person. In endemic areas, infection is characterized by perinatal transmission from mother to child. Therefore, although HBV is not easily transmitted, the virus can be readily spread through perinatal, percutaneous, or sexual exposure. Consequently, frequent person-to-person contact with infected individuals poses a serious risk to groups such as healthcare workers.

[0005] HBV infection can develop into acute viral hepatitis, which begins with general malaise, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, then progresses to jaundice. In most affected adults, the illness lasts for several weeks before gradually improving, although some may develop more severe, potentially fatal liver disease (fulminant hepatic failure). The infection can be completely asymptomatic and may go unnoticed.

[0006] Chronic hepatitis B virus infection can be asymptomatic or associated with chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of many years. This type of infection significantly increases the incidence of hepatocellular carcinoma (HCC) and can remain dormant for many years. Treatment of individuals with chronic HBV infection with antiviral drugs, such as nucleoside / nucleotide analogs (e.g., entecavir and tenofovir) or interferon (IFN) α, can effectively reduce serum viral load. However, antiviral therapy rarely produces a sustained virological response and resistance develops (Zoulim et al., 2012 BJ Hepatol 56 Supplement 1 S112; EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. [EASL 2017 Clinical Practice Guidelines on the Management of Hepatitis B Virus Infection] J. Hepatology 67:370). Furthermore, the vast majority of HBV carriers remain untreated.

[0007] Approximately 15%–40% of chronic HBV carriers will develop clinically significant liver disease during their lifetime, with a high risk of death from cirrhosis and related liver failure or hepatocellular carcinoma (Huang et al., 2011 Curr Opin Immunol [Current Views in Immunology] 23:237). Because antiviral drugs cannot eradicate the infection and therefore require long-term (if not lifelong) antiviral therapy, which has drawbacks such as toxic side effects and high costs, there is an urgent need for new treatment methods (Grimm et al., 2013 Clin Sci [Clinical Science] (London) 124:77).

[0008] Therapeutic vaccination constitutes a promising strategy for treating chronic hepatitis B. In addition to the humoral immune response against HBV (which is primarily involved in the protection against HBV infection provided by current prophylactic vaccines) (Lok, 2002 N Engl J Med [New England Journal of Medicine] 346:1682), the cellular immune response is clearly involved in the development of natural resistance to HBV infection.

[0009] Perinatal transmission of HBV from mother to newborn and infection during the first few years of life result in persistent infection in over 90% of children. In contrast, infection during adulthood clears spontaneously in over 90% of cases and leads to lifelong protective immunity (Rehermann et al., 2005 Nat Rev Immunol [Nature Review Immunology] 5:215).

[0010] In acute, self-limiting hepatitis B virus infection, strong polyclonal and multispecific CD8+ cytotoxic T cell (CTL) and CD4+ T-helper (Th) cell responses to many HBV antigens are readily observed in peripheral blood (Michel et al., 2011 J Hepatol [Journal of Hepatology] 54:1286).

[0011] These T cell responses are crucial in HBV clearance and control. Experiments in HBV-infected chimpanzees have demonstrated the essential role of HBV-specific CD8+ T cells as effector cells in this process (Thimme et al., 2003 J Virol [Journal of Virology] 77:68). Compared to the responses in patients with regressed HBV infection, T cell responses in patients with chronic hepatitis B are typically very weak, concentrated on only a few epitopes, and functionally impaired (Michel et al., 2011 J Hepatol [Journal of Hepatology] 54:1286). The goal of therapeutic vaccination is to establish strong and robust multivalent CTL and T helper cell responses against multiple HBV antigens, thereby achieving viral clearance, hepatitis control, and cure.

[0012] Despite significant progress in understanding the etiology and epidemiology of the disease, an effective therapeutic HBV vaccine is still needed.

[0013] HBV protein-derived peptides containing putative T-cell epitopes restricted to specific HLA types have been described in the art (WO 0219986, WO 2002020035, WO 2014102540, WO 15187009). However, many of these proposed antigens have one or more of the following disadvantages: ● Poor sequence conservation across HBV genotypes results in efficacy only for a (small) subset of HBV genotypes. ● The antigenic regions derived from these regions are not functionally linked to viral replication, reducing the likelihood that an immune response against these regions will interfere with viral replication. ● Due to excessive exposure to antigens, immune exhaustion occurs, resulting in an ineffective immune response. ● It only covers one or a few HLA types, resulting in a lack of efficacy in some populations that lack these HLA types. ● Difficult to manufacture (especially for longer peptides). ● It has no confirmed potential to induce a T-cell response. ● There is no indication regarding whether the immune response to the peptide antigen has the potential to resolve the infection. Summary of the Invention

[0014] This invention provides novel long peptide antigens derived from HBV-X and HBV polymerase proteins that overcome all or most of the drawbacks associated with previously described peptide antigens.

[0015] The HBV long peptide antigens described herein contain novel HLA-binding peptide sequences that are highly conserved across multiple HBV genotypes, originate from conserved regions of proteins essential for viral replication, and are therefore unlikely to escape from HBV-specific immune responses. Furthermore, the novel HBV-derived long peptide antigens possess multiple HLA-binding peptide sequences capable of being presented by various HLA types. In addition, synthetic long peptides (SLPs) containing these HLA-binding peptide sequences are described, which can be manufactured in sufficient yield and with sufficient purity. Furthermore, SLPs containing the newly identified HLA-binding peptide sequences have been found to elicit an IFNγ response in PBMCs of individuals whose HBV infection has regressed (HBV-regressed individuals).

[0016] In a first principal aspect, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the fragment is 20-34 amino acids in length, and wherein the fragment comprises: a) At least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, preferably comprising: - The amino acid sequence (x70-78) listed in SEQ ID NO: 1, and / or - The amino acid sequence (x67-75) listed in SEQ ID NO: 2, and / or - The amino acid sequence (x62-73) listed in SEQ ID NO: 3, and / or - The amino acid sequence (x58-66) listed in SEQ ID NO: 4, and / or - The amino acid sequence (x57-66) listed in SEQ ID NO: 5. or b) At least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, preferably comprising: - The amino acid sequence (x103-111) listed in SEQ ID NO: 6, and / or - The amino acid sequence (x104-113) listed in SEQ ID NO: 7, and / or - The amino acid sequence (x105-113) listed in SEQ ID NO: 8, and / or - The amino acid sequence (x110-120) listed in SEQ ID NO: 9. or c) The amino acid sequence listed in SEQ ID NO: 10 (x132-140). or d) The amino acid sequence listed in SEQ ID NO: 11 (p124-133). or e) The amino acid sequence listed in SEQ ID NO: 12 (p164-173). or f) The amino acid sequence listed in SEQ ID NO: 13 (p275-283). or g) At least 10 consecutive amino acids from the region at positions 403 to 415 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 14 (p403-412), and / or - The amino acid sequence listed in SEQ ID NO: 15 (p404-412), and / or - The amino acid sequence listed in SEQ ID NO: 16 (p407-415). or h) At least nine consecutive amino acids from the region at positions 509 to 523 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 17 (p509-517), and / or - The amino acid sequence listed in SEQ ID NO: 18 (p515-523). or i) At least 10 consecutive amino acids from the region at positions 649 to 658 of the HBV polymerase, comprising: - The amino acid sequence listed in SEQ ID NO: 19 (p649-658), and / or - The amino acid sequence listed in SEQ ID NO: 20 (p650-658). or j) At least 10 consecutive amino acids from the region at positions 693 to 706 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 21 (p693-701), and / or - The amino acid sequence listed in SEQ ID NO: 22 (p697-706). or k) The amino acid sequence listed in SEQ ID NO: 23 (p723-731). or l) At least 10 consecutive amino acids from the region at positions 755 to 765 of the HBV polymerase, comprising: - The amino acid sequence listed in SEQ ID NO: 24 (p755-764), and / or - The amino acid sequence listed in SEQ ID NO: 25 (p756-765). or m) The amino acid sequence listed in SEQ ID NO: 26 (p829-837).

[0017] In another aspect, the present invention relates to polynucleotides comprising nucleotide sequences encoding peptides according to the present invention, immunogenic compositions comprising immunogenic peptides or polynucleotides of the present invention, and the use of peptides, polynucleotides, recombinant viruses, or immunogenic compositions of the present invention in the treatment of HBV-related diseases. Attached Figure Description

[0018] Figure 1 Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for the protein conservation and function of HBx. A centered bar chart depicts the length of the HBx protein consortium sequence (method), where conservation scores across viral genotypes are indicated by grayscale codes for each amino acid (legend). Validated epitope sequences reported from the Hepitopes database are aligned with this consortium protein sequence and shown at the top. Below this, potential novel binders (9–11 amino acids) predicted by NetMHCpan are depicted for each HLA supertype representative. Below the aligned binders, we plot the frequency distribution of each amino acid within all predicted binders (8–14 amino acids in length) on the protein sequence. The conservation score for each amino acid (legend) is shown as a horizontal grayscale-coded bar chart. Essential amino acids whose mutations result in loss of viral persistence are indicated by arrows matching the color of the conservation score. Functional domains are depicted at the bottom according to the HBVdb nomenclature.

[0019] Figure 2A comparison of the protein conservation and function of validated HLA-I epitopes and predicted HLA-binding peptide sequences for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are depicted. Validated epitope sequences reported from the Hepitopes database are shown at the top. Below this, potential novel binders (9-11 amino acids) predicted by NetMHCpan are depicted for each HLA supertype (AD). Furthermore, we plotted the frequency distribution (EH) of predicted binders (8-14 amino acids in length) on the protein sequence. The conservation score (legend) for each amino acid is shown as a horizontal grayscale coded bar chart. Essential amino acids whose mutations, alone or in combination, result in loss of viral persistence (≥ 50%) are indicated by arrows matching the color of the conservation score for that particular amino acid. Amino acids predicted to be crucial for the correct folding of viral proteins are indicated by asterisks. The general domain is described according to the previously established nomenclature (Cao et al. 2014 J Viral Hepat [Journal of Viral Hepatitis] 21:882), which also indicates the T3 domain and the YMDD motif.

[0020] Figure 3 The in vitro binding capacity of selected predicted HLA-binding peptide sequences. Binding of the predicted HLA-binding peptide sequence is expressed as the percentage of binding to a positive control peptide (pos. ct.) with high affinity for the indicated HLA type. Mean and standard deviations are depicted for controls (black), bound peptides (>25% positive control; gray), and unbound peptides (≤25% positive control; white). Binding capacity was assessed for six supertype representatives, and also considered HLA... The high prevalence of 11:01 in the chronic hepatitis B patient population is included. Each of these is depicted in a separate figure (AG). As negative controls, we include known non-binding peptides (neg. ct.) for each HLA type and cases where no peptide is present. Peptides with closed underlines are rarely described as epitope sequences associated with the HLA type tested. Peptides with dashed underlines are epitope sequences that have so far been described only as associated with another HLA type; cross-reactive bindings are summarized in Table (H). An asterisk indicates which peptides do not meet our length and conservation thresholds.

[0021] Figure 4 The binding affinity of selected predicted HLA-binding peptide sequences is compared to a computer-predicted grading score. For example... Figure 3The plot depicts the binding capacity of predicted HLA binding sequences based on in vitro HLA binding assays against computer-predicted HLA binding capacity (expressed as a grading score) according to NetMHCpan. A 25% binding threshold is indicated for the positive control cutoff to separate confirmed binders from low / non-binding compounds.

[0022] Figure 5 Immunogenicity of HLA-binding peptide sequences. IFNγ production (minus DMSO) was measured via amplified PBMCs from nine HBV-regressed individuals in response to incubation with confirmed HLA-binding peptide sequences and well-established c18-27 and p549-557 epitopes (Figure B). Gray boxes represent the number of responsive donors as a fraction of the total number of subjects tested for each HLA-binding peptide sequence. Epitopes with closed underlines are rarely described for the HLA type used in the test. Epitopes with dashed underlines have so far been described only for another HLA type. Asterisks indicate which HLA-binding peptide sequences do not meet our length and conservation thresholds.

[0023] Figure 6 IFNγ response to SLP stimulation as measured by ELISpot. PBMCs from 15 donors with previously regressed HBV infection were incubated with 10 μM SLP or an equivalent concentration of DMSO for 20–24 hours, and IFNγ production was subsequently measured by ELISpot. The cumulative spot-forming units (SFU) from four replicate wells minus the cumulative SFU from four replicate wells under the DMSO control condition are plotted. Each spot represents one donor, and each donor is plotted with a different symbol. Negative SFU is plotted as 0. Figure 6 The donor number in the data is not necessarily the same as the donor number in the data. Figure 5 The donor number corresponds to the information in the database.

[0024] Figure 7The novel SLP can enhance functional CD8+ and CD4+ T cell responses in vitro from leukocytes derived from HBV-regressed individuals and patients with chronic HBV. PBMCs (A) isolated from the erythrocyte sedimentation rate (ESR) amber layer of previously HBV- and rHBV-cleared healthy donors, or PBMCs (B) isolated from whole blood of patients with chronic HBV (cHBV) visiting our outpatient clinic, were exposed to SLP pools containing indicated SLPs (1, 2, 4, and 6) and allowed to expand for 14 days in the presence of IL-2. After 14 days, the expanded cells were restimulated with the designated SLPs alone for 22 hours, after which surface markers and cytokines indicating functional T cell activation were assessed in both cells and culture supernatants. The percentages of CD4+ and CD8+ T cells expressing the activation marker CD69 and the percentage of CD8+ T cells expressing CD107a (minus the percentage observed against the DMSO-mediated control sample), which indicates recent CD8+ T cell cytotoxic activity, are shown. The secretion of T cell cytokines IFNα and TNFα in the supernatant induced by SLP restimulation was also plotted. Cytokine values ​​were subtracted from the mean observed for the DMSO / unrelated peptide control sample + 2 x the standard deviation of these controls.

[0025] Figure 8 A novel treatment regimen for clearing HBV infection. Viral load is reduced by nucleoside / nucleotide analogue (NA) treatment prior to initiating therapeutic vaccination (T). Therapeutic vaccination is administered when the viral load is stable at a low level. To further enhance T-cell effector function, therapeutic vaccination may optionally be combined with: myeloid dysplastic cell (MDSC) targeted agents (administered prior to therapeutic vaccination), siRNA (administered prior to therapeutic vaccination), T-cell metabolic modification agents (administered before or during therapeutic vaccination), or checkpoint blockade (administered during or after therapeutic vaccination). As a “natural” enhancer, NA treatment is discontinued to increase the presence of viral antigens, in situ enhancing HBV-specific T cells to drive the clearance of remaining infected hepatocytes. Proper monitoring of viral load is preferred to evaluate vaccine efficacy and determine subsequent (combination) therapies and / or NA discontinuation. Lines provide schematic indications of how parameters evolve over time. Arrows indicate preferred moments for intervention or monitoring. Detailed Implementation

[0026] definition The term "HBV" refers to the hepatitis B virus. Eight distinct HBV genotypes have been described, designated A through H. These genotypes share significant sequence homology but differ by at least 8% of their sequences. Within each genotype, subtypes have been described: these differ by 4%–8% of their genome.

[0027] The term "HBV polymerase," or simply "polymerase" or "Pol," refers to the polymerase encoded by the hepatitis B genome. GenBank NCBI reference NC_003977.2 describes an HBV polymerase sequence commonly used as a reference, which is also listed in SEQ ID NO: 27 herein. SEQ ID NO: 28 shows a common sequence of HBV polymerases based on 7489 genotypes. In one embodiment of the invention, the HBV polymerase fragment present in the peptide of the invention is more than 85%, such as more than 90%, such as more than 95%, such as more than 98%, identical to the corresponding sequence listed in SEQ ID NO: 27. In another embodiment of the invention, the HBV polymerase fragment is more than 85%, such as more than 90%, such as more than 95%, such as more than 98%, identical to the corresponding sequence listed in SEQ ID NO: 28. In yet another embodiment, the HBV polymerase mentioned is an HBV polymerase selected from the genotypes consisting of groups A, B, C, D, E, F, G, and H.

[0028] The terms “HBV-X,” “HBx,” “HBxAg,” “HBV-X protein,” or “X-protein,” etc., refer to the X protein encoded by the hepatitis B genome. GenBank NCBI reference NC_003977.2 describes the HBV-X sequence commonly used as a reference, which is also listed in SEQ ID NO: 29 herein. SEQ ID NO: 30 shows a common sequence of HBV-X protein based on 8127 genotypes. In one embodiment of the invention, the HBV-X fragment present in the peptide of the invention is more than 85%, such as more than 90%, such as more than 95%, such as more than 98%, identical to the corresponding sequence listed in SEQ ID NO: 29. In another embodiment of the invention, the HBV-X fragment is more than 85%, such as more than 90%, such as more than 95%, such as more than 98%, identical to the corresponding sequence listed in SEQ ID NO: 30. In another embodiment, the HBV-X protein mentioned is the HBV-X protein of the genotype selected from the group consisting of A, B, C, D, E, F, G, and H.

[0029] The position numbers within the HBV polymerase and HBV-X in this document are referenced to the common sequences listed in SEQ ID NO: 28 and SEQ ID NO: 30, respectively. In other words, the amino acid positions within the HBV polymerase and HBV-X proteins are numbered corresponding to the numbers in the common sequences listed in SEQ ID NO: 28 and SEQ ID NO: 30, respectively. The amino acid position in one sequence that “corresponds” to an amino acid position in another sequence is the amino acid position aligned to the other amino acid using a standard sequence alignment program (such as ALIGN, ClustalW, or a similar program) typically at default settings. How to align sequences and thereby determine the corresponding position in the common sequence for a specific position in the HBV polymerase or HBV-X sequence is considered well known in the art. For example, if a gap exists in a given HBV polymerase relative to the common sequence, the alignment may show that the fragment from amino acid positions 20 to 40 in that given HBV polymerase corresponds to positions 20 to 41 in the common sequence. To avoid ambiguity: Although the position numbering refers to the common sequence, the actual amino acid sequence of the fragment may differ from this common sequence and varies depending on the HBV genotype.

[0030] "Sequence identity" is defined in this paper as the relationship between two or more amino acid sequences, as determined by sequence comparison. Sequence identity can be determined by aligning two peptide sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch), which optimizes sequence alignment across the entire length, while sequences with very large length differences are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman).

[0031] "Treatment" refers to the administration of an effective amount of an immunogenic composition with the aim of reducing, alleviating, stopping, eradicating (curing), or preventing symptoms, disorders, or disease states. "Effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the necessary dosage and for the required time period.

[0032] When used herein, the term "immunogenic peptide" means a peptide capable of triggering or enhancing an immune response. The immunogenic peptides of the present invention may be unconjugated or unmodified, i.e., simple amino acid chains linked by peptide bonds, or they may be further modified, such as conjugated, covalently bound to another molecule (e.g., an adjuvant).

[0033] In the context of this invention, "20-34 amino acids in length" means that the number of amino acid residues is from 20 to 34, i.e., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acid residues. The peptides of this invention (also referred to as long peptides) exceed the length of the epitope peptide sequences presented by human leukocyte antigens (HLA) of types I and II. Preferably, the long peptides of this invention are synthetic peptides, also referred to herein as synthetic long peptides (SLPs).

[0034] In the context of this invention, the term "fragment of HBV protein" means an amino acid sequence corresponding to a portion of the HBV protein (i.e., identical to it). Therefore, it refers to a continuous sequence of the native HBV protein without insertions, deletions, or substitutions. If a peptide is specified to contain a fragment of HBV protein of a certain length, it means that the fragment is neither shorter nor longer. For example, if a fragment length of 20-34 amino acids is specified, it means that the fragment is not less than 20 amino acids or greater than 34 amino acids. Therefore, such a peptide, for example, does not contain a continuous sequence of the HBV protein of 35 amino acids or more. However, for the avoidance of ambiguity, "containing" has its usual meaning in the art, that is, a "peptide containing a fragment of HBV protein" may contain additional sequences beyond the specified fragment, such as sequences not derived from the HBV protein or other partial sequences of the HBV protein not adjacent to the fragment in the HBV protein.

[0035] In the context of this invention, the terms "HLA-binding peptide," "HLA conjugate," or "conjugate" refer to a short protein fragment of HBV-X or HBV polymerase that can specifically bind to HLA molecules.

[0036] In the context of this invention, an "epitope" is defined as a short HLA-binding peptide that binds to a specified HLA molecule and, when present on the cell surface, can elicit a T-cell response in an individual.

[0037] The use of the indefinite article "a" (or "an") to refer to an element does not preclude the possibility of more than one element, unless the context explicitly requires the presence of one and only one element. Therefore, the indefinite article "a" (or "an") generally means "at least one".

[0038] Other aspects and embodiments of the present invention As described above, in a first principal aspect, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the fragment is 20-34 amino acids in length, and wherein the fragment comprises: a) At least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, preferably comprising: - The amino acid sequence (x70-78) listed in SEQ ID NO: 1, and / or - The amino acid sequence (x67-75) listed in SEQ ID NO: 2, and / or - The amino acid sequence (x62-73) listed in SEQ ID NO: 3, and / or - The amino acid sequence (x58-66) listed in SEQ ID NO: 4, and / or - The amino acid sequence (x57-66) listed in SEQ ID NO: 5. or b) At least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, preferably comprising: - The amino acid sequence (x103-111) listed in SEQ ID NO: 6, and / or - The amino acid sequence (x104-113) listed in SEQ ID NO: 7, and / or - The amino acid sequence (x105-113) listed in SEQ ID NO: 8, and / or - The amino acid sequence (x110-120) listed in SEQ ID NO: 9. or c) The amino acid sequence listed in SEQ ID NO: 10 (x132-140). or d) The amino acid sequence listed in SEQ ID NO: 11 (p124-133). or e) The amino acid sequence listed in SEQ ID NO: 12 (p164-173). or f) The amino acid sequence listed in SEQ ID NO: 13 (p275-283). or g) At least 10 consecutive amino acids from the region at positions 403 to 415 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 14 (p403-412), and / or - The amino acid sequence listed in SEQ ID NO: 15 (p404-412), and / or - The amino acid sequence listed in SEQ ID NO: 16 (p407-415). or h) At least nine consecutive amino acids from the region at positions 509 to 523 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 17 (p509-517), and / or - The amino acid sequence listed in SEQ ID NO: 18 (p515-523). or i) At least 10 consecutive amino acids from the region at positions 649 to 658 of the HBV polymerase, comprising: - The amino acid sequence listed in SEQ ID NO: 19 (p649-658), and / or - The amino acid sequence listed in SEQ ID NO: 20 (p650-658). or j) At least 10 consecutive amino acids from the region at positions 693 to 706 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 21 (p693-701), and / or - The amino acid sequence listed in SEQ ID NO: 22 (p697-706). or k) The amino acid sequence listed in SEQ ID NO: 23 (p723-731). or l) At least 10 consecutive amino acids from the region at positions 755 to 765 of the HBV polymerase, comprising: - The amino acid sequence listed in SEQ ID NO: 24 (p755-764), and / or - The amino acid sequence listed in SEQ ID NO: 25 (p756-765). or m) The amino acid sequence listed in SEQ ID NO: 26 (p829-837).

[0039] In one embodiment, the peptide consists of the fragment of the HBV protein.

[0040] In another embodiment, the peptide is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids in length, such as 20-33 amino acids in length, such as 20-32 amino acids in length, such as 20-31 amino acids in length, such as 20-30 amino acids in length, such as 20-29 amino acids in length, such as 20-28 amino acids in length, such as 20-27 amino acids in length, such as 20-26 or 20-25 amino acids in length.

[0041] In another embodiment, the length of the fragment is 20-33 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, such as 20-32 amino acids, such as 20-31 amino acids, such as 20-30 amino acids, such as 20-29 amino acids, such as 20-28 amino acids, such as 20-27 amino acids, such as 20-26 or 20-25 amino acids.

[0042] In another aspect, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids in length, and wherein the fragment comprises one or more sequences selected from the group consisting of: - The amino acid sequence (x70-78) listed in SEQ ID NO: 1. - The amino acid sequence (x67-75) listed in SEQ ID NO: 2. - The amino acid sequence (x62-73) listed in SEQ ID NO: 3. - The amino acid sequence (x58-66) listed in SEQ ID NO: 4. - The amino acid sequence (x57-66) listed in SEQ ID NO: 5. - The amino acid sequence (x103-111) listed in SEQ ID NO: 6. - The amino acid sequence (x104-113) listed in SEQ ID NO: 7. - The amino acid sequence (x105-113) listed in SEQ ID NO: 8. - The amino acid sequence (x110-120) listed in SEQ ID NO: 9. - The amino acid sequence (x132-140) listed in SEQ ID NO: 10. - The amino acid sequence listed in SEQ ID NO: 11 (p124-133). - The amino acid sequence listed in SEQ ID NO: 12 (p164-173). - The amino acid sequence listed in SEQ ID NO: 13 (p275-283). - The amino acid sequence listed in SEQ ID NO: 14 (p403-412). - The amino acid sequence listed in SEQ ID NO: 15 (p404-412). - The amino acid sequence listed in SEQ ID NO: 16 (p407-415). - The amino acid sequence listed in SEQ ID NO: 17 (p509-517). - The amino acid sequence listed in SEQ ID NO: 18 (p515-523). - The amino acid sequence listed in SEQ ID NO: 19 (p649-658). - The amino acid sequence listed in SEQ ID NO: 20 (p650-658). - The amino acid sequence listed in SEQ ID NO: 21 (p693-701). - The amino acid sequence listed in SEQ ID NO: 22 (p697-706). - The amino acid sequence listed in SEQ ID NO: 23 (p723-731). - The amino acid sequence listed in SEQ ID NO: 24 (p755-764). - The amino acid sequence listed in SEQ ID NO: 25 (p756-765), and - The amino acid sequence listed in SEQ ID NO: 26 (p829-837). Preferably, the segment comprises: - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 2 (x67-75), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 3 (x62-73), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 5 (x57-66), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 3 (x62-73), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 5 (x57-66), or - The amino acid sequences listed in SEQ ID NO: 3 (x62-73) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 3 (x62-73) and SEQ ID NO: 5 (x57-66) or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 7 (x104-113), or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 8 (x105-113), or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 7 (x104-113) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 8 (x105-113) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 14 (p403-412) and SEQ ID NO: 16 (p407-415), or - The amino acid sequences listed in SEQ ID NO: 15 (p404-412) and SEQ ID NO: 16 (p407-415).

[0043] As described, in one embodiment, the immunogenic peptide comprises a fragment of HBV-X, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, comprising: - The amino acid sequence (x70-78) listed in SEQ ID NO: 1, and / or - The amino acid sequence (x67-75) listed in SEQ ID NO: 2, and / or - The amino acid sequence (x62-73) listed in SEQ ID NO: 3, and / or - The amino acid sequence (x58-66) listed in SEQ ID NO: 4, and / or - The amino acid sequence (x57-66) listed in SEQ ID NO: 5.

[0044] In another embodiment, the immunogenic peptide comprises a fragment of HBV-X, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, the fragment comprises at least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, and the fragment comprises: - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 2 (x67-75), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 3 (x62-73), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 5 (x57-66), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 3 (x62-73), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 5 (x57-66), or - The amino acid sequences listed in SEQ ID NO: 3 (x62-73) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 4 (x62-73) and SEQ ID NO: 5 (x57-66).

[0045] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 53 of HBV-X, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of HBV-X than 53 (i.e., position 54, position 55, position 56, etc.).

[0046] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 91 of HBV-X, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of HBV-X than 91 (i.e., position 90, position 89, position 88, etc.).

[0047] In even other embodiments: - The N-terminal amino acid of this fragment is either the amino acid at position 53 of HBV-X or the amino acid at a position further C-terminus of HBV-X than 53, and - The C-terminal amino acid of this fragment is either the amino acid at position 91 of HBV-X or the amino acid at a position further N-terminus of HBV-X than 91.

[0048] Therefore, in this latter embodiment, the HBV-X fragment contained in the peptide of the present invention does not extend beyond positions 53 and 91 of HBV-X.

[0049] In another embodiment, the immunogenic peptide comprises a fragment of HBV-X, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, comprising: - The amino acid sequence (x103-111) listed in SEQ ID NO: 6, and / or - The amino acid sequence (x104-113) listed in SEQ ID NO: 7, and / or - The amino acid sequence (x105-113) listed in SEQ ID NO: 8, and / or - The amino acid sequence (x110-120) listed in SEQ ID NO: 9.

[0050] In another embodiment, the immunogenic peptide comprises a fragment of HBV-X, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, the fragment comprises at least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, and the fragment comprises: - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 7 (x104-113), or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 8 (x105-113), or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 7 (x104-113) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 8 (x105-113) and SEQ ID NO: 9 (x110-120).

[0051] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 103 of HBV-X, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of HBV-X than 103 (i.e., position 104, position 105, position 106, etc.).

[0052] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 122 of HBV-X, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of HBV-X than 122 (i.e., position 121, position 120, position 119, etc.).

[0053] In even further embodiments, the HBV-X fragment contained within the peptide of the present invention does not extend beyond positions 103 and 122 of the HBV-X.

[0054] In another embodiment, the immunogenic peptide comprises a fragment of HBV-X, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, and the fragment comprises the amino acid sequence (x132-140) listed in SEQ ID NO: 10.

[0055] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 116 of HBV-X, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of HBV-X than 116 (i.e., position 117, position 118, position 119, etc.).

[0056] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 140 of HBV-X, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of HBV-X than 140 (i.e., position 139, position 138, position 137, etc.).

[0057] In even other embodiments: - The N-terminal amino acid of this fragment is either the amino acid at position 116 of HBV-X or the amino acid at a position further C-terminus of HBV-X than 116, and - The C-terminal amino acid of this fragment is either the amino acid at position 140 of HBV-X or the amino acid at a position further N-terminus of HBV-X than 140.

[0058] Therefore, in this latter embodiment, the HBV-X fragment contained in the peptide of the present invention does not extend beyond positions 116 and 140 of HBV-X.

[0059] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, and the fragment comprises the amino acid sequence (p124-133) listed in SEQ ID NO: 11.

[0060] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 155 of the HBV polymerase, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of the HBV polymerase than 155 (i.e., position 154, position 153, position 152, etc.).

[0061] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, and the fragment comprises the amino acid sequence (p164-173) listed in SEQ ID NO: 12.

[0062] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 151 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 151 (i.e., position 152, position 153, position 154, etc.).

[0063] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 174 of the HBV polymerase, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of the HBV polymerase than 174 (i.e., position 173, position 172, position 171, etc.).

[0064] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, and the fragment comprises the amino acid sequence (p275-283) listed in SEQ ID NO: 13.

[0065] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 262 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 262 (i.e., position 263, position 264, position 265, etc.).

[0066] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 10 consecutive amino acids from the region of HBV polymerase at positions 403 to 415, comprising: - The amino acid sequence listed in SEQ ID NO: 14 (p403-412), and / or - The amino acid sequence listed in SEQ ID NO: 15 (p404-412), and / or - The amino acid sequence listed in SEQ ID NO: 16 (p407-415).

[0067] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 10 consecutive amino acids from the region of HBV polymerase at positions 403 to 415, comprising: - The amino acid sequences listed in SEQ ID NO: 14 (p403-412) and SEQ ID NO: 16 (p407-415), or - The amino acid sequences listed in SEQ ID NO: 15 (p404-412) and SEQ ID NO: 16 (p407-415).

[0068] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 390 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 390 (i.e., position 391, position 392, position 393, etc.).

[0069] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 425 of the HBV polymerase, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of the HBV polymerase than 425 (i.e., position 424, position 423, position 422, etc.).

[0070] In even other embodiments: - The N-terminal amino acid of this fragment is either the amino acid at position 390 of the HBV polymerase or the amino acid at a position further C-terminus than 390 of the HBV polymerase, and - The C-terminal amino acid of this fragment is either the amino acid at position 425 of the HBV polymerase or the amino acid at a position further N-terminus than 425 of the HBV polymerase.

[0071] Therefore, in this latter embodiment, the HBV polymerase fragment contained in the peptide of the present invention does not extend beyond positions 390 and 425 of the HBV polymerase.

[0072] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 9 consecutive amino acids from the region of HBV polymerase at positions 509 to 523, comprising: - The amino acid sequence listed in SEQ ID NO: 17 (p509-517), and / or - The amino acid sequence listed in SEQ ID NO: 18 (p515-523).

[0073] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 503 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 503 (i.e., position 504, position 505, position 506, etc.).

[0074] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 532 of the HBV polymerase, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of the HBV polymerase than 532 (i.e., position 531, position 530, position 529, etc.).

[0075] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 10 consecutive amino acids from the region of HBV polymerase at positions 649 to 658, comprising: - The amino acid sequence listed in SEQ ID NO: 19 (p649-658), and / or - The amino acid sequence listed in SEQ ID NO: 20 (p650-658).

[0076] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 624 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 624 (i.e., position 625, position 626, position 627, etc.).

[0077] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 658 of the HBV polymerase, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of the HBV polymerase than 658 (i.e., position 657, position 656, position 655, etc.).

[0078] In even other embodiments: - The N-terminal amino acid of this fragment is either the amino acid at position 624 on the HBV polymerase or an amino acid at a position further C-terminus than 624 on the HBV polymerase, and - The C-terminal amino acid of this fragment is either the amino acid at position 658 of the HBV polymerase or the amino acid at a position further N-terminus than 658 of the HBV polymerase.

[0079] Therefore, in this latter embodiment, the HBV polymerase fragment contained in the peptide of the present invention does not extend beyond positions 624 and 658 of the HBV polymerase.

[0080] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 10 consecutive amino acids from the region of HBV polymerase at positions 693 to 706, comprising: - The amino acid sequence listed in SEQ ID NO: 21 (p693-701), and / or - The amino acid sequence listed in SEQ ID NO: 22 (p697-706).

[0081] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 672 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 672 (i.e., position 671, position 670, position 696, etc.).

[0082] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, and the fragment comprises the amino acid sequence (p723-731) listed in SEQ ID NO: 23.

[0083] In another embodiment, the C-terminal amino acid of the fragment is the amino acid at position 751 of the HBV polymerase, or the C-terminal amino acid of the fragment is the amino acid at a position further N-terminus of the HBV polymerase than 751 (i.e., position 750, position 749, position 748, etc.).

[0084] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids, and the fragment comprises at least 10 consecutive amino acids from the region of HBV polymerase at positions 755 to 765, comprising: - The amino acid sequence listed in SEQ ID NO: 24 (p755-764), and / or - The amino acid sequence listed in SEQ ID NO: 25 (p756-765).

[0085] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, and the fragment comprises the amino acid sequence (p829-837) listed in SEQ ID NO: 26.

[0086] In another embodiment, the N-terminal amino acid of the fragment is the amino acid at position 815 of the HBV polymerase, or the N-terminal amino acid of the fragment is the amino acid at a position further C-terminus of the HBV polymerase than 815 (i.e., position 816, position 817, position 818, etc.).

[0087] In another embodiment, the peptide of the present invention comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 31:HLSLRGLPVCAFSSAGPCALRFTSA (SLP1), SEQ ID NO: 32: LSAMSTTDLEAYFKDCLFKDWEELG (SLP2), SEQ ID NO: 33:ASSSSSCLHQSAVRKAAYSHLSTSK (SLP3), SEQ ID NO: 34: RKLHLYSHPIILGFRKIPMGVGLSP (SLP4), SEQ ID NO: 35: GFAAPFTQCGYPALMPLYACIQAKQA (SLP5), SEQ ID NO: 36: ARQRPGLCQVFADATPTGWGLAIGH (SLP6) AND SEQ ID NO: 37: SPSVPSHLPDRVHFASPLHVAWRPP (SLP7).

[0088] In another embodiment, the peptide of the present invention comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 121: KLHLYSHPIILGFRKIPMGVGLSPFLL (SLP8), SEQ ID NO: 122: GLLGFAAPFTQCGYPALMPLYACIQAKQAFT (SLP9), and SEQ ID NO: 123: ARQRPGLCQVFADATPTGWGLAIGHQRMR (SLP10).

[0089] SEQ ID NO: 121, 122 and 123 are variants of SLP4, SLP5 and SLP6, respectively. Potential improvements in these variants include increased manufacturability by introducing additional putative epitopes / ligands through the extension of the N-terminus / C-terminus, or by extending or shortening the N-terminus / C-terminus without losing the (putative) epitope / ligand.

[0090] In another embodiment, the peptides of the present invention do not contain or are not composed of any of the peptides described in SEQ ID NO: 53, 54, 59, 70, 76 and 79 in WO 15187009, i.e. SEQ ID NO: 38: VVNEKRRLKLIMPARFYPTHTKYLPLDKGIKPYY (SEQ ID NO: 53 in WO 15187009), SEQ ID NO: 39: YPTHTKYLPLDKGIKPYYPDQVVNHYFQTRHYL (SEQ ID NO: 54 in WO 15187009), SEQ ID NO: 40: TAESRLVVDFSQFSRGISRVSWPKFAVPNLQSL (SEQ ID NO: 59 in WO 15187009), SEQ ID NO: 41: QMRRGTFVAPLPIHTAELLAACFARSRSGAKL (SEQ ID NO: 70 in WO 15187009), SEQ ID NO: 42: ALPSPSPSAVPADHGAHLSLRGLPVCAFSSAGP (SEQ ID NO: 76 in WO 15187009), or SEQ ID NO: 43: LEAYFKDCVFKDWEELGEEIIRLKVFVLGGCRHKL (SEQ ID NO: 79 in WO 15187009).

[0091] In another key aspect, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the peptide is 20-34 amino acids in length, and wherein the fragment comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 26.

[0092] Preferably, when administered to human subjects, the immunogenic peptides of the present invention are capable of inducing potent, combined, antigen-targeting CD4+ T helper cell and CD8+ cytotoxic T cell responses. Preferably, these peptides can be effectively used for the prevention, partial clearance and / or treatment or complete clearance of HBV, HBV-related diseases or conditions in subjects, preferably as detected by the following: - An increase in the activation or induction of the immune system and / or antigen-specific activation of CD4+ and / or CD8+ T cells in peripheral blood or tissues, established by ELISpot or ELISA assays or related appropriate techniques or by HLA-multimer staining of CD4+ or CD8+ T cells; or an increase in cytokines produced by these T cells, established by intracellular cytokine staining or cytokine capture of CD4+ and CD8+ T cells via flow cytometry at least one week after treatment; and / or - Inhibition of proliferation in antigen-associated infections, or a detectable reduction in antigen-expressing cells, or a decrease in the viability of antigen-expressing cells; and / or - Induction or increased induction of cell death in antigen-expressing cells; and / or - Inhibition or prevention of increased antigen-expressing cells; and / or - A decrease in viral load during the precancerous stage of the disease (e.g., a decrease in serum viral DNA, viral RNA, or viral proteins); and / or - Reduction in hepatocytes possessing integrated viral DNA and / or covalently closed circular DNA (cccDNA). - A reduction in the size of the lesion; and / or - A decrease in tumor size; and / or - Increased survival rates (e.g., progression-free survival, overall survival).

[0093] In a preferred embodiment, the peptide used in this invention comprises a CTL epitope and a T helper epitope as described above, the T helper epitope exhibiting binding affinity, preferably at least moderate binding affinity, and more preferably high binding affinity, for class II HLA molecules encoded by a dominant HLA allele in the population of human subjects to be treated.

[0094] In a preferred embodiment, the peptide used in this invention does not have a cysteine ​​residue at its N-terminus or C-terminus.

[0095] Furthermore, in another preferred embodiment, the peptides used in this invention do not contain more than two cysteine ​​residues.

[0096] In another preferred embodiment, the peptides used in this invention do not contain more than three methionines.

[0097] In another preferred embodiment, the peptide used in this invention does not have glutamine at the N-terminus.

[0098] Preferably, the peptides used in this invention are isolated peptides, wherein “isolated” does not reflect the degree to which the peptide has been purified, but rather indicates that the peptide has been removed from its natural environment (i.e., has been artificially manipulated), and may be recombinant peptides or synthetic peptides.

[0099] Typically, peptides are synthesized. This can be done through solid-phase peptide synthesis or by any other suitable method.

[0100] In another aspect, the present invention relates to a polynucleotide comprising a nucleotide sequence encoding a peptide according to the present invention. As explained above, the term "fragment of HBV protein" refers to an amino acid sequence corresponding to a portion of the HBV protein sequence. If a peptide is specified to contain a fragment of HBV protein of a certain length, it means that the fragment is neither shorter nor longer. For example, if a fragment length of 20-34 amino acids is specified, it means that the peptide does not contain a continuous sequence of said HBV protein of 35 amino acids or more in length. Therefore, the peptide of the present invention will not contain a fragment of HBV protein longer than 34 amino acids. Therefore, it should be understood that the polynucleotide of the present invention comprising a nucleotide sequence encoding a peptide according to the present invention will also not encode a peptide containing a fragment of HBV protein longer than 34 amino acids.

[0101] In another aspect, the present invention relates to an immunogenic composition comprising: - Peptides as described herein according to the invention, or polynucleotides as described herein according to the invention, and - Pharmaceutically acceptable carriers Optionally, it may further include an adjuvant.

[0102] Suitable methods for polynucleotide-based vaccination have been described, for example, in Trimble et al. 2015 Lancet 386:2078; Kranz et al. 2016 Nature 534:396; WO 2011015656A2; Kratzer et al. 2018 AASLD, The Liver Meeting 2018, Abstract No. 426; WO 2017080920; and Boni et al. 2019 Int J Mol Sci 20(11):2754.

[0103] In another aspect, the present invention relates to a recombinant virus comprising the polynucleotides described in the present invention.

[0104] The immunogenic compositions used in this invention are preferably intended for administration to human subjects and are therefore formulated to be suitable for administration to human subjects. Preferably, the administration is parenteral, such as intravenous, subcutaneous, intramuscular, intradermal, intradermal, and / or intratumoral administration, i.e., by injection.

[0105] Immunogenic compositions are preferably chemically stable, meaning that the peptides in the composition do not chemically degrade or decompose. Therefore, preferably, after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the amount of undegraded, undecomposed, and / or unreacted peptides in the solution and / or composition, by weight, is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% compared to its original state. Chemical stability can be assessed using any suitable technique known in the art, such as UPLC / MS as exemplified herein. When using UPLC / MS, a solution / composition is defined as chemically stable if, after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the total area % of peaks not representing the desired peptide product in the UV spectrum, compared to its original state, is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0106] Immunogenic compositions are preferably also physically stable, meaning that the peptides in the composition do not precipitate or redisperse. Physical stability can be assessed using any suitable technique known in the art, such as by visual inspection or by particle distribution as exemplified herein using a Malvern Mastersizer, where the average particle size is expressed as D(0.5). When physical stability is assessed using a Malvern Mastersizer as exemplified herein, the solution / composition is defined as physically stable if, after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the average D(0.5) increases by at most 50%, 40%, 30%, 20%, 10%, or 5% compared to its original state (i.e., directly in a freshly prepared solution after preparation). Preferably, the solution / composition is defined as physically stable if, after storage at room temperature for 3 hours, the average D(0.5) increases by at most 50%, 40%, 30%, 20%, 10%, or 5%, preferably at most 20%, compared to its original state.

[0107] In one embodiment, the immunogenic composition comprises or consists of a mixture of dried or lyophilized peptides to be applied together.

[0108] Immunogenic compositions used in this invention can be prepared by any suitable method. In some embodiments, one or more immunogenic compositions are prepared from dried, preferably lyophilized, peptides.

[0109] For example, the composition can be prepared by a method including the following steps: a) Provide vials containing dried, preferably lyophilized, peptides; b) Thaw these peptides, preferably for about 5-30 minutes; c) Add the reconstituted composition to a vial containing these peptides, preferably a non-vortex vial; d) Mixing is permitted, preferably for about 0.5-5 minutes; and e) Rotate until a clear solution is obtained, preferably for about 1-3 minutes.

[0110] Preferably, steps b) to e) are performed at room temperature.

[0111] Preferably, in methods for prevention and / or treatment (preferably methods for treatment and / or prevention as defined herein), the vial contains a single volume of the peptide, i.e., a single drug dose unit, or a portion thereof (in the case of multiple injections at different locations on the subject’s body at substantially the same time points).

[0112] In one embodiment, the reconstructed composition of step c) comprises or is composed of DMSO and / or water for injection. In another embodiment, the reconstructed composition of step c) of the method for reconstructing peptides comprises or is composed of about 60%-80% v / v of an aqueous solution containing an organic acid, about 5%-10% v / v of propylene glycol (CAS No. 57-55-6), about 10%-20% v / v of a lower alcohol, and about 5%-10% v / v of a nonionic hydrophilic surfactant. In one embodiment, the organic acid is citric acid and the citric acid is present in the aqueous solution at a concentration of about 0.05-0.1 M. In one embodiment, the lower alcohol is ethanol. In one embodiment, the nonionic hydrophilic surfactant: a. It is a monoglyceride, diglyceride, or triglyceride, preferably an ethoxylated triglyceride, and / or b. Has a hydrophilic-lipophilic balance (HLB) value between 9 and 14. In another embodiment, the nonionic hydrophilic surfactant is ethoxylated castor oil, preferably polyoxyethylene glycerol triricinoleate 35 (CAS No. 61791-12-6).

[0113] In one embodiment, the composition comprises or consists of an aqueous solution of about 75% v / v, the aqueous solution comprising about 0.1 M citric acid, about 6.25% v / v propylene glycol (CAS No. 57-55-6), about 12.5% ​​v / v ethanol and about 6.25% v / v polyoxyethylene glycerol triricinoleate 35 (CAS No. 61791-12-6).

[0114] Preferably, the amount of the reconstituted composition in step c) is in the range of about 0.5 to 2 mL, preferably 1 mL. Preferably, the amount of the reconstituted peptide in step (a) is the total amount of the reconstituted peptide obtained after step e), i.e., in the clear solution obtained after step e).

[0115] In one embodiment, the reconstituted composition comprises or consists of the following: about 1-2 mg / mL of peptide in water, 0.038 M of citric acid, about 3.13% v / v of propylene glycol (CAS No. 57-55-6), about 6.25% v / v of ethanol, about 3.13% v / v of polyoxyethylene glycerol triricinoleate 35 (CAS No. 61791-12-6), and about 50% of an oil-based adjuvant (preferably Montanide ISA 51 VG (Seppic)).

[0116] The dried peptides may be peptides without additional components, but may also contain buffer components such as trifluoroacetic acid (TFA), salts (such as sodium salts, potassium salts, or phosphates (e.g., NaCl, KCl, and NaPO4)). The amount of additional components is preferably less than 30% of the total weight of the dried peptides to be reconstructed, more preferably less than 25%. The dried peptides to be reconstructed may be in a physically dried state, such as by a variety of processes including, but not limited to, rotor evaporation, lyophilization (freeze-drying), and spray drying.

[0117] adjuvant In one embodiment, the composition of the present invention further comprises an adjuvant, or the treatment or use according to the present invention further comprises administration of an adjuvant. The term "adjuvant" is used herein to refer to a substance having an immunomodulatory effect, and which is administered with an antigen, or added to or formulated with an antigen to enhance, induce, elicit, and / or modulate an immunological response against that antigen upon administration to a subject. In one embodiment, the adjuvant is physically linked, such as covalently linked, to one or more peptides to be reconstructed.

[0118] In one embodiment, the adjuvant is an emulsifying adjuvant. For example, in one embodiment, the adjuvant is an oil-based adjuvant. Oil-based adjuvants can be used to form emulsions (e.g., water-in-oil or oil-in-water emulsions) and are considered in the art to enhance and direct immune responses. Preferably, the oil-based adjuvant is a mineral oil-based adjuvant. Non-limiting examples of oil-based adjuvants are bio-based oil adjuvants (based on vegetable oils / fish oils, etc.), squalene-based adjuvants (e.g., MF59), Syntex adjuvant formulations (SAF; In the following Lidgate, Deborah M, Preparation of the Syntex Adjuvant Formulation (SAF, [Preparation of Syntex adjuvant formulations (SAF, SAF-m, and SAF-1)]: Vaccine Adjuvants, Volume 42 of the series Methods in Molecular Medicine™ [Molecular Medicine Methods™ Series, Volume 42, Vaccine Adjuvants, pp. 229-237, ISSN 1543-1894Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), peanut oil-based adjuvants (e.g., adjuvant 65), Lipovant ( Byars, NE, Allison, AC, 1990. Immunologic adjuvants include: general properties, advantages, and limitations. [Immune adjuvants: general characteristics, advantages, and limitations]: Zola, H. ( edit Laboratory Methods in Immunology (Pages 39-51) ASO4 A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dream becomes real. (Hum) Vaccine [human vaccine], 4 (5), 2008, (Pages 347-349) 、 Montanide adjuvant, which is based on purified squalene and squalene emulsified with high-purity mannitol monooleate (e.g., Montanide ISA 25 VG, 28 VG, 35 VG, 50 V, 50 V2, 51 VG, 61 VG, 70 VG, 70 M VG, 71 VG, 720 VG, 760 VG, 763 A VG, 775 VG, 780 VG, 201 VG, 206 VG, 207 VG). 。 More preferably, the oil-based adjuvant is Montanide ISA 51VG (Sebik), which is a mixture of Drakeol VR and mannitol monooleate.

[0119] Other suitable adjuvants are those that activate antigen-presenting cells, such as dendritic cells. For example, such adjuvants can be activated via Toll-like receptors and / or via RIG-I (retinoic acid-inducible gene-1) protein and / or via endothelin receptors. Immunomodulatory compounds capable of activating the innate immune system can be particularly well activated via Toll-like receptors (TLRs) (including TLRs 1-10). Compounds capable of activating TLR receptors, their modifications, and derivatives are well documented in the art. TLR1 can be activated by bacterial lipoproteins and their acetylated forms, and TLR2 can also be activated by Gram-positive bacterial glycolipids, LPS, LPA, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or the host, and mycobacterial lipoarabinomannan. TLR3 can be activated, particularly by viral-derived dsRNA, or by the chemical compound poly(I:C). TLR4 can be activated by: Gram-negative LPS, LTA, heat shock proteins from the host or bacteria, viral capsid or envelope proteins, paclitaxel or its derivatives, and hyaluronic acid containing oligosaccharides and fibronectin. TLR5 can be activated by bacterial flagella or flagellin proteins. TLR6 can be activated by: mycobacterial lipoproteins and heat-labile soluble factor (GBS-F) from group B streptococci or regulatory proteins from staphylococci. TLR7 can be activated by: imidazoquinolines, such as imiquimod, resiquimod, and derivatives of imiquimod or resiquimod (e.g., 3M-052). TLR9 can be activated by unmethylated CpG DNA or chromatin-IgG complexes. Particularly preferred adjuvants include, but are not limited to, synthetically produced compounds, including dsRNA, poly(I:C), polyI:CLC, unmethylated CpG DNA that triggers TLR3 and TLR9 receptors, IC31, TLR9 agonists, IMSAVAC, TLR4 agonists, MontanideISA-51, and MontanideISA 720 (adjuvants manufactured by Seppic, France). RIG-I protein is known to be activated by dsRNA, similar to TLR3. Kato people, (2005) Immunity [ [Immunity], 1: 19-28 ).

[0120] Further particularly preferred TLR ligands are pam3cys and / or its derivatives, preferably pam3cys lipopeptide or variants or derivatives thereof, preferably as described in WO 2013051936A1, more preferably U-Pam12 or U-Pam14 or AMPLIVANT®. Pam3cys and / or its derivatives may optionally be covalently linked to one or more peptide antigens.

[0121] Further preferred adjuvants are cyclic dinucleotides (CDNs), muramyl dipeptides (MDPs), and polyICLCs. In preferred embodiments, the adjuvants of the present invention are non-naturally occurring adjuvants, such as pam3cys lipopeptide derivatives (as described in WO2013051936A1), polyICLCs, imidazoquinolines such as imiquimod, rasimod, or derivatives thereof, CpG oligodeoxynucleotides having non-naturally occurring sequences (CpG-ODNs), and peptide adjuvants containing non-naturally occurring amino acids, such as muramyl dipeptides (MDPs) or tetanus toxoid peptides.

[0122] Further preferred adjuvants are those selected from the group consisting of: 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, ImuFact EV1P321, IS Patch, ISS, ISCMATRIX, Juvlmmune, lipid complexes, liposomes, LipoVac, MF59, monophospholipid A, MontanideIMS 1312, nanoparticles (such as nanoparticles in which the adjuvant has been integrated), OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, carrier systems, PLGA microparticles, SRL172, virions and other virus-like particles, Pam3Cys-GDPKHPKSF, YF-17D, VEGF traps, R848, β-glucan, and Aquila's QS21. Stimulon, vadimezan, AsA404 (DMXAA), STING (IFN gene stimulator) agonists (e.g., c-di-GMP VacciGrade™), PCI, NKT (natural killer T cell) agonists (e.g., α-galactosidase ceramide or α-GalCer, RNAdjuvant® (Curevac), retinoic acid inducible protein I ligands (e.g., 3pRNA or 5'-triphosphate RNA).

[0123] Treatment methods and uses The immunogenic peptides, polynucleotides, recombinant viruses, and immunogenic compositions of the present invention can be used to treat HBV infection (such as chronic HBV infection) and / or HBV-related diseases.

[0124] Examples of diseases that can be treated using the immunogenic peptides, polynucleotides, and immunogenic compositions of the present invention include, but are not limited to, hepatitis B infection, such as chronic hepatitis B infection, hepatitis B-related cirrhosis, and hepatitis B-related hepatocellular carcinoma.

[0125] Therefore, in another aspect, the present invention relates to peptides, polynucleotides, or immunogenic compositions according to the present invention, used as pharmaceuticals.

[0126] In another aspect, the present invention relates to peptides, polynucleotides, or immunogenic compositions according to the present invention for the treatment or prevention of HBV-related diseases.

[0127] In another aspect, the present invention relates to a method for treating or preventing HBV-related diseases, the method comprising administering to a human subject in need an immunogenic peptide, a polynucleotide, or an immunogenic composition according to the present invention.

[0128] Preferably, the administration is intravenous, subcutaneous, or intramuscular, although other routes of administration are conceivable, such as mucosal or intradermal and / or intradermal administration, for example by injection.

[0129] Preferably, administration of one or more immunogenic compositions induces a cytotoxic CD8+ T cell response against at least one HLA-binding peptide sequence presented by a class I HLA molecule contained in a long peptide. More preferably, administration of one or more immunogenic compositions induces a cytotoxic CD8+ T cell response against an HLA-binding peptide sequence presented by a class II HLA molecule contained in the one or more immunogenic compositions, together with a helper CD4+ T cell response. Preferably, the administration is for the prevention, partial clearance and / or treatment or complete clearance of HBV-related infection or disease in a subject, preferably as detectable by the following: - An increase in the activation or induction of the immune system and / or antigen-specific activation of CD4+ and / or CD8+ T cells in peripheral blood or tissues, established by ELISpot or ELISA assays or related appropriate techniques or by HLA-multimer staining of CD4+ or CD8+ T cells; or an increase in cytokines produced by these T cells, established by intracellular cytokine staining or cytokine capture of CD4+ and CD8+ T cells via flow cytometry at least one week after treatment; and / or - Inhibition of proliferation in antigen-associated infections, or a detectable reduction in antigen-expressing cells, or a decrease in the viability of antigen-expressing cells; and / or - Induction or increased induction of cell death in antigen-expressing cells; and / or - Inhibition or prevention of increased antigen-expressing cells; and / or - Reduced viral load (precancerous stage of disease); and / or - A reduction in the size of the lesion; and / or - A decrease in tumor size; and / or - Increased survival rate (e.g., PFS, OS).

[0130] combination In some embodiments, treatment includes administering a combination of the immunogenic peptides, polynucleotides, or recombinant viruses of the present invention with other immunogenic peptides, polynucleotides, or recombinant viruses. For example, treatment may include administering two or more, such as three, four, five, six, seven, eight, or more immunogenic peptides.

[0131] Two or more peptides may be contained entirely in one immunogenic composition, or multiple peptides may be separated into two or more compositions. If these peptides are separated into two or more compositions, the compositions may be mixed before administration and thus administered together, or they may be administered separately. Typically, all compositions, and therefore all peptides in multiple compositions, will be administered to the subject within a 24-hour timeframe (preferably within 4, 2, or 1 hour).

[0132] If two or more compositions are applied, the application may be at the same site (e.g., on the same limb) or at two or more different sites. During treatment, the application of one or more compositions may be performed once, or alternatively may be repeated (enhanced) subsequently, such as, but not limited to, two or three times.

[0133] In a preferred embodiment, the treatment method comprises a combination of long peptides, wherein the combination of long peptides comprises an HLA-binding peptide sequence capable of binding to at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of class I HLA molecules encoded by a dominant HLA allele in the population of subjects to be treated. Preferred class I HLA-binding peptide sequences in the long peptides according to the invention are HLA-binding peptide sequences capable of binding to class I HLA allele supertype HLA-A. 01. HLA-A 02, HLA-A 03. HLA-A 24. HLA-B 07. HLA-B 08. HLA-B 27. HLA-B 44. HLA-B 58. HLA-B 62. and HLA supertype A 01 Combination HLA-A 01 / A 03 and HLA-A 01 / A 24. and their respective subtypes (Sidney et al. 2008, BMC Immunology) 9), preferably HLA-A0101, HLA-A0201, HLA-A0206, HLA-A0301, HLA-A1101, HLA-A2301, HLA-A2402, HLA-A2501, HLA-A2601, HLA-A2902, HLA-A3001, HLA-A3002, HLA-A3101, HLA-A3201, HLA-A3303, HLA-A6801, HLA-A6802, HLA-A7401, HLA-B0702, HLA-B0801, HLA-B1301, HLA-B1302, HLA-B1402, HLA-B1501, HLA-B15 02. HLA-B1525, HLA-B1801, HLA-B2702, HLA-B2705, HLA-B3501, HLA-B3503, HLA-B3701, HLA-B3801, HLA-B3901, HLA-B4001, HLA-B4002, HLA-B4402, HLA -B4403, HLA-B4601, HLA-B4801, HLA-B4901, HLA-B5001, HLA-B5101, HLA-B5201, HLA-B5301, HLA-B5501, HLA-B5601, HLA-B5701, HLA-B5801 and HLA-B5802. In a preferred embodiment, the treatment method comprises a combination of long peptides, wherein the combination of long peptides comprises an HLA-binding peptide sequence capable of binding to at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of class I HLA molecules and an HLA-binding peptide sequence capable of binding to at least 20%, 30%, 40%, 42%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of class II HLA molecules encoded by a dominant HLA allele in the population of subjects to be treated.

[0134] In a preferred embodiment, the long peptide used in this invention comprises an HLA-binding peptide sequence that exhibits binding affinity, preferably at least moderate, and more preferably high, for class I HLA molecules, and elicits a cytotoxic CD8+ T cell response, wherein the class I HLA molecules are encoded by a dominant HLA allele in the population of human subjects to be treated. Preferably, the long peptide used in this invention comprises an HLA-binding peptide sequence that exhibits binding affinity, preferably at least moderate, and more preferably high, for at least one class I HLA molecule from the group consisting of: HLA-A01 (i.e., A) 0101 A 0112 A 2601 A 0114 A 2610 A 2602 A 0115 A 2611 A 2603 A 2604 A 2612 A 3002 A 2613 A 3003 A 0109 A 2606 A 3004 A 0110 A 2615 A 3201 A 0111 A 2618 A 3012 A 2619 A 3202 A 2621 A 3205 A 2623 A 3206 A 2624 A 3207 A 2626 A 3009 A 3601 A 2501 A 2502 A 2504 A 8001 A 2622 A 3110 A 0103 A 2609 A 0104 A 2605 A 2617 A 0106 A 3006 A 3210 A 0107 A 3204 A 3208 A 0108 A 2614 A 3203 A 2607 A 3603 A 7410 A 2608 A 3209 A 3602 A 3604); HLA-A02 (i.e., A 0201 A 0240 A 0271 A 0202 A 0243 A 0258 A 0203 A 0212 A 0259 A 0274 A 0204 A 0213 A 0227 A 0205 A 0215 A 0228 A 0206 A 0207 A 0218 A 0214 A 0219 A 0236 A 0267 A 0217 A 0220 A 0251 A 6802 A 0269 A 0285 A 6901 A 0256 A 0241 A 0260 A 0284 A 0209 A 0257 A 6827 A 0211 A 0224 A 0272 A 0225 A 0226 A 6828 A 0244 A 0261 A 0275 A 0245 A 0262 A 0230 A 0246 A 0216 A 0263 A 0231 A 0248 A 0266 A 0249 A 0282 A 0279 A 0237 A 0268 A 0221 A 0277 A 0247 A 0222 A 0270 A 0242 A 0273 A 6815 A 0283 A 0238 A 0254 A 0278 A 0239 A 0286 A 0250); HLA-A03 (i.e., A 0301 A 0317 A 3101 A 0307 A 6601 A 1107 A 7401 A 0312 A 1108 A 0313 A 1109 A 0314 A 1110 A 1112 A 1114 A 1116 A 1121 A 3103 A 1104 A 7402 A 3106 A 0280 A 3109 A 3111 A 6603 A 6816 A 7405 A 1106 A 3304 A 6604 A 6819 A 7407 A 1122 A 3305 A 6803 A 6821 A 3306 A 3307 A 6808 A 7411 A 3402 A 6825 A 6823 A 3403 A 6810 A 7406 A 0305 A 1102 A 3301 A 0306 A 1103 A 3303 A 0308 A 1105 A 6801 A 0310 A 0302 A 0316 A 1101 A 0304 A 6602 A 6814 A 7404 A 0309 A 6813 A 7403 A 3404 A 6812 A 6822 A 7409 A 6805 A 7408 A 6804 A 3112 A 6824 A 6820 A 6809 A 6826 A 1113 A 1115 A 1120 A 1123 A 3104 A 3105 A 0265 A 3406); HLA-A24 (i.e., A 2301 A 2410 A 2422 A 2402 A 2411 A 2423 A 2304 A 2427 A 2307 A 2408 A 2308 A 2421 A 2429 A 2305 A 2417 A 2452 A 2425 A 2405 A 2413 A 2426 A 2435 A 2446 A 2306 A 2406 A 2418 A 2302 A 2310 A 2433 A 2440 A 2303 A 2403 A 2434 A 2443 A 2420 A 2428 A 2438 A 2448 A 2430 A 2441 A 2442 A 2312 A 2444 A 2439 A 2449 A 2409 A 2437 A 2447); HLA-B07 (i.e., B 0702 B 0741 B 5134 B 5510 B 0703 B 0706 B 5135 B 5515 B 0705 B 0715 B 0743 B 3542 B 5136 B 1508 B 3507 B 3543 B 5116 B 5138 B 5519 B 3501 B 5117 B 5302 B 3503 B 0721 B 3511 B 3545 B 5118 B 5306 B 4201 B 3514 B 3546 B 5308 B 5101 B 3515 B 3554 B 5121 B 5310 B 5615 B 5102 B 3521 B 3555 B 5123 B 5403 B 5616 B 5103 B 3557 B 5124 B 5301 B 3561 B 5126 B 5406 B 5401 B 5128 B 5407 B 5501 B 3532 B 5129 B 5503 B 5502 B 3533 B 4204 B 5130 B 5601 B 3535 B 4205 B 5131 B 6701 B 3536 B 5132 B 7801 B 5133 B 5509 B 0707 B 3518 B 5112 B 0709 B 3529 B 5113 B 0712 B 3530 B 0714 B 3534 B 5120 B 0716 B 3537 B 5137 B 0717 B 3539 B 5304 B 0718 B 3551 B 0723 B 3553 B 5511 B 0736 B 5514 B 3502 B 3504 B 3807 B 5604 B 3505 B 3917 B 5609 B 3506 B 3509 B 4406 B 3512 B 5104 B 5612 B 3517 B 5106 B 8101 B 8102 B 5508 B 5602 B 5513 B 0737 B 3558 B 3560 B 3806 B 5610 B 4206 B 5611 B 0726 B 3522 B 5404 B 0730 B 3531 B 3910 B 7804 B 0731 B 7802 B 3524 B 0733 B 3916 B 5108 B 3538 B 0734 B 5114 B 0725 B 5504 B 0735 B 5505 B 0739 B 5105 B 5507 B 0740 B 0704 B 3540 B 5109 B 3541 B 5110 B 0742 B 3508 B 3544 B 5603 B 5111 B 5517 B 0719 B 5605 B 0722 B 0720 B 5119 B 5613 B 0724); HLA-B08 (i.e., B 0801 B 0815 B 0819 B 0821 B 0823 B 0818 B 0820 B 0822 B 0824 B 0825 B 0811 B 0813 B 0807 B 0802 B 0809 B 0803 B 0812 B 0808 B 0816).

[0135] As mentioned, treatment may involve administering two or more antigens of the present invention. In one embodiment, the present invention provides a method for treating or preventing HBV-related disease, the method comprising administering to a human subject: - The peptide as described in claim 1a) and the peptide as described in claim 1b), or - The peptide as described in claim 1a) and the peptide as described in claim 1c), or - The peptide as described in claim 1a) and the peptide as described in claim 1d), or - The peptide as described in claim 1a) and the peptide as described in claim 1e), or - The peptide as described in claim 1a) and the peptide as described in claim 1f), or - The peptide as described in claim 1a) and the peptide as described in claim 1g), or - The peptide as described in claim 1a) and the peptide as described in claim 1h), or -The peptide as described in claim 1a) and the peptide as described in claim 1i, or - The peptide as described in claim 1a) and the peptide as described in claim 1j), or - The peptide as described in claim 1a) and the peptide as described in claim 1k), or - The peptide as described in claim 1a) and the peptide as described in claim 1l), or - The peptide as described in claim 1a) and the peptide as described in claim 1m), or - The peptide as described in claim 1b) and the peptide as described in claim 1c), or - The peptide as described in claim 1b) and the peptide as described in claim 1d), or - The peptide as described in claim 1b) and the peptide as described in claim 1e), or - The peptide as described in claim 1b) and the peptide as described in claim 1f), or - The peptide as described in claim 1b) and the peptide as described in claim 1g, or - The peptide as described in claim 1b) and the peptide as described in claim 1h), or - The peptide as described in claim 1b) and the peptide as described in claim 1i, or - The peptide as described in claim 1b) and the peptide as described in claim 1j), or - The peptide as described in claim 1b) and the peptide as described in claim 1k), or - The peptide as described in claim 1b) and the peptide as described in claim 11), or - The peptide as described in claim 1b) and the peptide as described in claim 1m), or - The peptide as described in claim 1c) and the peptide as described in claim 1d), or - The peptide as described in claim 1c) and the peptide as described in claim 1e), or - The peptide as described in claim 1c) and the peptide as described in claim 1f), or - The peptide as described in claim 1c) and the peptide as described in claim 1g), or - The peptide as described in claim 1c) and the peptide as described in claim 1h), or - The peptide as described in claim 1c) and the peptide as described in claim 1i, or - The peptide as described in claim 1c) and the peptide as described in claim 1j), or - The peptide as described in claim 1c) and the peptide as described in claim 1k), or - The peptide as described in claim 1c) and the peptide as described in claim 11), or - The peptide as described in claim 1c) and the peptide as described in claim 1m), or - The peptide as described in claim 1d) and the peptide as described in claim 1e), or - The peptide as described in claim 1d) and the peptide as described in claim 1f), or - The peptide as described in claim 1d) and the peptide as described in claim 1g), or - The peptide as described in claim 1d) and the peptide as described in claim 1h), or - The peptide as described in claim 1d) and the peptide as described in claim 1i), or - The peptide as described in claim 1d) and the peptide as described in claim 1j), or - The peptide as described in claim 1d) and the peptide as described in claim 1k), or - The peptide as described in claim 1d) and the peptide as described in claim 1l), or - The peptide as described in claim 1d) and the peptide as described in claim 1m), or - The peptide as described in claim 1e) and the peptide as described in claim 1f), or - The peptide as described in claim 1e) and the peptide as described in claim 1g), or - The peptide as described in claim 1e) and the peptide as described in claim 1h), or - The peptide as described in claim 1e) and the peptide as described in claim 1i, or - The peptide as described in claim 1e) and the peptide as described in claim 1j), or - The peptide as described in claim 1e) and the peptide as described in claim 1k), or - The peptide as described in claim 1e) and the peptide as described in claim 11), or - The peptide as described in claim 1e) and the peptide as described in claim 1m), or - The peptide as described in claim 1f) and the peptide as described in claim 1g), or - The peptide as described in claim 1f) and the peptide as described in claim 1h), or - The peptide as described in claim 1f) and the peptide as described in claim 1i), or - The peptide as described in claim 1f) and the peptide as described in claim 1j), or - The peptide as described in claim 1f) and the peptide as described in claim 1k), or - The peptide as described in claim 1f) and the peptide as described in claim 1l), or - The peptide as described in claim 1f) and the peptide as described in claim 1m), or - The peptide as described in claim 1g) and the peptide as described in claim 1h), or - The peptide as described in claim 1g) and the peptide as described in claim 1i), or - The peptide as described in claim 1g) and the peptide as described in claim 1j), or - The peptide as described in claim 1g) and the peptide as described in claim 1k), or - The peptide as described in claim 1g) and the peptide as described in claim 1l), or - The peptide as described in claim 1g) and the peptide as described in claim 1m), or - The peptide as described in claim 1h) and the peptide as described in claim 1i), or - The peptide as described in claim 1h) and the peptide as described in claim 1j), or - The peptide as described in claim 1h) and the peptide as described in claim 1k), or - The peptide as described in claim 1h) and the peptide as described in claim 1l), or - The peptide as described in claim 1h) and the peptide as described in claim 1m), or - The peptide as described in claim 1i) and the peptide as described in claim 1j), or - The peptide as described in claim 1i) and the peptide as described in claim 1k), or - The peptide as described in claim 1i) and the peptide as described in claim 1l), or - The peptide as described in claim 1i) and the peptide as described in claim 1m), or - The peptide as described in claim 1j) and the peptide as described in claim 1k), or - The peptide as described in claim 1j) and the peptide as described in claim 1l), or - The peptide as described in claim 1j) and the peptide as described in claim 1m), or - The peptide as described in claim 1k) and the peptide as described in claim 1l), or - The peptide as described in claim 1k) and the peptide as described in claim 1m), or - The peptide as described in claim 1l) and the peptide as described in claim 1m), or - A polynucleotide sequence comprising one or more of the above combinations encoding a peptide.

[0136] Preferably, the immunogenic composition comprises or consists of the amount of peptides constituting a drug dose. A drug dose is defined herein as the amount of active ingredient applied to a subject at a given time point (i.e., the total amount of peptides in a peptide-based immunogenic composition). The drug dose may be administered to the subject in a single volume (i.e., a single injection), or in 2, 3, 4, 5, or more individual volumes, preferably applied at different locations on the body, such as the right and left limbs. The reasons for applying a single drug dose in individual volumes can be multiple, such as avoiding negative side effects, avoiding antigen competition, and / or compositional analysis considerations.

[0137] The dosage of a drug can be an effective amount or a portion thereof. "Effective amount" is to be understood herein as the amount or dose of the active ingredient required, relative to an untreated patient, to prevent and / or alleviate symptoms of a disease (e.g., chronic infection, precancerous condition, and / or cancer). The effective amount of one or more active compounds used in the practice of this invention for the preventive and / or therapeutic treatment of a disease or condition varies depending on the manner of administration, the subject's age, weight, and general health. Ultimately, the physician or veterinarian in charge of treatment will determine the appropriate amount and dosage regimen. This amount is referred to as the "effective" amount. The effective amount can also be an amount capable of inducing an effective cellular T-cell response, or more preferably an effective systemic cellular T-cell response, in the subject to be treated.

[0138] Preferably, the drug dose or total amount of peptide administered to the subject at a given time point (by a single or multiple injections or administrations at a given time point) comprises peptide amounts within the following ranges: from 0.1 μg to 20 mg, such as about 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, etc. The preferred dosage ranges are 1 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 15 mg, or about 20 mg, or any value between these ranges. Preferred dosage ranges are from 0.1 μg to 20 mg, 1 μg to 10 mg, 10 μg to 5 mg, 0.5 mg to 2 mg, 0.5 mg to 10 mg, or 1 mg to 5 mg, or 2 to 4 mg.

[0139] In one embodiment, the immunogenic composition used in this invention is administered at a dose of between 1 microgram and 300 micrograms (e.g., between 50 micrograms and 150 micrograms, such as approximately 100 micrograms) for each peptide.

[0140] The method of the present invention can be part of a combination therapy with other forms of HBV treatment, which can be provided as a standalone treatment or added to the immunogenic composition of the present invention. The method of the present invention can be combined with: drugs that inhibit viral replication (e.g., nucleoside or nucleotide analogs, including entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide), and / or drugs that prevent HBV from entering cells (e.g., myrcludex), and / or drugs that inhibit viral protein production (e.g., siRNA, shRNA, CRISPR / CAS9-based) and / or drugs that modulate immune responses (e.g., PEG-interferon α), activate innate immune responses (e.g., αGalCer), and / or hepatitis B immunoglobulin (HBIG) to support... Drugs that induce an immune response to vaccines and / or HBV prophylactic vaccines to induce antibodies against HBsAg, and / or liver resection or transplantation and tumor ablation therapy (e.g., transendothelial embolization, radiofrequency ablation) and / or drugs that inhibit VEGFR and / or kinases, and / or drugs that inhibit or block immune checkpoint molecules (e.g., A2AR (adenosine A2A receptor), B7-H3 / CD276, B7-H4 / VTCN1, BTLA / CD272, CTLA-4 / CD152, IDO (indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin)). Drugs that stimulate stimulating checkpoint molecules (e.g., selected tumor necrosis factor (TNF) receptor superfamily members) such as protein-like receptors, LAG3 (lymphocyte activation gene 3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), PD-1 (programmed cell death 1), PD-L1, TIM-3 (T cell immunoglobulin domain and mucin domain 3), VISTA (T cell activation V domain Ig inhibitor), SIGLEC7 / CD328 and SIGLEC9 / CD329, NKG2A, and / or other drugs that stimulate stimulating checkpoint molecules (e.g., selected tumor necrosis factor (TNF) receptor superfamily members). (e.g., CD27, CD40, CD122, 4-1BB / CD137, OX-40 / CD134 and GITR (associated with the glucocorticoid-induced TNFR family)), CD28 and ICOS / CD278), immunosuppressive cytokines (e.g., IL-10, TGF-β and IL-6) and / or γC cytokines (e.g., IL-7, IL-15 and IL-21 or IL-2), thalidomide and / or its derivatives, and other immunomodulators (e.g., compounds known to deplete immunosuppressive Tregs and / or MDSCs).

[0141] In one embodiment, one or more immunogenic peptides or compositions of the present invention may be combined with antiviral compounds (such as nucleoside / nucleotide analogs (NA)) in a treatment regimen comprising the following steps: (i) Administer antiviral (e.g., NA) treatment to patients infected with HBV to reduce viral load. (ii) When the viral load is significantly reduced (e.g., more than 2, 5, or 10 times), one or more immunogenic peptides or compositions of the present invention are applied (preferably intradermally or subcutaneously), and (iii) When a sufficiently long period of time has elapsed for an initial T-cell response to occur in the patient (e.g., between 2 and 16 weeks after the last administration of the immunogenic composition, such as between 2 and 12 weeks, or for example between 2 and 8 weeks), optional immune surveillance is performed (e.g., characterization of peripheral and intrahepatic T cells, particularly by, for example, IFNγ ELISpot analysis or antigen-specific T-cell proliferation and / or FACS-based phenotypic analysis of antigen-specific T cells to determine the vaccine-specific T-cell response to one or more immunogenic peptides of the administered composition of the invention. Optionally, this is accompanied by determining the T-cell response to the target non-vaccine antigen and unrelated control microbial antigens) to assess whether the vaccine has elicited an appropriate T-cell response (see, for example, Rivino et al. (2018) J Clin Invest [Journal of Clinical Research] 128:668). Subsequently, antiviral therapy is interrupted or stopped to increase viral antigen presentation, thereby further enhancing HBV-specific T cells in situ and exposing infected hepatocytes to the immune system (i.e., by increasing hepatocyte HBV protein expression and antigen presentation) to drive the clearance of those remaining infected hepatocytes. To further improve T-cell effector function, therapeutic vaccination may optionally be combined with: myeloid dysplastic cell (MDSC) targeted drugs (administered prior to therapeutic vaccination), siRNA (prior to therapeutic vaccination), T-cell metabolic modification drugs (before or during therapeutic vaccination), or checkpoint blockade (during or after therapeutic vaccination). Preferably, adequate monitoring of viral load is performed to evaluate vaccine efficacy and determine subsequent (combination) therapies and / or NA discontinuation. Figure 8A non-limiting illustration of a treatment regimen according to this embodiment is provided. This treatment regimen is particularly suitable for patients receiving antiviral therapy as recommended by treatment guidelines. For patients with persistent but low viral load infections, antiviral therapy may not be necessary prior to vaccination. Such patients are eligible to receive a therapeutic vaccination at any given time, and optional immune monitoring may be performed to assess whether the vaccine has elicited an appropriate T-cell response once sufficient time has elapsed for an initial T-cell response to occur in the patient. To further improve T-cell effector function, the therapeutic vaccination may optionally be combined with: myeloid phagocytic suppressor (MDSC) targeted agents (administered prior to therapeutic vaccination), siRNA (prior to therapeutic vaccination), T-cell metabolic modification agents (pre- or during therapeutic vaccination), or checkpoint blockade (during or after therapeutic vaccination). Adequate monitoring of viral load is preferably performed to evaluate vaccine efficacy and determine subsequent (combination) therapies.

[0142] In another aspect, the immunogenic peptide or composition can be used in ex vivo immunization protocols. In these protocols, the peptide or composition can be used to generate antigen-presenting cells (APCs) loaded with antigens (such as activated dendritic cells (DCs) loaded with antigens), and subsequently stimulate the expansion of antigen-specific T cells (e.g., CD4 and CD8 positive circulating T cells, tumor-infiltrating lymphocytes (TILs)). The antigen-loaded APCs or expanded antigen-specific T cells are then administered to a human subject.

[0143] Therefore, in another aspect, the present invention relates to peptides according to the invention or peptides comprising sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 26, for in vitro stimulation of activated antigen-presenting cells or expanded antigen-specific T cells loaded with antigens.

[0144] Similarly, in another embodiment, the method of the present invention involves treating a human subject with a population of activated antigen-presenting cells (APCs) loaded with antigens or expanded antigen-specific T cells, wherein said cells have been generated ex vivo (i.e., in vitro) using one or more immunogenic compositions described herein. This can be achieved, for example, by culturing a patient's PBMCs to generate autologous activated APCs (e.g., DCs) loaded with an immunogenic composition (i.e., antigen-loaded APCs) and subsequently stimulating and expanding T cells derived from the PBMCs or tumor-infiltrating lymphocytes. Alternatively, antigen-specific T cells can be expanded by incubating with activated APCs cultured from HLA-matched healthy donor PBMCs loaded with an immunogenic composition. Suitable techniques have been described in the field, for example, McCormack et al. (2018) Cytotherapy 20:385; Stevanovic et al. (2015) J Clin Oncol 33:1543; and Stevanovic et al. (2018) Clin Cancer Research, doi:10.1158 / 1078-0432.

[0145] In another embodiment, treatment with a population of activated antigen-presenting cells (APCs) or expanded antigen-specific T cells is combined with direct immunization of a human subject using the immunogenic composition described herein. This combination regimen may involve sequential and / or simultaneous administration.

[0146] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0147] Example Example 1: Comparison of common sequences and functional domains between HBV-X and HBV polymerase HBV sequences for all genotypes of HBV-X (n = 8127) and HBV polymerase (n = 7489) were used, and frequency tables were downloaded from HBVdbV42.0 (Hayer et al., 2013 Nucleic Acid Res [Nucleic Acid Research] 41:566). The most frequent nicks (indicated by "-") were removed, and the dominant amino acid at each position was determined. The percentage of sequences containing the dominant amino acid was calculated as a conservation score.

[0148] A common sequence (SEQ ID NO: 28) was generated by combining all the dominant amino acids of HBV polymerase: The common sequence of HBV-X was determined to be (SEQ ID NO: 30): In addition, individual or combined amino acids previously associated with loss of viral replication were compared with the common sequence. Figure 1 and Figure 2 Compared to HBV-X, Pol has a greater number of function-related amino acids. Therefore, an additional threshold of ≥ 50% loss of viral persistence was introduced for Pol to select the most critical amino acids. Tables 1 and 2 present references regarding functional domains and amino acids.

[0149] Table 1: References for experimental evidence of the functional domains and essential amino acids of HBV-x in reports or reviews.

[0150] Table 2: References for reporting or reviewing experimental evidence on the functional domains and essential amino acids of HBV polymerase.

[0151] Example 2: Prediction of novel class I HLA-binding peptides derived from HBV-x and HBV polymerase We set out to identify novel peptides that can bind to at least one of the six HLA supertypes commonly found in Caucasians, Africans, or Asians, and these peptides can be readily confirmed by in vitro assays (i.e., supertype HLA-A). 01, A 02, A 03, A 24. B 07, B 08). We first used the established computer prediction tool NetMHCpan (Nielsen and Andreatta 2016, Genome Med [Genomic Medicine] 8:33) to predict binding compounds spanning 8–14 amino acids for supertype-representative HLA types, in order to construct the frequency distribution of the predicted binding compounds ( Figure 1 and Figure 2(Gray bar graph). The densities of all predicted bindings / amino acids were similar between Pol and HBx (mean ± SD: Pol 16.36 ± 12.62, HBx 15.60 ± 9.49; Mann-Whitney; p = 0.57). Since the 9-11 polymer is most likely to represent the associated epitope (Trolle et al. 2016 J Immunol [Journal of Immunology] 196:1480), the predicted bindings of more than 9-11 amino acids were subsequently compared with our graph, which outlines the conservation and function described in Example 1 (). Figure 1 and Figure 2 ).

[0152] This prediction yielded a total of 251 potential new HLA conjugates against HBx and 1655 potential new HLA conjugates against Pol. Of these, we selected the most promising peptides for validation in an in vitro HLA binding assay based on UV-induced peptide exchange (Toebes et al. 2006 Nat Med [Nature Medicine] 12:246). For practical and economic reasons, we aimed to test the binding of 96 unique peptide sequences to both proteins and across HLA types. We included two well-described epitopes (core 18–27 and Pol 549–557) to contextualize the binding ability of our newly identified conjugates. The 96 potential conjugates were selected based on peptide length (preferably 9mers), predicted HLA binding strength, conservation, and functional importance of the included amino acids. Not all 96 potential conjugates consistently met all the criteria. For HLA-A… 01 and HLA-A 24. The predicted number of binding compounds for HBx is unsatisfactory and cannot maintain our strict thresholds for conservation and peptide length. Therefore, for these conditions, we also include some less conserved peptides or peptides spanning 8-12 amino acids (in... Figure 3 (Indicated by an asterisk, see Example 3). In addition, we selected several peptides that were not frequently reported as epitopes in the literature (once or twice) and were therefore considered unestablished. Figure 3 (Closed underscore, see example 3). This includes c123-130 because it is registered in Hepitopes with HLA-B. The only HBV-derived epitope associated with 08. Furthermore, peptides predicted to bind to several HLA types were preferentially considered throughout the selection process, resulting in a total of 113 potential conjugates tested. Overall, the selection process yielded 45 potential conjugates against HBx and 68 potential conjugates against Pol for validation via in vitro binding assays. The majority of these mapped to highly conserved regions with established functional importance. For HBx, the median conservation among the selected peptides was greater than 93%, while for Pol, the median was even greater than 96%.

[0153] Example 3: In vitro binding capacity of selected peptides derived from HBx and polymerase 3.1 Method As previously described (Karimzadeh et al. 2018 J Virol [Journal of Virology] 92:e01891), synthetic peptides (Peptide 2.0 Inc.) were used in the in vitro binding assay to select potential HLA conjugates. Briefly, the peptide exchange reaction was performed by exposing a conditional peptide-HLA complex (pHLA) (0.53 μM) to long-wave UV light for 30 min using a 366 nm UV lamp (Camag), with or without the indicated peptide (50 μM). The efficiency of the peptide exchange was then analyzed using a class I HLA enzyme-linked immunosorbent assay (ELISA), which detects β-2 microglobulins of the peptide-stabilized class I HLA complex in the exchange reaction mixture. For this purpose, streptavidin (2 μg / ml) was bound to polystyrene microtiter wells (NuncMaxiSorp). After washing and blocking, HLA complexes in the exchange reaction mixture or control were captured by streptavidin on a microtiter plate via their biotinylated heavy chains (incubated at 37°C for 1 h). Unbound material was removed by washing. Subsequently, an antibody conjugated to human β-2-microglobulin and horseradish peroxidase (HRP) (0.6 μg / ml; Sanquin Reagents BV) was added (incubated at 37°C for 1 h). After removing unbound HRP conjugates by washing, a substrate solution of ABTS [2,2'-azono-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] (Sanquin Reagents) was added to the wells. The reaction was terminated after 8 min (incubated at room temperature) by adding 2% (w / v) oxalate dihydrate stop solution (Sanquin Reagents), and read at 414 nm on a Thermo Electron Multiskan Ascent ELISA reader. Each peptide was exchanged twice independently. In a class I HLA ELISA, each exchange mixture was measured in duplicate. The absorbance of all peptides was normalized to the absorbance of a known HLA allele-specific ligand with high affinity for each corresponding allele (representing 100%; Table 3). Negative controls included HLA allele-specific non-binding compounds and conditional class I HLA complexes irradiated with UV in the absence of rescue peptides.

[0154] Table 3: Reference peptides for in vitro binding assays, categorized by HLA type.

[0155] 3.2 Results As described in section 3.1, the binding affinity of selected peptides was tested in a plate-based in vitro binding assay. Peptides with binding affinity greater than 25% of known high-affinity peptides were classified as HLA conjugates. HLA-A 11:01 and HLA-A 03:01 Both are HLA-A Members of the 03 supertype were tested because many patients infected with HBV are Asian, and the supertype is more representative of HLA-A than it is in Caucasians. 03:01, HLA-A 11:01 is more common in this group (Chang et al. 2013 Eur J Immunol [European Journal of Immunology] 43:1109).

[0156] We identified 13 conjugates targeting HBx and 33 conjugates targeting Pol in HLA supertypes. Figure 3 (Gray bar). This includes new conjugates previously described in the context of another HLA type ( Figure 3 A-3G (dashed underline and 3H). Notably, for each HLA supertype tested, HBx and Pol-derived conjugates were identified. For HLA-A 02, well-established epitopes c18-27 and p549-557 scored even better than the positive control ( Figure 3 B). In contrast, the combination of epitopes that are not frequently reported ( Figure 3 (Closed underscore) is not always confirmed.

[0157] Typically, binding predictions are found to be quite poor, as only about one-third of the predicted conjugates actually exhibit binding ability above the threshold. Many peptides predicted to bind strongly (low-scoring in computer predictions) do not show binding above the threshold in in vitro assays. Figure 4 ).

[0158] Table 4 compares the predicted score (grade) of the peptide for each HLA type test with the assay result (binding %).

[0159] Table 4:

[0160] Example 4: Immunogenicity of selected HLA-binding peptides 4.1 Methods Immunogenicity of peptides scoring above 25% in in vitro HLA binding assays was assessed. Briefly, PBMCs were isolated from erythrocyte sedimentation rate (ESR) amber layers from nine previously HBV-regressed donors by Ficoll density centrifugation. The ESR amber layers, provided by local blood banks, contained corresponding two-digit HLA types. Four-digit HLA typing was performed on seven of the nine donors using a global screening array (GSA) (Illumina, via the Human Genomics Facility Erasmus MC Rotterdam) (Table 5).

[0161] Table 5: HLA-I type of HBV-regressed donors used for immunogenicity testing of HLA conjugates:

[0162] All donors provided written informed consent. Based on HLA matching at 10 μg / ml / peptide, PBMCs were cultured in IMDM (Lonza) + 2% human serum (Sankun) + 50 IU / ml hIL-2 (Miltenyi) in the presence of a peptide pool of up to 5 target peptides. After 14 days, 200,000 cells were restimulated with the target peptide at 37°C at 10 μg / ml / peptide for 48 hours, in triplicate. The supernatant from the restimulation was then used for hIFNγ ELISA (BioLegend) according to the manufacturer's instructions. The plate was read at 450 nm using an Infinite 200Pro ELISA reader. Using the supernatant derived from a previously successfully restimulated c18-27 (which was quantified in a separate ELISA using an hIFNγ standard provided by the manufacturer), hIFNγ levels were calculated from the OD value minus the background (average of three replicates). HLA conjugates with an average OD value at least equal to the average of the DMSO control plus 2 x SD were quantified.

[0163] 4.2 Results Subsequently, the immunogenicity of the HBx and Pol-derived conjugates was tested to confirm their efficacy. PBMCs from previously HBV-infected, regressed blood donors were amplified for 2 weeks in the presence of a peptide pool, followed by single-peptide restimulation and IFNγ ELISA as described in section 4.1. As expected, IFNγ production in response to well-established epitopes c18-27 and p549-557 was detected. Figure 5 B). IFNγ production varies considerably, and some donors generally appear to respond better than others. Figure 5 In summary, we observed responses to 5 novel HBx-derived peptides and 17 novel Pol-derived peptides. Additionally, we observed IFNγ production in response to 1 HBx-derived epitope and 3 Pol-derived epitopes, which have not been frequently described previously, although these responses were not very high. Figure 5 ; closed underscore). Importantly, four additional Pol-derived epitopes elicited a response in HLA-type-negative donors, epitopes previously described ( Figure 5 (The dashed underline and Table 3) indicate that these peptide sequences are epitopes in multiple HLA types. No measurable response was observed in any of the donors tested for the six Pol-derived HLA conjugates and the seven HBx-derived HLA conjugates. Figure 5 (gray box), but this may be due to the small number of donors. Therefore, future testing on additional donors may confirm the immunogenicity of these HLA conjugates.

[0164] Example 5: 5.1 Method To assess whether the claimed peptide fragments are immunogenic in human cases, seven SLPs containing one or more epitope peptide sequences were designed, manufactured, and tested using PBMC samples from fifteen different donors who had previously cleared HBV infection.

[0165] The seven SLPs were designed based on: 1) the naturally occurring HBV-X genotype sequence of HBV polymerase; 2) good manufacturability predicted using a computer machine learning algorithm based on general synthetic principles (especially those described above) and trained on a large set of actual peptide synthesis yields; and 3) containing one or more epitope peptide sequences (see Table 6). The preferred length of the SLP was set to 25 AA. Where deemed necessary, flanking regions along the corresponding HBV-X or HBV polymerase sequence were included to improve manufacturability, given poor manufacturability predicted based on established peptide synthesis experience. Six SLPs with a length of 25 AA and one SLP with a length of 26 AA were produced.

[0166] Using solid-phase Fmoc / tBu chemically synthesized single SLPs (Table 6), treated them with a lysis mixture, purified them by HPLC, and analyzed them by UPLC-MS according to established methods. All reagents and solvents used in solid-phase peptide synthesis (SPPS) were purchased from Merck, Sigma-Aldrich, Actu-All, Bachem, Biosolve, and GL Biochem and used as is. Peptide synthesis was performed on a Tetras peptide synthesizer (Advanced ChemTech). The resin was dried, cooled, and treated with a trifluoroacetic acid (TFA)-based lysis mixture. After filtering the resin, the reaction mixture was shaken at room temperature. Subsequently, the peptide was precipitated in an ether-based solution, centrifuged, and the supernatant was removed. The solid precipitate was resuspended in an ether-based solution, centrifuged, and the supernatant was removed. The resulting precipitate was dissolved in an H2O-based mixture containing acetonitrile (ACN) and TFA, or acetic acid, and lyophilized overnight. After HPLC purification, selected purified fractions were collected and lyophilized overnight using either TFA in H2O and TFA in ACN, or TFA in H2O and TFA in ACN in a solvent system with tert-butanol. The identity and purity of the purified peptide were determined by UPLC-MS. Prior to use, the SLP was reconstituted in 10% DMSO and 90% H2O to achieve a concentration of 2 mM.

[0167] The ability of SLP to induce IFNγ production in PBMCs after 24 hours of stimulation was tested using an IFNγ ELISpot assay. Briefly, PBMCs were isolated from the erythrocyte sedimentation rate (ESR) amber layer of 15 HLA-typed donors (Sankun Reagents Blood Bank) by density gradient centrifugation. PBMCs were cultured in PVDF plates (MSIPS 4510, Millipore) coated with an IFNγ capture antibody (5 μg / ml, Mab-1-D1K, Mabtech) in the presence of 10 μM SLP or an equivalent DMSO control. Cells were seeded at a density of 200,000 cells / well in four replicate wells of IMDM + 8% human serum. After incubation for 20–24 hours, IFNγ detection antibody (0.3 μg / ml, Mab-7-B6-1-Biotin, Antibody Technology) was added, followed by streptavidin-ALP (1 μg / ml, Antibody Technology). Spots were visualized by adding BCIP / NBT-plus substrate (100 μl / well, Antibody Technology) and counted using a CTL Immunospot S6 Ultimate analyzer (Immunospot). The number of spot-forming units (SFUs) from the four replicate wells was summed and the cumulative number of spots from the four replicate DMSO control wells was subtracted.

[0168] 5.2 Results To test the ability of SLPs to induce an IFNγ response, IFNγ ELISpot assays were performed on PBMCs from 15 HBV-regressed donors. These donors had previously regressed HBV infection and were therefore expected to have HBV-specific T-cell responses. All SLPs derived from both polymerase and HBx were able to induce an IFNγ response ( Figure 6 ).

[0169] Table 6:

[0170] Example 6. The novel SLP can enhance the functional CD8+ and CD4+ T cell responses in vitro from leukocytes derived from HBV-regressed individuals and patients with chronic HBV. 6.1 Method The functional enhancement capabilities of the novel SLP were tested in amplification experiments. Briefly, PBMCs were isolated from erythrocyte sedimentation rate (ESR) amber layers derived from previously HBV-cleared healthy donors (n = 6) or from blood of chronic HBV patients visiting the Erasmus Medical Center Rotterdam (n = 5). PBMCs were cultured for 14 days in IMDM (Lonza) + 2% human serum (Sanqun Reagents) in the presence of an SLP pool (3 uM / SLP). After 2 days, 50 IU / ml of IL-2 was added to the culture, repeated 3 times per week until day 14. After 14 days, 200,000 cells / well were restimulated in quadruplicate with either a single SLP (10 uM / SLP) or DMSO as a control. After 22 hours, the supernatant was harvested for cytokine analysis, and the cells were used for flow cytometry analysis. Cells were pooled and stained in the dark for 30 minutes at 4°C with the following members: CD3 (SK7) and CD8 (RPA-T8) from eBiosciences, CD4 (SK3) from BD, CD69 (FN50) and CD107a (H4A3) from Biolegend, and LIVE / DEAD Green from Invitrogen. Acquisition was performed on a BD FACSCanto instrument and analyzed using FlowJo v10 (BD). The percentage of marker expression was determined by subtracting the percentage of the corresponding marker observed in the DMSO control. Secreted cytokines in the culture supernatant were determined using Luminex technology. Cytokines were analyzed using a custom Procarta plate from Thermo Fisher Scientific and an MAGPIX instrument (Merck Millipore). The number of secreted cytokines was calculated using standards. Background is subtracted by subtracting the following from the calculated value: mean (DMSO and unrelated peptides) + 2 x SD (DMSO and unrelated peptides).

[0171] 6.2 Results In vitro amplification experiments simulating vaccination indicated that all four novel SLPs (SLP1, SLP2, SLP4, and SLP6) were able to enhance functional CD8+ and CD4+ T cell responses in vitro from leukocytes of HBV-regressed individuals (rHBV1-6) and chronic HBV patients (cHBV1-5), as each SLP triggered a response in at least one donor. T cell activation was indicated by the presence of increased CD69 in response to SLPs in both cell types. Figure 7This expansion and activation of donor T cells also yielded functional T cells, as demonstrated by the fact that CD8+ T cells exhibited CD107a in response to SLP, indicating the secretion of recent cytotoxic agents; and the presence of type I T cell cytokines IFNγ and TNFα, which are essential for T cell effector function.

Claims

1. An immunogenic peptide comprising a fragment of HBV protein, wherein the fragment is 20-34 amino acids in length, and wherein the fragment comprises: a) At least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, preferably comprising: - The amino acid sequence (x70-78) listed in SEQ ID NO: 1, and / or - The amino acid sequence (x67-75) listed in SEQ ID NO: 2, and / or - The amino acid sequence (x62-73) listed in SEQ ID NO: 3, and / or - The amino acid sequence (x58-66) listed in SEQ ID NO: 4, and / or - The amino acid sequence (x57-66) listed in SEQ ID NO:

5. or b) At least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, preferably comprising: - The amino acid sequence (x103-111) listed in SEQ ID NO: 6, and / or - The amino acid sequence (x104-113) listed in SEQ ID NO: 7, and / or - The amino acid sequence (x105-113) listed in SEQ ID NO: 8, and / or - The amino acid sequence (x110-120) listed in SEQ ID NO:

9. or c) The amino acid sequence listed in SEQ ID NO: 10 (x132-140). or d) The amino acid sequence listed in SEQ ID NO: 11 (p124-133). or e) The amino acid sequence listed in SEQ ID NO: 12 (p164-173). or f) The amino acid sequence listed in SEQ ID NO: 13 (p275-283). or g) At least 10 consecutive amino acids from the region at positions 403 to 415 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 14 (p403-412), and / or - The amino acid sequence listed in SEQ ID NO: 15 (p404-412), and / or - The amino acid sequence listed in SEQ ID NO: 16 (p407-415). or h) At least nine consecutive amino acids from the region at positions 509 to 523 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 17 (p509-517), and / or - The amino acid sequence listed in SEQ ID NO: 18 (p515-523). or i) At least 10 consecutive amino acids from the region at positions 649 to 658 of the HBV polymerase, comprising: - The amino acid sequence listed in SEQ ID NO: 19 (p649-658), and / or - The amino acid sequence listed in SEQ ID NO: 20 (p650-658). or j) At least 10 consecutive amino acids from the region at positions 693 to 706 of the HBV polymerase, preferably comprising: - The amino acid sequence listed in SEQ ID NO: 21 (p693-701), and / or - The amino acid sequence listed in SEQ ID NO: 22 (p697-706). or k) The amino acid sequence listed in SEQ ID NO: 23 (p723-731). or l) At least 10 consecutive amino acids from the region at positions 755 to 765 of the HBV polymerase, comprising: - The amino acid sequence listed in SEQ ID NO: 24 (p755-764), and / or - The amino acid sequence listed in SEQ ID NO: 25 (p756-765). or m) The amino acid sequence listed in SEQ ID NO: 26 (p829-837). The amino acid positions within HBV polymerase and HBV-X are numbered according to the numbers in the common sequences listed in SEQ ID NO: 28 and SEQ ID NO: 30, respectively.

2. The peptide of claim 1, wherein the peptide comprises the fragment of HBV protein.

3. The peptide according to claim 1 or 2, wherein the peptide is 20-34 amino acids in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids in length, such as 20-33 amino acids in length, such as 20-32 amino acids in length, such as 20-31 amino acids in length, such as 20-30 amino acids in length, such as 20-29 amino acids in length, such as 20-28 amino acids in length, such as 20-27 amino acids in length, such as 20-26 or 20-25 amino acids in length.

4. The peptide according to any one of the preceding claims, wherein the length of said fragment is 20-33 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids, such as 20-32 amino acids, such as 20-31 amino acids, such as 20-30 amino acids, such as 20-29 amino acids, such as 20-28 amino acids, such as 20-27 amino acids, such as 20-26 or 20-25 amino acids.

5. The peptide according to any one of the preceding claims, wherein... a) The fragment contains at least 10 consecutive amino acids from the region at positions 57 to 78 of HBV-X, and the fragment contains: - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 2 (x67-75), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 3 (x62-73), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 1 (x70-78) and SEQ ID NO: 5 (x57-66), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 3 (x62-73), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 2 (x67-75) and SEQ ID NO: 5 (x57-66), or - The amino acid sequences listed in SEQ ID NO: 3 (x62-73) and SEQ ID NO: 4 (x58-66), or - The amino acid sequences listed in SEQ ID NO: 3 (x62-73) and SEQ ID NO: 5 (x57-66) or b) The fragment contains at least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, and the fragment contains: - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 7 (x104-113), or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 8 (x105-113), or - The amino acid sequences listed in SEQ ID NO: 6 (x103-111) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 7 (x104-113) and SEQ ID NO: 9 (x110-120), or - The amino acid sequences listed in SEQ ID NO: 8 (x105-113) and SEQ ID NO: 9 (x110-120). or c) The fragment contains at least 10 consecutive amino acids from the region at positions 403 to 415 of the HBV polymerase, and the fragment contains: - The amino acid sequences listed in SEQ ID NO: 14 (p403-412) and SEQ ID NO: 16 (p407-415), or - The amino acid sequences listed in SEQ ID NO: 15 (p404-412) and SEQ ID NO: 16 (p407-415).

6. The peptide according to any one of the preceding claims, wherein the peptide comprises or consists of a sequence selected from the group consisting of: SEQ ID NO: 31 (SLP1), SEQ ID NO: 32 (SLP2), SEQ ID NO: 33 (SLP3), SEQ ID NO: 34 (SLP4), SEQ ID NO: 35 (SLP5), SEQ ID NO: 36 (SLP6) and SEQ ID NO: 37 (SLP7).

7. An immunogenic peptide comprising a fragment of HBV protein, wherein the peptide is 20-34 amino acids in length, and wherein the fragment comprises a sequence selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO:

26.

8. A polynucleotide comprising a nucleotide sequence encoding a peptide according to any one of the preceding claims.

9. A recombinant virus comprising the polynucleotide according to claim 8.

10. An immunogenic composition comprising: - The peptide according to any one of claims 1 to 7, the polynucleotide according to claim 8, or the recombinant virus according to claim 9, and - Pharmaceutically acceptable carriers Optionally, it may further include an adjuvant.

11. A method for treating or preventing HBV-related diseases, the method comprising administering to a human subject: - The peptide as described in claim 1a) and the peptide as described in claim 1b), or - The peptide as described in claim 1a) and the peptide as described in claim 1c), or - The peptide as described in claim 1a) and the peptide as described in claim 1d), or - The peptide as described in claim 1a) and the peptide as described in claim 1e), or - The peptide as described in claim 1a) and the peptide as described in claim 1f), or - The peptide as described in claim 1a) and the peptide as described in claim 1g), or - The peptide as described in claim 1a) and the peptide as described in claim 1h), or - The peptide as described in claim 1a) and the peptide as described in claim 1i, or - The peptide as described in claim 1a) and the peptide as described in claim 1j), or - The peptide as described in claim 1a) and the peptide as described in claim 1k), or - The peptide as described in claim 1a) and the peptide as described in claim 1l), or - The peptide as described in claim 1a) and the peptide as described in claim 1m), or - The peptide as described in claim 1b) and the peptide as described in claim 1d), or - The peptide as described in claim 1b) and the peptide as described in claim 1e), or - The peptide as described in claim 1b) and the peptide as described in claim 1f), or - The peptide as described in claim 1b) and the peptide as described in claim 1g, or - The peptide as described in claim 1b) and the peptide as described in claim 1h), or - The peptide as described in claim 1b) and the peptide as described in claim 1i, or - The peptide as described in claim 1b) and the peptide as described in claim 1j), or - The peptide as described in claim 1b) and the peptide as described in claim 1k), or - The peptide as described in claim 1b) and the peptide as described in claim 11), or - The peptide as described in claim 1b) and the peptide as described in claim 1m), or - The peptide as described in claim 1c) and the peptide as described in claim 1d), or - The peptide as described in claim 1c) and the peptide as described in claim 1e), or - The peptide as described in claim 1c) and the peptide as described in claim 1f), or - The peptide as described in claim 1c) and the peptide as described in claim 1g), or - The peptide as described in claim 1c) and the peptide as described in claim 1h), or - The peptide as described in claim 1c) and the peptide as described in claim 1i, or - The peptide as described in claim 1c) and the peptide as described in claim 1j), or - The peptide as described in claim 1c) and the peptide as described in claim 1k), or - The peptide as described in claim 1c) and the peptide as described in claim 11), or - The peptide as described in claim 1c) and the peptide as described in claim 1m), or - The peptide as described in claim 1d) and the peptide as described in claim 1e), or - The peptide as described in claim 1d) and the peptide as described in claim 1f), or - The peptide as described in claim 1d) and the peptide as described in claim 1g), or - The peptide as described in claim 1d) and the peptide as described in claim 1h), or - The peptide as described in claim 1d) and the peptide as described in claim 1i), or - The peptide as described in claim 1d) and the peptide as described in claim 1j), or - The peptide as described in claim 1d) and the peptide as described in claim 1k), or - The peptide as described in claim 1d) and the peptide as described in claim 1l), or - The peptide as described in claim 1d) and the peptide as described in claim 1m), or - The peptide as described in claim 1e) and the peptide as described in claim 1f), or - The peptide as described in claim 1e) and the peptide as described in claim 1g), or - The peptide as described in claim 1e) and the peptide as described in claim 1h), or - The peptide as described in claim 1e) and the peptide as described in claim 1i, or - The peptide as described in claim 1e) and the peptide as described in claim 1j), or - The peptide as described in claim 1e) and the peptide as described in claim 1k), or - The peptide as described in claim 1e) and the peptide as described in claim 11), or - The peptide as described in claim 1e) and the peptide as described in claim 1m), or - The peptide as described in claim 1f) and the peptide as described in claim 1g), or - The peptide as described in claim 1f) and the peptide as described in claim 1h), or - The peptide as described in claim 1f) and the peptide as described in claim 1i), or - The peptide as described in claim 1f) and the peptide as described in claim 1j), or - The peptide as described in claim 1f) and the peptide as described in claim 1k), or - The peptide as described in claim 1f) and the peptide as described in claim 1l), or - The peptide as described in claim 1f) and the peptide as described in claim 1m), or - The peptide as described in claim 1g) and the peptide as described in claim 1h), or - The peptide as described in claim 1g) and the peptide as described in claim 1i), or - The peptide as described in claim 1g) and the peptide as described in claim 1j), or - The peptide as described in claim 1g) and the peptide as described in claim 1k), or - The peptide as described in claim 1g) and the peptide as described in claim 1l), or - The peptide as described in claim 1g) and the peptide as described in claim 1m), or - The peptide as described in claim 1h) and the peptide as described in claim 1i), or - The peptide as described in claim 1h) and the peptide as described in claim 1j), or - The peptide as described in claim 1h) and the peptide as described in claim 1k), or - The peptide as described in claim 1h) and the peptide as described in claim 1l), or - The peptide as described in claim 1h) and the peptide as described in claim 1m), or - The peptide as described in claim 1i) and the peptide as described in claim 1j), or - The peptide as described in claim 1i) and the peptide as described in claim 1k), or - The peptide as described in claim 1i) and the peptide as described in claim 1l), or - The peptide as described in claim 1i) and the peptide as described in claim 1m), or - The peptide as described in claim 1j) and the peptide as described in claim 1k), or - The peptide as described in claim 1j) and the peptide as described in claim 1l), or - The peptide as described in claim 1j) and the peptide as described in claim 1m), or - The peptide as described in claim 1k) and the peptide as described in claim 1l), or - The peptide as described in claim 1k) and the peptide as described in claim 1m), or - The peptide as described in claim 1l) and the peptide as described in claim 1m), or - A polynucleotide sequence comprising one or more of the above combinations encoding a peptide.

12. The peptide according to any one of claims 1 to 7, the polynucleotide according to claim 8, the recombinant virus according to claim 9, or the immunogenic composition according to claim 10, used as a pharmaceutical.

13. The peptide according to any one of claims 1 to 7, the polynucleotide according to claim 8, the recombinant virus according to claim 9, or the immunogenic composition according to claim 10, for the treatment or prevention of HBV-related diseases.

14. The peptide according to any one of claims 1 to 7 or a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 26, for in vitro stimulation of activated antigen-presenting cells or expanded antigen-specific T cells loaded with antigens.

15. The peptide according to any one of claims 1 to 7, the polynucleotide according to claim 8, the recombinant virus according to claim 9, or the immunogenic composition according to claim 10, for treating HBV-related diseases, wherein the treatment comprises the following steps: (i) Administer antiviral therapy to patients infected with HBV to reduce viral load. (ii) When the viral load is significantly reduced, administer one or more of the immunogenic peptides, polynucleotides, recombinant viruses, or immunogenic compositions and (iii) Interrupt or stop antiviral therapy when a sufficient amount of time has passed for an initial T-cell response to occur in the patient.

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