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

Technical Field

[0001] 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

[0003] 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).

[0004] 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).

[0005] 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.

[0006] 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.

[0007] Chronic hepatitis B virus (HBV) 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 drug resistance occurs (Zoulim et al., 2012B J Hepatol, 56 Supplement 1S112; 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.

[0008] 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).

[0009] 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.

[0010] 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).

[0011] 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).

[0012] 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.

[0013] Despite significant progress in understanding the etiology and epidemiology of the disease, an effective therapeutic HBV vaccine is still needed.

[0014] 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:

[0015] • Poor sequence conservation across HBV genotypes results in efficacy only for a (small) subset of HBV genotypes.

[0016] 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.

[0017] • Due to excessive exposure to antigens leading to immune depletion, the immune response becomes ineffective.

[0018] • Covering only one or a few HLA types in total results in a lack of efficacy in certain populations that lack these HLA types.

[0019] • Difficult to manufacture (especially for longer peptides),

[0020] • No confirmed potential to induce T cell response.

[0021] There is no indication regarding whether the immune response to the peptide antigen has the potential to resolve the infection. Summary of the Invention

[0022] 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.

[0023] 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).

[0024] 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:

[0025] a) At least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, preferably comprising:

[0026] -The amino acid sequence (x70-78) listed in SEQ ID NO:1, and / or

[0027] -The amino acid sequence (x67-75) listed in SEQ ID NO:2, and / or

[0028] -The amino acid sequence (x62-73) listed in SEQ ID NO:3, and / or

[0029] -The amino acid sequence (x58-66) listed in SEQ ID NO:4, and / or

[0030] -The amino acid sequence (x57-66) listed in SEQ ID NO:5,

[0031] or

[0032] b) At least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, preferably comprising:

[0033] -The amino acid sequence (x103-111) listed in SEQ ID NO:6, and / or

[0034] -The amino acid sequence (x104-113) listed in SEQ ID NO:7, and / or

[0035] -The amino acid sequence (x105-113) listed in SEQ ID NO:8, and / or

[0036] -The amino acid sequence (x110-120) listed in SEQ ID NO:9,

[0037] or

[0038] c) The amino acid sequence (x132-140) listed in SEQ ID NO:10,

[0039] or

[0040] d) The amino acid sequence listed in SEQ ID NO:11 (p124-133),

[0041] or

[0042] e) The amino acid sequence listed in SEQ ID NO:12 (p164-173),

[0043] or

[0044] f) The amino acid sequence listed in SEQ ID NO:13 (p275-283),

[0045] or

[0046] g) At least 10 consecutive amino acids from the region at positions 403 to 415 of the HBV polymerase, preferably comprising:

[0047] -The amino acid sequence (p403-412) listed in SEQ ID NO:14, and / or

[0048] -The amino acid sequence (p404-412) listed in SEQ ID NO:15, and / or

[0049] -The amino acid sequence listed in SEQ ID NO:16 (p407-415),

[0050] or

[0051] h) At least nine consecutive amino acids from the region at positions 509 to 523 of the HBV polymerase, preferably comprising:

[0052] -The amino acid sequence listed in SEQ ID NO:17 (p509-517), and / or

[0053] -The amino acid sequence listed in SEQ ID NO:18 (p515-523),

[0054] or

[0055] i) At least 10 consecutive amino acids from the region at positions 649 to 658 of the HBV polymerase, comprising:

[0056] -The amino acid sequence listed in SEQ ID NO:19 (p649-658), and / or

[0057] -The amino acid sequence (p650-658) listed in SEQ ID NO:20,

[0058] or

[0059] j) At least 10 consecutive amino acids from the region at positions 693 to 706 of the HBV polymerase, preferably comprising:

[0060] -The amino acid sequence listed in SEQ ID NO:21 (p693-701), and / or

[0061] -The amino acid sequence listed in SEQ ID NO:22 (p697-706),

[0062] or

[0063] k) The amino acid sequence listed in SEQ ID NO:23 (p723-731),

[0064] or

[0065] l) At least 10 consecutive amino acids from the region at positions 755 to 765 of the HBV polymerase, comprising:

[0066] -The amino acid sequence listed in SEQ ID NO:24 (p755-764), and / or

[0067] -The amino acid sequence listed in SEQ ID NO:25 (p756-765),

[0068] or

[0069] m) The amino acid sequence listed in SEQ ID NO:26 (p829-837).

[0070] 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

[0071] 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.

[0072] Figure 2 A comparison of the protein conservation and function of validated HLA-I epitopes and predicted HLA-binding peptide sequences against the polymerase. 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 the viral protein 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.

[0073] Figure 3The in vitro binding capacity of selected predicted HLA-binding peptide sequences. Binding of predicted HLA-binding peptide sequences is expressed as the percentage of binding to a positive control peptide (pos.ct.) with high affinity for the indicated HLA type. The mean and standard deviation of controls (black), bound peptides (>25% positive control; gray), and unbound peptides (≤25% positive control; white) are depicted. The binding capacity of six supertype representatives was assessed, and HLA*11:01 was also included, taking into account its high prevalence in the chronic hepatitis B patient population. Each of these is depicted in a separate plot (AG). As negative controls, we included known unbound peptides (neg.ct.) for each HLA type and cases where no peptide was 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, and cross-reactive bindings are summarized in Table (H). An asterisk indicates which peptides do not meet our length and conservation thresholds.

[0074] Figure 4 The binding affinity of selected predicted HLA-binding peptide sequences is compared to a computer-predicted grading score. For example... Figure 3 The 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.

[0075] 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.

[0076] Figure 6IFNγ 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.

[0077] Figure 7 The 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 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 IFNγ and TNFα in the supernatant induced by SLP restimulation was also plotted. Cytokine values ​​were calculated by subtracting the mean observed for the DMSO / unrelated peptide control samples and adding 2x the standard deviation of those controls.

[0078] Figure 8A 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

[0079] definition

[0080] 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.

[0081] 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 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.

[0082] 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 an 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.

[0083] The position numbering within HBV polymerase and HBV-X in this document refers to the common sequences listed in SEQ ID NO:28 and SEQ ID NO:30, respectively. In other words, the amino acid positions within 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.

[0084] "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).

[0085] "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.

[0086] 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).

[0087] 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).

[0088] 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 sequence (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 that is 35 amino acids or longer. 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.

[0089] 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.

[0090] 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.

[0091] 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".

[0092] Other aspects and embodiments of the present invention

[0093] 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:

[0094] a) At least 10 consecutive amino acids from the region of HBV-X at positions 57 to 78, preferably comprising:

[0095] -The amino acid sequence (x70-78) listed in SEQ ID NO:1, and / or

[0096] -The amino acid sequence (x67-75) listed in SEQ ID NO:2, and / or

[0097] -The amino acid sequence (x62-73) listed in SEQ ID NO:3, and / or

[0098] -The amino acid sequence (x58-66) listed in SEQ ID NO:4, and / or

[0099] -The amino acid sequence (x57-66) listed in SEQ ID NO:5,

[0100] or

[0101] b) At least 11 consecutive amino acids from the region of HBV-X at positions 103 to 120, preferably comprising:

[0102] -The amino acid sequence (x103-111) listed in SEQ ID NO:6, and / or

[0103] -The amino acid sequence (x104-113) listed in SEQ ID NO:7, and / or

[0104] -The amino acid sequence (x105-113) listed in SEQ ID NO:8, and / or

[0105] -The amino acid sequence (x110-120) listed in SEQ ID NO:9,

[0106] or

[0107] c) The amino acid sequence (x132-140) listed in SEQ ID NO:10,

[0108] or

[0109] d) The amino acid sequence listed in SEQ ID NO:11 (p124-133),

[0110] or

[0111] e) The amino acid sequence listed in SEQ ID NO:12 (p164-173),

[0112] or

[0113] f) The amino acid sequence listed in SEQ ID NO:13 (p275-283),

[0114] or

[0115] g) At least 10 consecutive amino acids from the region at positions 403 to 415 of the HBV polymerase, preferably comprising:

[0116] -The amino acid sequence (p403-412) listed in SEQ ID NO:14, and / or

[0117] -The amino acid sequence (p404-412) listed in SEQ ID NO:15, and / or

[0118] -The amino acid sequence listed in SEQ ID NO:16 (p407-415),

[0119] or

[0120] h) At least nine consecutive amino acids from the region at positions 509 to 523 of the HBV polymerase, preferably comprising:

[0121] -The amino acid sequence listed in SEQ ID NO:17 (p509-517), and / or

[0122] -The amino acid sequence listed in SEQ ID NO:18 (p515-523),

[0123] or

[0124] i) At least 10 consecutive amino acids from the region at positions 649 to 658 of the HBV polymerase, comprising:

[0125] -The amino acid sequence listed in SEQ ID NO:19 (p649-658), and / or

[0126] -The amino acid sequence (p650-658) listed in SEQ ID NO:20,

[0127] or

[0128] j) At least 10 consecutive amino acids from the region at positions 693 to 706 of the HBV polymerase, preferably comprising:

[0129] -The amino acid sequence listed in SEQ ID NO:21 (p693-701), and / or

[0130] -The amino acid sequence listed in SEQ ID NO:22 (p697-706),

[0131] or

[0132] k) The amino acid sequence listed in SEQ ID NO:23 (p723-731),

[0133] or

[0134] l) At least 10 consecutive amino acids from the region at positions 755 to 765 of the HBV polymerase, comprising:

[0135] -The amino acid sequence listed in SEQ ID NO:24 (p755-764), and / or

[0136] -The amino acid sequence listed in SEQ ID NO:25 (p756-765),

[0137] or

[0138] m) The amino acid sequence listed in SEQ ID NO:26 (p829-837).

[0139] In one embodiment, the peptide consists of the fragment of the HBV protein.

[0140] 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.

[0141] 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.

[0142] 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:

[0143] -The amino acid sequence (x70-78) listed in SEQ ID NO:1,

[0144] -The amino acid sequence (x67-75) listed in SEQ ID NO:2,

[0145] -The amino acid sequence (x62-73) listed in SEQ ID NO:3,

[0146] -The amino acid sequence (x58-66) listed in SEQ ID NO:4,

[0147] -The amino acid sequence (x57-66) listed in SEQ ID NO:5,

[0148] -The amino acid sequence (x103-111) listed in SEQ ID NO:6,

[0149] -The amino acid sequence (x104-113) listed in SEQ ID NO:7,

[0150] -The amino acid sequence (x105-113) listed in SEQ ID NO:8,

[0151] -The amino acid sequence (x110-120) listed in SEQ ID NO:9,

[0152] -The amino acid sequence (x132-140) listed in SEQ ID NO:10,

[0153] -The amino acid sequence (p124-133) listed in SEQ ID NO:11,

[0154] -The amino acid sequence listed in SEQ ID NO:12 (p164-173),

[0155] -The amino acid sequence listed in SEQ ID NO:13 (p275-283),

[0156] -The amino acid sequence listed in SEQ ID NO:14 (p403-412),

[0157] -The amino acid sequence listed in SEQ ID NO:15 (p404-412),

[0158] -The amino acid sequence listed in SEQ ID NO:16 (p407-415),

[0159] -The amino acid sequence listed in SEQ ID NO:17 (p509-517),

[0160] -The amino acid sequence listed in SEQ ID NO:18 (p515-523),

[0161] -The amino acid sequence listed in SEQ ID NO:19 (p649-658),

[0162] -The amino acid sequence (p650-658) listed in SEQ ID NO:20,

[0163] -The amino acid sequence listed in SEQ ID NO:21 (p693-701),

[0164] -The amino acid sequence listed in SEQ ID NO:22 (p697-706),

[0165] -The amino acid sequence listed in SEQ ID NO:23 (p723-731),

[0166] -The amino acid sequence listed in SEQ ID NO:24 (p755-764),

[0167] -The amino acid sequence listed in SEQ ID NO:25 (p756-765), and

[0168] -The amino acid sequence listed in SEQ ID NO:26 (p829-837),

[0169] Preferably, the segment comprises:

[0170] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:2 (x67-75), or

[0171] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:3 (x62-73), or

[0172] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:4 (x58-66), or

[0173] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:5 (x57-66), or

[0174] -The amino acid sequences listed in SEQ ID NO:2 (x67-75) and SEQ ID NO:3 (x62-73), or

[0175] -The amino acid sequences listed in SEQ ID NO:2 (x67-75) and SEQ ID NO:4 (x58-66), or

[0176] -The amino acid sequences listed in SEQ ID NO:2 (x67-75) and SEQ ID NO:5 (x57-66), or

[0177] -The amino acid sequences listed in SEQ ID NO:3 (x62-73) and SEQ ID NO:4 (x58-66), or

[0178] - The amino acid sequences listed in SEQ ID NO:3 (x62-73) and SEQ ID NO:5 (x57-66) or

[0179] - The amino acid sequences listed in SEQ ID NO:6 (x103-111) and SEQ ID NO:7 (x104-113), or

[0180] -The amino acid sequences listed in SEQ ID NO:6 (x103-111) and SEQ ID NO:8 (x105-113), or

[0181] - The amino acid sequences listed in SEQ ID NO:6 (x103-111) and SEQ ID NO:9 (x110-120), or

[0182] - The amino acid sequences listed in SEQ ID NO:7 (x104-113) and SEQ ID NO:9 (x110-120), or

[0183] - The amino acid sequences listed in SEQ ID NO:8 (x105-113) and SEQ ID NO:9 (x110-120), or

[0184] - The amino acid sequences listed in SEQ ID NO:14 (p403-412) and SEQ ID NO:16 (p407-415), or

[0185] - The amino acid sequences listed in SEQ ID NO:15 (p404-412) and SEQ ID NO:16 (p407-415).

[0186] 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:

[0187] -The amino acid sequence (x70-78) listed in SEQ ID NO:1, and / or

[0188] -The amino acid sequence (x67-75) listed in SEQ ID NO:2, and / or

[0189] -The amino acid sequence (x62-73) listed in SEQ ID NO:3, and / or

[0190] -The amino acid sequence (x58-66) listed in SEQ ID NO:4, and / or

[0191] -The amino acid sequence (x57-66) listed in SEQ ID NO:5.

[0192] 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:

[0193] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:2 (x67-75), or

[0194] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:3 (x62-73), or

[0195] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:4 (x58-66), or

[0196] -The amino acid sequences listed in SEQ ID NO:1 (x70-78) and SEQ ID NO:5 (x57-66), or

[0197] -The amino acid sequences listed in SEQ ID NO:2 (x67-75) and SEQ ID NO:3 (x62-73), or

[0198] -The amino acid sequences listed in SEQ ID NO:2 (x67-75) and SEQ ID NO:4 (x58-66), or

[0199] -The amino acid sequences listed in SEQ ID NO:2 (x67-75) and SEQ ID NO:5 (x57-66), or

[0200] -The amino acid sequences listed in SEQ ID NO:3 (x62-73) and SEQ ID NO:4 (x58-66), or

[0201] - The amino acid sequences listed in SEQ ID NO:4 (x62-73) and SEQ ID NO:5 (x57-66).

[0202] 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.).

[0203] 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.).

[0204] In even other embodiments:

[0205] - 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

[0206] - 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.

[0207] 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.

[0208] 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:

[0209] -The amino acid sequence (x103-111) listed in SEQ ID NO:6, and / or

[0210] -The amino acid sequence (x104-113) listed in SEQ ID NO:7, and / or

[0211] -The amino acid sequence (x105-113) listed in SEQ ID NO:8, and / or

[0212] - The amino acid sequence (x110-120) listed in SEQ ID NO:9.

[0213] 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:

[0214] - The amino acid sequences listed in SEQ ID NO:6 (x103-111) and SEQ ID NO:7 (x104-113), or

[0215] -The amino acid sequences listed in SEQ ID NO:6 (x103-111) and SEQ ID NO:8 (x105-113), or

[0216] - The amino acid sequences listed in SEQ ID NO:6 (x103-111) and SEQ ID NO:9 (x110-120), or

[0217] - The amino acid sequences listed in SEQ ID NO:7 (x104-113) and SEQ ID NO:9 (x110-120), or

[0218] - The amino acid sequences listed in SEQ ID NO:8 (x105-113) and SEQ ID NO:9 (x110-120).

[0219] 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.).

[0220] 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.).

[0221] 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.

[0222] 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.

[0223] 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.).

[0224] 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.).

[0225] In even other embodiments:

[0226] - 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

[0227] - 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.

[0228] 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.

[0229] 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.

[0230] 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.).

[0231] 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.

[0232] 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.).

[0233] 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.).

[0234] 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.

[0235] 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.).

[0236] 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:

[0237] -The amino acid sequence (p403-412) listed in SEQ ID NO:14, and / or

[0238] -The amino acid sequence (p404-412) listed in SEQ ID NO:15, and / or

[0239] - The amino acid sequence listed in SEQ ID NO:16 (p407-415).

[0240] 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:

[0241] - The amino acid sequences listed in SEQ ID NO:14 (p403-412) and SEQ ID NO:16 (p407-415), or

[0242] - The amino acid sequences listed in SEQ ID NO:15 (p404-412) and SEQ ID NO:16 (p407-415).

[0243] 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.).

[0244] 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.).

[0245] In even other embodiments:

[0246] - The N-terminal amino acid of this fragment is either the amino acid at position 390 on the HBV polymerase or an amino acid at a position further C-terminus than 390 on the HBV polymerase, and

[0247] - 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.

[0248] 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.

[0249] 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:

[0250] -The amino acid sequence listed in SEQ ID NO:17 (p509-517), and / or

[0251] -The amino acid sequence listed in SEQ ID NO:18 (p515-523).

[0252] 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.).

[0253] 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.).

[0254] 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:

[0255] -The amino acid sequence listed in SEQ ID NO:19 (p649-658), and / or

[0256] - The amino acid sequence listed in SEQ ID NO:20 (p650-658).

[0257] 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.).

[0258] 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.).

[0259] In even other embodiments:

[0260] - 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

[0261] - 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.

[0262] 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.

[0263] 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:

[0264] -The amino acid sequence listed in SEQ ID NO:21 (p693-701), and / or

[0265] -The amino acid sequence listed in SEQ ID NO:22 (p697-706).

[0266] 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.).

[0267] 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.

[0268] 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.).

[0269] 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:

[0270] -The amino acid sequence listed in SEQ ID NO:24 (p755-764), and / or

[0271] -The amino acid sequence listed in SEQ ID NO:25 (p756-765).

[0272] 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.

[0273] 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.).

[0274] In another embodiment, the peptide of the present invention comprises or consists of a sequence selected from the group consisting of:

[0275] SEQ ID NO:31:HLSLRGLPVCAFSSAGPCALRFTSA(SLP1),

[0276] SEQ ID NO:32:LSAMSTTDLEAYFKDCLFKDWEELG(SLP2),

[0277] SEQ ID NO:33:ASSSSSCLHQSAVRKAAYSHLSTSK(SLP3),

[0278] SEQ ID NO:34: RKLHLYSHPIILGFRKIPMGVGLSP(SLP4),

[0279] SEQ ID NO:35: GFAAPFTQCGYPALMPLYACIQAKQA(SLP5),

[0280] SEQ ID NO:36: ARQRPGLCQVFADATPTGWGLAIGH(SLP6) and

[0281] SEQ ID NO: 37: SPSVPSHLPDRVHFASPLHVAWRPP (SLP7).

[0282] In another embodiment, the peptide of the present invention comprises or consists of a sequence selected from the group consisting of:

[0283] SEQ ID NO:121: KLHLYSHPIILGFRKIPMGVGLSPFLL(SLP8),

[0284] SEQ ID NO: 122: GLLGFAAPFTQCGYPALMPLYACIQAKQAFT (SLP9), and

[0285] SEQ ID NO: 123: ARQRPGLCQVFADATPTGWGLAIGHQRMR(SLP10).

[0286] 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.

[0287] In other embodiments, 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 of WO 15187009, i.e.

[0288] SEQ ID NO:38: VVNEKRRLKLIMPARFYPTHTKYLPLDKGIKPYY (SEQ ID NO:53 in WO 15187009),

[0289] SEQ ID NO:39: YPTHTKYLPLDKGIKPYYPDQVVNHYFQTRHYL (SEQ ID NO:54 in WO 15187009),

[0290] SEQ ID NO:40: TAESRLVVDFSQFSRGISRVSWPKFAVPNLQSL (SEQ ID NO:59 in WO 15187009),

[0291] SEQ ID NO: 41: QMRRGTFVAPLPIHTAELLAACFARSRSGAKL (SEQ ID NO: 70 in WO 15187009),

[0292] SEQ ID NO:42: ALPSPSPSAVPADHGAHLSLRGLPVCAFSSAGP (SEQ ID NO:76 in WO 15187009), or

[0293] SEQ ID NO:43: LEAYFKDCVFKDWEELGEEIIRLKVFVLGGCRHKL (SEQ ID NO:79 in WO 15187009).

[0294] 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.

[0295] 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 determined by the following assays:

[0296] - 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

[0297] - Inhibition of antigen-associated infection proliferation, or detectable reduction of antigen-expressing cells, or decreased cell viability of antigen-expressing cells; and / or

[0298] - Induction or increased induction of cell death in antigen-expressing cells; and / or

[0299] - Inhibition or prevention of increased antigen-expressing cells; and / or

[0300] - 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

[0301] - Reduction in hepatocytes possessing integrated viral DNA and / or covalently closed circular DNA (cccDNA).

[0302] - A reduction in the size of the lesion; and / or

[0303] - A reduction in tumor size; and / or

[0304] - Increased survival rates (e.g., progression-free survival, overall survival).

[0305] 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.

[0306] In a preferred embodiment, the peptide used in this invention does not have a cysteine ​​residue at its N-terminus or C-terminus.

[0307] Furthermore, in another preferred embodiment, the peptides used in this invention do not contain more than two cysteine ​​residues.

[0308] In another preferred embodiment, the peptides used in this invention do not contain more than three methionines.

[0309] In another preferred embodiment, the peptide used in this invention does not have glutamine at the N-terminus.

[0310] 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.

[0311] Typically, peptides are synthesized. This can be done through solid-phase peptide synthesis or by any other suitable method.

[0312] 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.

[0313] In another aspect, the present invention relates to an immunogenic composition comprising:

[0314] - Peptides as described herein or polynucleotides as described herein, and

[0315] - A pharmaceutically acceptable carrier

[0316] Optionally, it may further include an adjuvant.

[0317] 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.

[0318] In another aspect, the present invention relates to a recombinant virus comprising the polynucleotides described in the present invention.

[0319] 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.

[0320] 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 being stored at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the total area % of the peaks that do not represent the desired peptide product in the UV spectrum is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% compared to its original state.

[0321] 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.

[0322] In one embodiment, the immunogenic composition comprises or consists of a mixture of dried or lyophilized peptides to be applied together.

[0323] 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.

[0324] For example, the composition can be prepared by a method including the following steps:

[0325] a) Provide vials containing dried, preferably lyophilized, peptides;

[0326] b) Thaw these peptides, preferably for about 5-30 minutes;

[0327] c) Add the reconstituted composition to a vial containing these peptides, preferably a non-vortex vial;

[0328] d) Mixing is permitted, preferably for about 0.5-5 minutes; and

[0329] e) Rotate until a clear solution is obtained, preferably for about 1-3 minutes.

[0330] Preferably, steps b) to e) are performed at room temperature.

[0331] Preferably, in methods for prevention and / or treatment (preferably methods for treatment and / or prevention as defined herein), the vial contains an injection volume of 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).

[0332] 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:

[0333] a. is a monoglyceride, diglyceride, or triglyceride, preferably an ethoxylated triglyceride, and / or

[0334] 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).

[0335] 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).

[0336] 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).

[0337] 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 51VG (Seppic)).

[0338] 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.

[0339] adjuvant

[0340] 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.

[0341] 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, SAF-m, and SAF-1) [Preparation of Syntex Adjuvant Formulations (SAF, SAF-m, and SAF-1)]: Vaccine Adjuvants, Volume 42 of the series Methods in Molecular Medicine TM Molecular medicine approach TMVolume 42 of the series, Vaccine Adjuvants] pp. 229-237, ISSN 1543-1894), Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), peanut oil-based adjuvants (e.g., Adjuvant 65), Lipovant (Byars, NE, Allison, AC, 1990. In the following Immunologic adjuvants: general properties, advantages, and limitations. [Immunologic Adjuvants: General Properties, Advantages, and Limitations]: Zola, H. (ed.), Laboratory Methods in Immunology [Immunologic Laboratory Methods]. pp. 39-51), ASO4 (A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dream becomes real [Vaccine Adjuvants: Dreams Come True] Hum. Vaccine [Human Vaccines], 4(5), 2008, pp. 347-349), Montanide adjuvant, which is based on purified squalene and squalene emulsified with high-purity mannitol monooleate (e.g., Montanide ISA 25VG, 28VG, 35VG, 50V, 50V2, 51VG, 61VG, 70VG, 70M VG, 71VG, 720VG, 760VG, 763A VG, 775VG, 780VG, 201VG, 206VG, ​​207VG). More preferably, the oil-based adjuvant is Montanide ISA 51VG (Sebik), which is a mixture of Drakeol VR and mannitol monooleate.

[0342] 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, Montanide ISA-51, and Montanide ISA 720 (an adjuvant manufactured by Seppic, France). RIG-I proteins are known to be activated by dsRNA, similar to TLR3 (Kato et al., (2005) Immunity, 1:19-28).

[0343] 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 Pam3cys and / or their derivatives may optionally be covalently linked to one or more peptide antigens.

[0344] 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.

[0345] Further preferred adjuvants are those selected from the group consisting of: 1018ISS, 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 adjuvants have been integrated), OK-432, OM-174, OM-197-MP-EC, ONTAK, Vector systems, PLGA microparticles, SRL172, virions and other virus-like particles, Pam3Cys-GDPKHPKSF, YF-17D, VEGF traps, R848, β-glucan, Aquila's QS21 stimulon, vadimezan, AsA404 (DMXAA), STING (an IFN gene stimulator), agonists (e.g., c-di-GMP VacciGrade) TM PCI, NKT (natural killer T cell) agonists (e.g., α-galactosidase ceramide or α-GalCer), (Curevac), retinoic acid-inducible protein I ligands (e.g., 3pRNA or 5'-triphosphate RNA).

[0346] Treatment methods and uses

[0347] 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.

[0348] 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.

[0349] Therefore, in another aspect, the present invention relates to peptides, polynucleotides, or immunogenic compositions according to the present invention, which are used as pharmaceuticals.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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:

[0354] - 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

[0355] - Inhibition of antigen-associated infection proliferation, or detectable reduction of antigen-expressing cells, or decreased cell viability of antigen-expressing cells; and / or

[0356] - Induction or increased induction of cell death in antigen-expressing cells; and / or

[0357] - Inhibition or prevention of increased antigen-expressing cells; and / or

[0358] - A decrease in viral load (precancerous stage of disease); and / or

[0359] - A reduction in the size of the lesion; and / or

[0360] - A reduction in tumor size; and / or

[0361] - Increased survival rate (e.g., PFS, OS).

[0362] combination

[0363] 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.

[0364] 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).

[0365] 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.

[0366] 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 human subjects to be treated. Preferred class I HLA-binding peptide sequences in the long peptides described according to the present invention are HLA-binding peptide sequences capable of binding to the following: class I HLA allele supertypes 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 combinations HLA-A*01 / A*03 and HLA-A*01 / A*24, and their respective subtypes (Sidney et al. 2008 BMC). Immunology [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-A30 01. HLA-A3002, HLA-A3101, HLA-A3201, HLA-A3303, HLA-A6801, HLA-A6802, HL A-A7401, HLA-B0702, HLA-B0801, HLA-B1301, HLA-B1302, HLA-B1402, HLA-B150 1. HLA-B1502, 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 human subjects to be treated.

[0367] 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:

[0368] 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),

[0369] 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),

[0370] 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),

[0371] 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),

[0372] 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*540, 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*5504B*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),

[0373] 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).

[0374] 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:

[0375] -The peptide as described in claim 1a) and the peptide as described in claim 1b), or

[0376] - The peptide as described in claim 1a) and the peptide as described in claim 1c), or

[0377] -The peptide as described in claim 1a) and the peptide as described in claim 1d), or

[0378] -The peptide as described in claim 1a) and the peptide as described in claim 1e), or

[0379] - The peptide as described in claim 1a) and the peptide as described in claim 1f), or

[0380] - The peptide as described in claim 1a) and the peptide as described in claim 1g), or

[0381] -The peptide as described in claim 1a) and the peptide as described in claim 1h), or

[0382] -The peptide as described in claim 1a) and the peptide as described in claim 1i), or

[0383] - The peptide as described in claim 1a) and the peptide as described in claim 1j), or

[0384] - The peptide as described in claim 1a) and the peptide as described in claim 1k), or

[0385] -The peptide as described in claim 1a) and the peptide as described in claim 11), or

[0386] -The peptide as described in claim 1a) and the peptide as described in claim 1m), or

[0387] -The peptide as described in claim 1b) and the peptide as described in claim 1c), or

[0388] -The peptide as described in claim 1b) and the peptide as described in claim 1d), or

[0389] - The peptide as described in claim 1b) and the peptide as described in claim 1e), or

[0390] -The peptide as described in claim 1b) and the peptide as described in claim 1f), or

[0391] - The peptide as described in claim 1b) and the peptide as described in claim 1g), or

[0392] -The peptide as described in claim 1b) and the peptide as described in claim 1h), or

[0393] - The peptide as described in claim 1b) and the peptide as described in claim 1i), or

[0394] - The peptide as described in claim 1b) and the peptide as described in claim 1j), or

[0395] - The peptide as described in claim 1b) and the peptide as described in claim 1k), or

[0396] -The peptide as described in claim 1b) and the peptide as described in claim 11), or

[0397] -The peptide as described in claim 1b) and the peptide as described in claim 1m), or

[0398] -The peptide as described in claim 1c) and the peptide as described in claim 1d), or

[0399] -The peptide as described in claim 1c) and the peptide as described in claim 1e), or

[0400] -The peptide as described in claim 1c) and the peptide as described in claim 1f), or

[0401] - The peptide as described in claim 1c) and the peptide as described in claim 1g), or

[0402] -The peptide as described in claim 1c) and the peptide as described in claim 1h), or

[0403] -The peptide as described in claim 1c) and the peptide as described in claim 1i), or

[0404] -The peptide as described in claim 1c) and the peptide as described in claim 1j), or

[0405] - The peptide as described in claim 1c) and the peptide as described in claim 1k), or

[0406] - The peptide as described in claim 1c) and the peptide as described in claim 11), or

[0407] -The peptide as described in claim 1c) and the peptide as described in claim 1m), or

[0408] -The peptide as described in claim 1d) and the peptide as described in claim 1e), or

[0409] -The peptide as described in claim 1d) and the peptide as described in claim 1f), or

[0410] -The peptide as described in claim 1d) and the peptide as described in claim 1g), or

[0411] -The peptide as described in claim 1d) and the peptide as described in claim 1h), or

[0412] -The peptide as described in claim 1d) and the peptide as described in claim 1i), or

[0413] -The peptide as described in claim 1d) and the peptide as described in claim 1j), or

[0414] -The peptide as described in claim 1d) and the peptide as described in claim 1k), or

[0415] -The peptide as described in claim 1d) and the peptide as described in claim 11), or

[0416] -The peptide as described in claim 1d) and the peptide as described in claim 1m), or

[0417] - The peptide as described in claim 1e) and the peptide as described in claim 1f), or

[0418] - The peptide as described in claim 1e) and the peptide as described in claim 1g), or

[0419] -The peptide as described in claim 1e) and the peptide as described in claim 1h), or

[0420] - The peptide as described in claim 1e) and the peptide as described in claim 1i), or

[0421] - The peptide as described in claim 1e) and the peptide as described in claim 1j), or

[0422] - The peptide as described in claim 1e) and the peptide as described in claim 1k), or

[0423] -The peptide as described in claim 1e) and the peptide as described in claim 11), or

[0424] - The peptide as described in claim 1e) and the peptide as described in claim 1m), or

[0425] - The peptide as described in claim 1f) and the peptide as described in claim 1g), or

[0426] -The peptide as described in claim 1f) and the peptide as described in claim 1h), or

[0427] -The peptide as described in claim 1f) and the peptide as described in claim 1i), or

[0428] -The peptide as described in claim 1f) and the peptide as described in claim 1j), or

[0429] - The peptide as described in claim 1f) and the peptide as described in claim 1k), or

[0430] -The peptide as described in claim 1f) and the peptide as described in claim 1l), or

[0431] -The peptide as described in claim 1f) and the peptide as described in claim 1m), or

[0432] -The peptide as described in claim 1g) and the peptide as described in claim 1h), or

[0433] - The peptide as described in claim 1g) and the peptide as described in claim 1i), or

[0434] - The peptide as described in claim 1g) and the peptide as described in claim 1j), or

[0435] - The peptide as described in claim 1g) and the peptide as described in claim 1k), or

[0436] - The peptide as described in claim 1g) and the peptide as described in claim 1l), or

[0437] - The peptide as described in claim 1g) and the peptide as described in claim 1m), or

[0438] -The peptide as described in claim 1h) and the peptide as described in claim 1i), or

[0439] -The peptide as described in claim 1h) and the peptide as described in claim 1j), or

[0440] -The peptide as described in claim 1h) and the peptide as described in claim 1k), or

[0441] -The peptide as described in claim 1h) and the peptide as described in claim 1l), or

[0442] -The peptide as described in claim 1h) and the peptide as described in claim 1m), or

[0443] -The peptide as described in claim 1i) and the peptide as described in claim 1j), or

[0444] -The peptide as described in claim 1i) and the peptide as described in claim 1k), or

[0445] -The peptide as described in claim 1i) and the peptide as described in claim 1l), or

[0446] -The peptide as described in claim 1i) and the peptide as described in claim 1m), or

[0447] -The peptide as described in claim 1j) and the peptide as described in claim 1k), or

[0448] -The peptide as described in claim 1j) and the peptide as described in claim 1l), or

[0449] -The peptide as described in claim 1j) and the peptide as described in claim 1m), or

[0450] -The peptide as described in claim 1k) and the peptide as described in claim 1l), or

[0451] -The peptide as described in claim 1k) and the peptide as described in claim 1m), or

[0452] -The peptide as described in claim 1l) and the peptide as described in claim 1m), or

[0453] - A polynucleotide sequence comprising one or more of the above combinations encoding a peptide.

[0454] 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.

[0455] The dosage of a drug can be an effective amount or a portion thereof. "Effective amount" should 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 practicing 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 responsible for 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.

[0456] 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 range: from 0.1 micrograms to 20 mg, such as about 0.1 micrograms, 0.5 micrograms, 1 microgram, 5 micrograms, 10 micrograms, 15 micrograms, 20 micrograms, 30 micrograms, 40 micrograms, 50 micrograms, 60 micrograms, 70 micrograms, 80 micrograms, 90 micrograms, 100 micrograms, 150 micrograms, 200 micrograms, 250 micrograms, 300 micrograms, 350 micrograms, 400 micrograms, 450 micrograms, 500 micrograms, 650 micrograms, etc. 0 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, 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 values. Preferred drug 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.

[0457] In one embodiment, the immunogenic composition used in this invention is administered at a dose of between 1 microgram and 300 micrograms for each peptide (e.g., between 50 micrograms and 150 micrograms, such as about 100 micrograms).

[0458] 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) 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 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).

[0459] 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:

[0460] (i) Administer antiviral (e.g., NA) treatment to patients infected with HBV to reduce viral load.

[0461] (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

[0462] (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, IFNγELISpot 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 (pre- 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). Preferably, adequate monitoring of viral load is performed to evaluate vaccine efficacy and determine subsequent (combination) therapies.

[0463] 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 antigen-loaded activated dendritic cells (DCs)) 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.

[0464] Therefore, in another aspect, the present invention relates to peptides according to the present 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.

[0465] Similarly, in another embodiment, the method of the present invention relates to 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.

[0466] 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.

[0467] All patents and references cited in this specification are incorporated herein by reference in their entirety.

[0468] Example

[0469] Example 1: Comparison of common sequences and functional domains between HBV-X and HBV polymerase

[0470] HBV sequences for all genotypes of HBV-X (n=8127) and HBV polymerase (n=7489) were obtained, 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.

[0471] The common sequence (SEQ ID NO:28) is generated by combining all the dominant amino acids of HBV polymerase:

[0472]

[0473] The common sequence of HBV-X obtained was determined to be (SEQ ID NO:30):

[0474]

[0475] 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.

[0476] Table 1: References for experimental evidence of the functional domains and essential amino acids of HBV-x in reports or reviews.

[0477]

[0478]

[0479] Table 2: References for reporting or reviewing experimental evidence on the functional domains and essential amino acids of HBV polymerase.

[0480]

[0481]

[0482]

[0483]

[0484] Example 2: Prediction of novel class I HLA-binding peptides derived from HBV-x and HBV polymerase

[0485] We set out to identify novel peptides that can bind to at least one of six HLA supertypes prevalent in Caucasians, Africans, or Asians, and whose binding can be readily confirmed by in vitro assays (i.e., supertypes HLA-A*01, A*02, A*03, A*24, B*07, and B*08). We first used the established computer prediction tool NetMHCpan (Nielsen and Andreatta 2016, Genome Med [Genomic Medicine] 8:33) to predict bindings spanning 8–14 amino acids against representative supertype HLA types, in order to construct a frequency distribution of the predicted bindings. 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 ).

[0486] 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 9mer), predicted HLA binding strength, conservation, and functional importance of the included amino acids. Not all 96 potential conjugates consistently met all criteria. For HLA-A*01 and HLA-A*24, the number of predicted conjugates against HBx was unsatisfactory, failing to maintain our stringent 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 (See Example 3). This includes c123-130, as this is the only HBV-derived epitope associated with HLA-B*08 registered in Hepitopes. 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 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%.

[0487] Example 3: In vitro binding capacity of selected peptides derived from HBx and polymerase

[0488] 3.1 Method

[0489] 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, the 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 by adding 2% (w / v) oxalate dihydrate stop solution (Sanquin Reagents) after 8 min (incubated at room temperature) and read at 414 nm on a Thermo Electron Multiskan Ascent ELISA reader. Each peptide was exchanged twice independently. Each exchange mixture was measured in duplicate in a class I HLA ELISA. 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.

[0490] Table 3: Reference peptides for in vitro binding assays, categorized by HLA type.

[0491]

[0492] 3.2 Results

[0493] 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. Both HLA-A*11:01 and HLA-A*03:01 were tested as members of the HLA-A*03 supertype because many HBV-infected patients are Asian, and HLA-A*11:01 is more prevalent in this population than the supertype-representative HLA-A*03:01, which is more prevalent in Caucasians (Chang et al. 2013 Eur J Immunol 43:1109).

[0494] 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, HBx and Pol-derived conjugates were identified for each HLA supertype tested. 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 (A closed underscore) is not always confirmed.

[0495] 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 as strongly binding (low-scoring in computer predictions) do not show binding above the threshold in in vitro assays. Figure 4 ).

[0496] Table 4 compares the predicted score (grade) of the peptide for each HLA type test with the assay result (binding %).

[0497] Table 4:

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509] Example 4: Immunogenicity of selected HLA-binding peptides

[0510] 4.1 Methods

[0511] 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).

[0512] Table 5: HLA-I type of HBV-regressed donors used for immunogenicity testing of HLA conjugates:

[0513]

[0514] *Available only with 2-digit HLA types

[0515] 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 five target peptides. After 14 days, 200,000 cells were restimulated with the target peptide at 10 μg / ml / peptide at 37°C for 48 hours, in triplicate. The restimulated supernatant 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 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 (average of triplicate) were calculated from the OD value after subtracting the background. HLA conjugates with an average OD value at least equal to the average of the DMSO control plus 2x SD were quantified.

[0516] 4.2 Results

[0517] 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 Importantly, four additional Pol-derived epitopes elicited a response in HLA-type-negative donors; these epitopes had been 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.

[0518] Example 5:

[0519] 5.1 Method

[0520] 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.

[0521] 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) the presence of 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.

[0522] Using solid-phase Fmoc / t Bu 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 for 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 a mixture of acetonitrile (ACN) and TFA or an H2O-based mixture of acetic acid and lyophilized overnight. Following HPLC purification, selected purified fractions were pooled 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 peptides 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.

[0523] 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 20–24 hours of incubation, an IFNγ detection antibody (0.3 μg / ml, Mab-7-B6-1-Biotin, Mabtech) was added, followed by streptavidin-ALP (1 μg / ml, Mabtech). Spots were visualized by adding BCIP / NBT-plus substrate (100 μL / well, Antibody Technologies) 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.

[0524] 5.2 Results

[0525] 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 ).

[0526] Table 6:

[0527]

[0528]

[0529] 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.

[0530] 6.1 Method

[0531] 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 μM / 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 μM / 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 from 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) + 2x SD (DMSO and unrelated peptides).

[0532] 6.2 Results

[0533] 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 demonstrated by the presence of increased CD69 in response to SLPs in both cell types. Figure 7This expansion and activation of donor T cells also produced 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. sequence list <110> ISA Pharmaceutical Co., Ltd. Erasmus University Medical Center, Rotterdam <120> Treatment of Hepatitis B Virus-Related Diseases <130> P9003 <160> 123 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x70-78 <400> 1 Ala Leu Arg Phe Thr Ser Ala Arg Arg 1 5 <210> 2 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x67-75 <400> 2 Gly Pro Cys Ala Leu Arg Phe Thr Ser 1 5 <210> 3 <211> 12 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x62-73 <400> 3 Ala Phe Ser Ser Ala Gly Pro Cys Ala Leu Arg Phe 1 5 10 <210> 4 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x58-66 <400> 4 Leu Pro Val Cys Ala Phe Ser Ser Ala 1 5 <210> 5 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x57-66 <400> 5 Gly Leu Pro Val Cys Ala Phe Ser Ser Ala 1 5 10 <210> 6 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x103-111 <400> 6 Met Ser Thr Thr Asp Leu Glu Ala Tyr 1 5 <210> 7 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x104-113 <400> 7 Ser Thr Thr Asp Leu Glu Ala Tyr Phe Lys 1 5 10 <210> 8 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x105-113 <400> 8 Thr Thr Asp Leu Glu Ala Tyr Phe Lys 1 5 <210> 9 <211> 11 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x110-120 <400> 9 Ala Tyr Phe Lys Asp Cys Val Phe Lys Asp Trp 1 5 10 <210> 10 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x132-140 <400> 10 Phe Val Leu Gly Gly Cys Arg His Lys 1 5 <210> 11 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p124-133 <400> 11 Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr 1 5 10 <210> 12 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p164-173 <400> 12 Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr 1 5 10 <210> 13 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p275-283 <400> 13 Cys Leu His Gln Ser Ala Val Arg Lys 1 5 <210> 14 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p403-412 <400> 14 Ser Trp Pro Lys Phe Ala Val Pro Asn Leu 1 5 10 <210> 15 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p404-412 <400> 15 Trp Pro Lys Phe Ala Val Pro Asn Leu 1 5 <210> 16 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p407-415 <400> 16 Phe Ala Val Pro Asn Leu Gln Ser Leu 1 5 <210> 17 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p509-517 <400> 17 Ile Leu Gly Phe Arg Lys Ile Pro Met 1 5 <210> 18 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p515-523 <400> 18 Ile Pro Met Gly Val Gly Leu Ser Pro 1 5 <210> 19 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p649-658 <400> 19 Cys Gly Tyr Pro Ala Leu Met Pro Leu Tyr 1 5 10 <210> 20 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p650-658 <400> 20 Gly Tyr Pro Ala Leu Met Pro Leu Tyr 1 5 <210> twenty one <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p693-701 <400> twenty one Gly Leu Cys Gln Val Phe Ala Asp Ala 1 5 <210> twenty two <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p697-706 <400> twenty two Val Phe Ala Asp Ala Thr Pro Thr Gly Trp 1 5 10 <210> twenty three <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p723-731 <400> twenty three Leu Pro Ile His Thr Ala Glu Leu Leu 1 5 <210> twenty four <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p755-764 <400> twenty four Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu 1 5 10 <210> 25 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p756-765 <400> 25 Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly 1 5 10 <210> 26 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p829-837 <400> 26 Arg Val His Phe Ala Ser Pro Leu His 1 5 <210> 27 <211> 832 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> HBV polymerase sequence <400> 27 Met Pro Leu Ser Tyr Gln His Phe Arg Arg Leu Leu Leu Leu Asp Asp 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser 50 55 60 Ser Thr Val Pro Val Phe Asn Pro His Trp Lys Thr Pro Ser Phe Pro 65 70 75 80 Asn Ile His Leu His Gln Asp Ile Ile Lys Lys Cys Glu Gln Phe Val 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Gln Leu Ile Met Pro 100 105 110 Ala Arg Phe Tyr Pro Lys Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Leu Val Asn His Tyr Phe Gln Thr 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg 145 150 155 160 Glu Thr Thr His Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 165 170 175 Gln Asp Leu Gln His Gly Ala Glu Ser Phe His Gln Gln Ser Ser Gly 180 185 190 Ile Leu Ser Arg Pro Pro Val Gly Ser Ser Leu Gln Ser Lys His Arg 195 200 205 Lys Ser Arg Leu Gly Leu Gln Ser Gln Gln Gly His Leu Ala Arg Arg 210 215 220 Gln Gln Gly Arg Ser Trp Ser Ile Arg Ala Gly Phe His Pro Thr Ala 225 230 235 240 Arg Arg Pro Phe Gly Val Glu Pro Ser Gly Ser Gly His Thr Thr Asn 245 250 255 Phe Ala Ser Lys Ser Ala Ser Cys Leu His Gln Ser Pro Val Arg Lys 260 265 270 Ala Ala Tyr Pro Ala Val Ser Thr Phe Glu Lys His Ser Ser Ser Gly 275 280 285 His Ala Val Glu Phe His Asn Leu Pro Pro Asn Ser Ala Arg Ser Gln 290 295 300 Ser Glu Arg Pro Val Phe Pro Cys Trp Trp Leu Gln Phe Arg Asn Ser 305 310 315 320 Lys Pro Cys Ser Asp Tyr Cys Leu Ser Leu Ile Val Asn Leu Leu Glu 325 330 335 Asp Trp Gly Pro Cys Ala Glu His Gly Glu His His Ile Arg Ile Pro 340 345 350 Arg Thr Pro Ser Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn 355 360 365 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe 370 375 380 Ser Arg Gly Asn Tyr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn 385 390 395 400 Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser 405 410 415 Leu Asp Val Ser Ala Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Ala 420 425 430 Met Pro His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala 435 440 445 Arg Leu Ser Ser Asn Ser Arg Ile Leu Asn Asn Gln His Gly Thr Met 450 455 460 Pro Asp Leu His Asp Tyr Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu 465 470 475 480 Leu Leu Tyr Gln Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro 485 490 495 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro 500 505 510 Phe Leu Leu Ala Gln Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg 515 520 525 Ala Phe Pro His Cys Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu 530 535 540 Gly Ala Lys Ser Val Gln His Leu Glu Ser Leu Phe Thr Ala Val Thr 545 550 555 560 Asn Phe Leu Leu Ser Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys 565 570 575 Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Cys Tyr 580 585 590 Gly Ser Leu Pro Gln Glu His Ile Ile Gln Lys Ile Lys Glu Cys Phe 595 600 605 Arg Lys Leu Pro Ile Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg 610 615 620 Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr 625 630 635 640 Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe 645 650 655 Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn 660 665 670 Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala 675 680 685 Asp Ala Thr Pro Thr Gly Trp Gly Leu Val Met Gly His Gln Arg Met 690 695 700 Arg Gly Thr Phe Ser Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu 705 710 715 720 Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Asn Ile Ile Gly Thr 725 730 735 Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu 740 745 750 Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr 755 760 765 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu 770 775 780 Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly 785 790 795 800 Arg Thr Ser Leu Tyr Ala Asp Ser Pro Ser Val Pro Ser His Leu Pro 805 810 815 Asp Arg Val His Phe Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 820 825 830 <210> 28 <211> 843 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> HBV polymerase common sequence <400> 28 Met Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Asp 1 5 10 15 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly 20 25 30 Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val 35 40 45 Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser 50 55 60 Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro 65 70 75 80 Asp Ile His Leu Gln Glu Asp Ile Ile Asn Arg Cys Gln Gln Phe Val 85 90 95 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro 100 105 110 Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly 115 120 125 Ile Lys Pro Tyr Tyr Pro Glu His Val Val Asn His Tyr Phe Gln Thr 130 135 140 Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg 145 150 155 160 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 165 170 175 Gln Glu Leu Gln His Gly Arg Leu Val Phe Gln Thr Ser Lys Arg His 180 185 190 Gly Asp Glu Ser Phe Cys Ser Gln Ser Ser Gly Ile Leu Ser Arg Ser 195 200 205 Pro Val Gly Pro Cys Ile Gln Ser Gln Leu Lys Gln Ser Arg Leu Gly 210 215 220 Leu Gln Pro Gln Gln Gly Ser Leu Ala Arg Arg Gln Gln Gly Arg Ser 225 230 235 240 Gly Ser Ile Arg Ala Arg Val His Pro Thr Thr Arg Arg Ser Phe Gly 245 250 255 Val Glu Pro Ser Gly Ser Gly His Ile Asp Asn Ser Ala Ser Ser Ser 260 265 270 Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala Ala Tyr Ser His 275 280 285 Leu Ser Thr Ser Lys Arg Gln Ser Ser Ser Gly His Ala Val Glu Leu 290 295 300 His Asn Ile Pro Pro Ser Ser Ala Arg Ser Gln Ser Glu Gly Pro Val 305 310 315 320 Phe Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Asp 325 330 335 Tyr Cys Leu Ser His Ile Val Asn Leu Leu Glu Asp Trp Gly Pro Cys 340 345 350 Thr Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg 355 360 365 Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Thr 370 375 380 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Asn Thr 385 390 395 400 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr 405 410 415 Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala 420 425 430 Ala Phe Tyr His Leu Pro Leu His Pro Ala Ala Met Pro His Leu Leu 435 440 445 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn 450 455 460 Ser Arg Ile Ile Asn Asn Gln His Gly Thr Met Gln Asn Leu His Asp 465 470 475 480 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr 485 490 495 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Phe 500 505 510 Arg Lys Ile Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu Ala Gln 515 520 525 Phe Thr Ser Ala Ile Cys Ser Val Val Arg Arg Ala Phe Pro His Cys 530 535 540 Leu Ala Phe Ser Tyr Met Asp Asp Val Val Leu Gly Ala Lys Ser Val 545 550 555 560 Gln His Leu Glu Ser Leu Tyr Thr Ala Val Thr Asn Phe Leu Leu Ser 565 570 575 Leu Gly Ile His Leu Asn Pro Asn Lys Thr Lys Arg Trp Gly Tyr Ser 580 585 590 Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Thr Leu Pro Gln 595 600 605 Glu His Ile Val Gln Lys Ile Lys Gln Cys Phe Arg Lys Leu Pro Val 610 615 620 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Leu 625 630 635 640 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pro 645 650 655 Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr Phe Ser Pro Thr 660 665 670 Tyr Lys Ala Phe Leu Cys Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala 675 680 685 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr 690 695 700 Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg Gly Thr Phe Val 705 710 715 720 Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala 725 730 735 Arg Ser Arg Ser Gly Ala Lys Leu Ile Gly Thr Asp Asn Ser Val Val 740 745 750 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala 755 760 765 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu 770 775 780 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Tyr Arg Pro 785 790 795 800 Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Tyr 805 810 815 Ala Val Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Phe 820 825 830 Ala Ser Pro Leu His Val Ala Trp Arg Pro Pro 835 840 <210> 29 <211> 154 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> HBV-X sequence <400> 29 Met Ala Ala Arg Leu Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu 1 5 10 15 Cys Leu Arg Pro Val Gly Ala Glu Ser Cys Gly Arg Pro Phe Ser Gly 20 25 30 Ser Leu Gly Thr Leu Ser Ser Pro Ser Pro Ser Ala Val Pro Thr Asp 35 40 45 His Gly Ala His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser 50 55 60 Ser Ala Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg Met Glu 65 70 75 80 Thr Thr Val Asn Ala His Gln Ile Leu Pro Lys Val Leu His Lys Arg 85 90 95 Thr Leu Gly Leu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe 100 105 110 Lys Asp Cys Leu Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg 115 120 125 Leu Lys Val Phe Val Leu Gly Gly Cys Arg His Lys Leu Val Cys Ala 130 135 140 Pro Ala Pro Cys Asn Phe Phe Thr Ser Ala 145 150 <210> 30 <211> 154 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> HBV-X Common Sequence <400> 30 Met Ala Ala Arg Leu Cys Cys Gln Leu Asp Pro Ala Arg Asp Val Leu 1 5 10 15 Cys Leu Arg Pro Val Gly Ala Glu Ser Arg Gly Arg Pro Leu Ser Gly 20 25 30 Pro Leu Gly Thr Leu Pro Ser Pro Ser Pro Ser Ala Val Pro Ala Asp 35 40 45 His Gly Ala His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser 50 55 60 Ser Ala Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg Met Glu 65 70 75 80 Thr Thr Val Asn Ala His Gln Val Leu Pro Lys Val Leu His Lys Arg 85 90 95 Thr Leu Gly Leu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe 100 105 110 Lys Asp Cys Val Phe Lys Asp Trp Glu Glu Leu Gly Glu Glu Ile Arg 115 120 125 Leu Lys Val Phe Val Leu Gly Gly Cys Arg His Lys Leu Val Cys Ser 130 135 140 Pro Ala Pro Cys Asn Phe Phe Thr Ser Ala 145 150 <210> 31 <211> 25 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP1 <400> 31 His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser Ser Ala Gly 1 5 10 15 Pro Cys Ala Leu Arg Phe Thr Ser Ala 20 25 <210> 32 <211> 25 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP2 <400> 32 Leu Ser Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe Lys Asp Cys 1 5 10 15 Leu Phe Lys Asp Trp Glu Glu Leu Gly 20 25 <210> 33 <211> 25 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP3 <400> 33 Ala Ser Ser Ser Ser Ser Cys Leu His Gln Ser Ala Val Arg Lys Ala 1 5 10 15 Ala Tyr Ser His Leu Ser Thr Ser Lys 20 25 <210> 34 <211> 25 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP4 <400> 34 Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys 1 5 10 15 Ile Pro Met Gly Val Gly Leu Ser Pro 20 25 <210> 35 <211> 26 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP5 <400> 35 Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pro 1 5 10 15 Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala 20 25 <210> 36 <211> 25 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP6 <400> 36 Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro 1 5 10 15 Thr Gly Trp Gly Leu Ala Ile Gly His 20 25 <210> 37 <211> 25 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> SLP7 <400> 37 Ser Pro Ser Val Pro Ser His Leu Pro Asp Arg Val His Phe Ala Ser 1 5 10 15 Pro Leu His Val Ala Trp Arg Pro Pro 20 25 <210> 38 <211> 34 <212> PRT <213> Hepatitis B virus <400> 38 Val Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro Ala Arg Phe 1 5 10 15 Tyr Pro Thr His Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro 20 25 30 Tyr Tyr <210> 39 <211> 33 <212> PRT <213> Hepatitis B virus <400> 39 Tyr Pro Thr His Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro 1 5 10 15 Tyr Tyr Pro Asp Gln Val Val Asn His Tyr Phe Gln Thr Arg His Tyr 20 25 30 Leu <210> 40 <211> 33 <212> PRT <213> Hepatitis B virus <400> 40 Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly 1 5 10 15 Ile Ser Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser 20 25 30 Leu <210> 41 <211> 32 <212> PRT <213> Hepatitis B virus <400> 41 Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala 1 5 10 15 Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Leu 20 25 30 <210> 42 <211> 33 <212> PRT <213> Hepatitis B virus <400> 42 Ala Leu Pro Ser Pro Ser Pro Ser Ala Val Pro Ala Asp His Gly Ala 1 5 10 15 His Leu Ser Leu Arg Gly Leu Pro Val Cys Ala Phe Ser Ser Ala Gly 20 25 30 Pro <210> 43 <211> 34 <212> PRT <213> Hepatitis B virus <400> 43 Leu Glu Ala Tyr Phe Lys Asp Cys Val Phe Lys Asp Trp Glu Glu Leu 1 5 10 15 Gly Glu Glu Ile Arg Leu Lys Val Phe Val Leu Gly Gly Cys Arg His 20 25 30 Lys Leu <210> 44 <211> 8 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p166-173 <400> 44 Ala Ser Phe Cys Gly Ser Pro Tyr 1 5 <210> 45 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p549-557 <400> 45 Tyr Met Asp Asp Val Val Leu Gly Ala 1 5 <210> 46 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x63-71 <400> 46 Phe Ser Ser Ala Gly Pro Cys Ala Leu 1 5 <210> 47 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x102-111 <400> 47 Ala Met Ser Thr Thr Asp Leu Glu Ala Tyr 1 5 10 <210> 48 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p642-651 <400> 48 Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr 1 5 10 <210> 49 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p149-158 <400> 49 His Thr Leu Trp Lys Ala Gly Ile Leu Tyr 1 5 10 <210> 50 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x104-112 <400> 50 Ser Thr Thr Asp Leu Glu Ala Tyr Phe 1 5 <210> 51 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x103-112 <400> 51 Met Ser Thr Thr Asp Leu Glu Ala Tyr Phe 1 5 10 <210> 52 <211> 11 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x63-73 <400> 52 Phe Ser Ser Ala Gly Pro Cys Ala Leu Arg Phe 1 5 10 <210> 53 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p646-655 <400> 53 Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met 1 5 10 <210> 54 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p165-173 <400> 54 Ser Ala Ser Phe Cys Gly Ser Pro Tyr 1 5 <210> 55 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p55-63 <400> 55 Lys Val Gly Asn Phe Thr Gly Leu Tyr 1 5 <210> 56 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p54-63 <400> 56 His Lys Val Gly Asn Phe Thr Gly Leu Tyr 1 5 10 <210> 57 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> c18-27 <400> 57 Phe Leu Pro Ser Asp Phe Phe Pro Ser Val 1 5 10 <210> 58 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p368-376 <400> 58 Arg Val Thr Gly Gly Val Phe Leu Val 1 5 <210> 59 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x15-23 <400> 59 Val Leu Cys Leu Arg Pro Val Gly Ala 1 5 <210> 60 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p730-738 <400> 60 Leu Leu Ala Ala Cys Phe Ala Arg Ser 1 5 <210> 61 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p411-419 <400> 61 Asn Leu Gln Ser Leu Thr Asn Leu Leu 1 5 <210> 62 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x102-110 <400> 62 Ala Met Ser Thr Thr Asp Leu Glu Ala 1 5 <210> 63 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x91-100 <400> 63 Lys Val Leu His Lys Arg Thr Leu Gly Leu 1 5 10 <210> 64 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p646-654 <400> 64 Phe Thr Gln Cys Gly Tyr Pro Ala Leu 1 5 <210> 65 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x132-141 <400> 65 Phe Val Leu Gly Gly Cys Arg His Lys Leu 1 5 10 <210> 66 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x51-60 <400> 66 Ala His Leu Ser Leu Arg Gly Leu Pro Val 1 5 10 <210> 67 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x133-142 <400> 67 Val Leu Gly Gly Cys Arg His Lys Leu Val 1 5 10 <210> 68 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p59-67 <400> 68 Phe Thr Gly Leu Tyr Ser Ser Thr Val 1 5 <210> 69 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p547-555 <400> 69 Phe Ser Tyr Met Asp Asp Val Val Leu 1 5 <210> 70 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x97-106 <400> 70 Thr Leu Gly Leu Ser Ala Met Ser Thr Thr 1 5 10 <210> 71 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p545-553 <400> 71 Leu Ala Phe Ser Tyr Met Asp Asp Val 1 5 <210> 72 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p665-674 <400> 72 Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys 1 5 10 <210> 73 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p150-159 <400> 73 Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 1 5 10 <210> 74 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p549-558 <400> 74 Tyr Met Asp Asp Val Val Leu Gly Ala Lys 1 5 10 <210> 75 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p106-114 <400> 75 Arg Leu Lys Leu Ile Met Pro Ala Arg 1 5 <210> 76 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p369-378 <400> 76 Val Thr Gly Gly Val Phe Leu Val Asp Lys 1 5 10 <210> 77 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x69-78 <400> 77 Cys Ala Leu Arg Phe Thr Ser Ala Arg Arg 1 5 10 <210> 78 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p730-739 <400> 78 Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg 1 5 10 <210> 79 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p108-116 <400> 79 Lys Leu Ile Met Pro Ala Arg Phe Tyr 1 5 <210> 80 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p107-116 <400> 80 Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr 1 5 10 <210> 81 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p771-779 <400> 81 Ile Leu Arg Gly Thr Ser Phe Val Tyr 1 5 <210> 82 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p756-764 <400> 82 Lys Tyr Thr Ser Phe Pro Trp Leu Leu 1 5 <210> 83 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p403-412 <400> 83 Ser Phe Cys Gly Ser Pro Tyr Ser Trp 1 5 <210> 84 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p166-175 <400> 84 Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 1 5 10 <210> 85 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x111-120 <400> 85 Tyr Phe Lys Asp Cys Val Phe Lys Asp Trp 1 5 10 <210> 86 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p752-760 <400> 86 Val Leu Ser Arg Lys Tyr Thr Ser Phe 1 5 <210> 87 <211> 8 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x110-117 <400> 87 Ala Tyr Phe Lys Asp Cys Val Phe 1 5 <210> 88 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p406-415 <400> 88 Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 1 5 10 <210> 89 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p146-154 <400> 89 His Tyr Leu His Thr Leu Trp Lys Ala 1 5 <210> 90 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p548-556 <400> 90 Ser Tyr Met Asp Asp Val Val Leu Gly 1 5 <210> 91 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p387-395 <400> 91 Arg Leu Val Val Asp Phe Ser Gln Phe 1 5 <210> 92 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x143-151 <400> 92 Cys Ser Pro Ala Pro Cys Asn Phe Phe 1 5 <210> 93 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p365-374 <400> 93 Thr Pro Ala Arg Val Thr Gly Gly Val Phe 1 5 10 <210> 94 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p651-659 <400> 94 Tyr Pro Ala Leu Met Pro Leu Tyr Ala 1 5 <210> 95 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p365-373 <400> 95 Thr Pro Ala Arg Val Thr Gly Gly Val 1 5 <210> 96 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x52-60 <400> 96 His Leu Ser Leu Arg Gly Leu Pro Val 1 5 <210> 97 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x92-100 <400> 97 Val Leu His Lys Arg Thr Leu Gly Leu 1 5 <210> 98 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x94-102 <400> 98 His Lys Arg Thr Leu Gly Leu Ser Ala 1 5 <210> 99 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x95-103 <400> 99 Lys Arg Thr Leu Gly Leu Ser Ala Met 1 5 <210> 100 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x89-97 <400> 100 Leu Pro Lys Val Leu His Lys Arg Thr 1 5 <210> 101 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p712-720 <400> 101 His Gln Arg Met Arg Gly Thr Phe Val 1 5 <210> 102 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x50-58 <400> 102 Gly Ala His Leu Ser Leu Arg Gly Leu 1 5 <210> 103 <211> 8 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> c123-130 <400> 103 Gly Leu Lys Ile Leu Gln Leu Leu 1 5 <210> 104 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p500-508 <400> 104 Lys Leu His Leu Tyr Ser His Pro Ile 1 5 <210> 105 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p502-510 <400> 105 His Leu Tyr Ser His Pro Ile Ile Leu 1 5 <210> 106 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x89-98 <400> 106 Leu Pro Lys Val Leu His Lys Arg Thr Leu 1 5 10 <210> 107 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p751-760 <400> 107 Val Val Leu Ser Arg Lys Tyr Thr Ser Phe 1 5 10 <210> 108 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x54-63 <400> 108 Ser Leu Arg Gly Leu Pro Val Cys Ala Phe 1 5 10 <210> 109 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x67-76 <400> 109 Gly Pro Cys Ala Leu Arg Phe Thr Ser Ala 1 5 10 <210> 110 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x75-83 <400> 110 Ser Ala Arg Arg Met Glu Thr Thr Val 1 5 <210> 111 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> x134-142 <400> 111 Leu Gly Gly Cys Arg His Lys Leu Val 1 5 <210> 112 <211> 10 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p508-517 <400> 112 Ile Ile Leu Gly Phe Arg Lys Ile Pro Met 1 5 10 <210> 113 <211> 9 <212> PRT <213> Hepatitis B virus <220> <221> Unclassified features <223> p482-490 <400> 113 Cys Ser Arg Asn Leu Tyr Val Ser Leu 1 5 <210> 114 <211> 9 <212> PRT <213> Influenza A virus <220> <221> Unclassified features <223> NP 44-52 <400> 114 Cys Thr Glu Leu Lys Leu Ser Asp Tyr 1 5 <210> 115 <211> 9 <212> PRT <213> RSV <220> <221> Unclassified features <223> NP 306-314 <400> 115 Asn Pro Lys Ala Ser Leu Leu Ser Leu 1 5 <210> 116 <211> 9 <212> PRT <213> CMV <220> <221> Unclassified features <223> pp65 495-503 <400> 116 Asn Leu Val Pro Met Val Ala Thr Val 1 5 <210> 117 <211> 9 <212> PRT <213> EBNA3B <220> <221> Unclassified features <223> 416-424 <400> 117 Ile Val Thr Asp Phe Ser Val Ile Lys 1 5 <210> 118 <211> 9 <212> PRT <213> gp100 <220> <221> Unclassified features <223> 614-622 <400> 118 Leu Ile Tyr Arg Arg Arg Leu Met Lys 1 5 <210> 119 <211> 9 <212> PRT <213> GPR143 <220> <221> Unclassified features <223> 126-134 <400> 119 Leu Tyr Ser Ala Cys Phe Trp Trp Leu 1 5 <210> 120 <211> 9 <212> PRT <213> Influenza A virus <220> <221> Unclassified features <223> NP 380-388 <400> 120 Glu Leu Arg Ser Arg Tyr Trp Ala Ile 1 5 <210> 121 <211> 27 <212> PRT <213> Hepatitis B virus <400> 121 Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Gly Phe Arg Lys Ile 1 5 10 15 Pro Met Gly Val Gly Leu Ser Pro Phe Leu Leu 20 25 <210> 122 <211> 31 <212> PRT <213> Hepatitis B virus <400> 122 Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala 1 5 10 15 Leu Met Pro Leu Tyr Ala Cys Ile Gln Ala Lys Gln Ala Phe Thr 20 25 30 <210> 123 <211> 29 <212> PRT <213> Hepatitis B virus <400> 123 Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro 1 5 10 15 Thr Gly Trp Gly Leu Ala Ile Gly His Gln Arg Met Arg 20 25

Claims

1. An immunogenic peptide comprising the amino acid sequence shown in SEQ ID NO:

31.

2. An immunogenic composition comprising: - The peptide according to claim 1, and - Pharmaceutically acceptable carriers Optionally, it may further include an adjuvant.

Images