Treatment of diseases related to hepatitis B virus

Abstract

The present invention provides novel immunogenic peptides derived from the X and polymerase proteins of hepatitis B virus (HBV). These peptides contain epitopes that are well conserved among multiple HBV variants and are derived from regions of the proteins essential for viral replication. Furthermore, these novel HBV antigens bind to multiple HLA types and induce IFN-glucan synthesis in PBMCs from HBV-resolved individuals. γ Epitopes that elicit a response were identified.

Description

Technical Field

[0001] The present invention relates to the field of infectious diseases. Specifically, the present invention relates to immunogenic peptides, polynucleotides, immunogenic compositions and methods for treating diseases associated with hepatitis B virus (HBV).

Background Art

[0002] Chronic infections caused by hepatitis B virus (HBV) are a major global health problem. HBV is the prototype member of the Hepadnaviridae family and has a strong tropism for hepatocytes (Ganem et al, 2004 N Engl J Med 350:1118).

[0003] For more than 30 years, despite the availability of an effective prophylactic vaccine for the prevention of hepatitis B, an estimated 2 billion people are still infected with HBV, and currently, the number of chronic (long-term) hepatitis B infected individuals exceeds 240 million, and is prevalent geographically in regions excluding Western Europe and North America (World Health Organization, July 2013).

[0004] Person-to-person transmission of this virus occurs either by direct contact between blood or through the semen or vaginal fluid of an infected person. Characteristically, in endemic areas, perinatal transmission results in mother-to-child infection. Thus, HBV is not an accidental transmitter, but this virus can be easily transmitted by perinatal, percutaneous or sexual exposure. Along with this, for groups such as medical workers, due to the high frequency of personal contact with infected individuals, a serious risk arises.

[0005] HBV infection can manifest as acute viral hepatitis, beginning with general malaise, loss of appetite, nausea, vomiting, body aches, low-grade fever, and dark urine, progressing to jaundice. This illness lasts for several weeks, then gradually improves in most affected adults, although some develop severe liver disease (fulminant hepatic failure) that can be fatal. The infection can also be completely asymptomatic and may progress unnoticed.

[0006] Chronic infection with hepatitis B virus can be asymptomatic or involve chronic inflammation of the liver (chronic hepatitis), and can progress to cirrhosis over many years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (liver cancer), and the incubation period can be several years. Treatment of chronic HBV-infected individuals with antiviral drugs such as nucleoside / nucleotide analogs (e.g., entecavir and tenofovir) or interferon (IFN)α effectively reduces serum viral load. However, antiviral therapy rarely leads to a sustained virological response, and drug resistance develops (Zoulim et al, 2012 BJ Hepatol 56 suppl.1 S112; EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J. Hepatology 67:370). Furthermore, the vast majority of HBV carriers remain untreated.

[0007] Approximately 15-40% of chronic HBV carriers will develop clinically significant liver disease during their lifetime, with a high risk of death from cirrhosis and associated liver failure or hepatocellular carcinoma (Huang et al, 2011 Curr Opin Immunol 23:237). Antiviral drugs cannot eradicate the infection, resulting in the need for long-term antiviral therapy, although not lifelong. However, this therapy has drawbacks such as toxic side effects and high costs, making new treatment methods urgently needed (Grimm et al., 2013 Clin Sci(Lond) 124:77).

[0008] Therapeutic vaccination is a promising strategy for treating chronic hepatitis B. Following the humoral immune response to HBV, which is primarily involved in protection against HBV infection by current preventive vaccines (Lok, 2002 N Engl J Med 346:1682), the cellular immune response is clearly involved in the body's natural resistance to HBV infection.

[0009] Perinatal transmission of HBV from mother to newborn and infection during the first year of life result in persistent infection in over 90% of children. In contrast, infection during adulthood is spontaneously cleared in over 90% of cases, and protective immunity lasts a lifetime (Rehermann et al., 2005 Nat Rev Immunol 5:215).

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

[0011] These T cell responses are crucial in the clearance and control of HBV. Experiments in HBV-infected chimpanzees have shown that HBV-specific CD8+ T cells play an essential role as effector cells in this process (Thimme et al, 2003 J Virol 77:68). In contrast to the response in patients who have cleared HBV infection, the T cell response in patients with chronic hepatitis B is usually very weak, concentrated on only a few epitopes, and functionally impaired (Michel et al, 2011 J Hepatol 54:1286). The goal of therapeutic vaccination is to build an active and robust multivalent CTL and T helper cell response targeting many HBV antigens, thereby promoting viral clearance, hepatitis management, and cure.

[0012] Despite significant advances in our understanding of the etiology and epidemiology of this disease, an effective therapeutic HBV vaccine remains necessary.

[0013] In this technical field, peptides derived from HBV proteins containing putative T cell epitopes limited to specific HLA types have been described (International Publication No. 0219986, International Publication No. 2002020035, International Publication No. 2014102540, International Publication No. 15187009). However, many of these proposed antigens have one or more of the following drawbacks: • Low sequence conservation among HBV genotypes means that effectiveness is limited to (a very small) number of HBV genotypes. • There is no functional link between the antigenic region from which it originates and viral replication, making it less likely that an immune response to that region would interfere with viral replication. • Due to immune depletion caused by excessive exposure to the antigen, the effectiveness of the immune response is lost. • Because it covers only one or a few HLA types, it may be ineffective in certain populations that lack those HLA types. • Difficult to manufacture (especially for long-chain peptides), • The potential ability to induce a T cell response has not been confirmed. • There are no indicators of whether the immune response to that peptide antigen has the potential to resolve the infection. [Overview of the project] [Means for solving the problem]

[0014] The present invention provides a novel long peptide antigen derived from HBV-X and HBV polymerase protein that eliminates all or most of the drawbacks associated with peptide antigens described to date.

[0015] The HBV long peptide antigen described herein contains a novel HLA-binding peptide sequence that is well conserved across multiple HBV genotypes, originates from a conserved region of a protein essential for viral replication, and is therefore less likely to evade the HBV-specific immune response. Furthermore, this novel HBV-derived long peptide antigen has multiple HLA-binding peptide sequences capable of being presented by diverse HLA types. In addition, synthetic long peptides (SLPs) containing such HLA-binding peptide sequences that can be produced in sufficient yield and purity are described. Moreover, it has been found that SLPs containing the newly identified HLA-binding peptide sequences elicit an IFNγ response in PBMCs of individuals who have recovered from HBV infection (HBV-cleared individuals).

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

[0017] In a further embodiment, the present invention relates to a polynucleotide comprising a nucleotide sequence encoding the peptide according to the present invention, an immunogenic composition comprising the immunogenic peptide or polynucleotide of the present invention, and the use of the peptide, polynucleotide, recombinant virus or immunogenic composition of the present invention in the treatment of HBV-related diseases. [Brief explanation of the drawing]

[0018] [Figure 1]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function of HBx. The central bar chart displays the length of the consensus sequence of the HBx protein (Method), and here, the conservation score between viral genotypes for each amino acid is shown in grayscale (Legend). Previously reported validated epitope sequences obtained from the Hepitopes database are aligned with this consensus protein sequence and shown at the top. Below that, for representative examples of each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown. Below the aligned conjugates, the inventors plotted the frequency distribution of each amino acid within the range of all predicted conjugates (8-14 amino acid lengths) across the protein sequence. The conservation score for each amino acid (Legend) is shown as a horizontal grayscale bar chart. Essential amino acids whose mutations lead to loss of viral persistence are indicated by arrows corresponding to the conservation score color. At the bottom, the functional domains following the HBVdb naming convention are displayed. [Figure 2-1]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-2]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-3]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-4]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-5]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-6]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-7]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 2-8]Alignment of validated HLA-I epitopes and predicted HLA-binding peptide sequences for protein conservation and function for polymerases. The first 200 amino acids (A, E), amino acids 201-474 (B, F), 475-631 (C, G), and amino acids 632-843 (D, H) are shown. Previously reported validated epitope sequences obtained from the Hepitopes database are shown at the top. Below that, for each HLA supertype, novel conjugate candidates (9-11 amino acids) predicted by NetMHCpan are shown (A-D). In addition, the inventors plotted the frequency distribution of predicted conjugates (8-14 amino acid lengths) across protein sequences (E-H). The conservation score (legend) for each amino acid is shown as a horizontal grayscale bar chart. Essential amino acids whose single or compound mutations lead to loss of viral persistence (50% or more) are indicated by arrows corresponding to the conservation score color for that particular amino acid. Amino acids expected to be essential for the correct folding of the viral protein are indicated with an asterisk. The general domain is shown according to a predetermined nomenclature (Cao et al. 2014 J Viral Hepat 21:882), and this also includes the T3 domain and the YMDD motif. [Figure 3-1]In vitro binding ability of selected predicted HLA-binding peptide sequences. Binding of predicted HLA-binding peptide sequences is expressed as percentage binding to a positive control peptide (pos.ct.) that shows high affinity for the indicated HLA type. Mean and standard deviation are shown for control (black), conjugated (greater than 25% of positive control; gray), and unconjugated (less than 25% of positive control; white). Binding ability was evaluated for six representative supertypes, and in addition, HLA*11:01 was included considering its high prevalence among chronic hepatitis B patient populations. These are each shown in separate graphs (A-G). As negative controls, the inventors included known unconjugated peptides (neg.ct.) and conditions where no peptides were present for each HLA type. Peptides underlined with a solid line are rarely described as epitope sequences in relation to the HLA type being validated. Peptides underlined with a dashed line are epitope sequences that have so far only been described in relation to other HLA types, and the table below summarizes the cross-reactive conjugates among them (H). An asterisk indicates which peptides did not meet the inventors' length and conservation thresholds. [Figure 3-2]In vitro binding ability of selected predicted HLA-binding peptide sequences. Binding of predicted HLA-binding peptide sequences is expressed as percentage binding to a positive control peptide (pos.ct.) that shows high affinity for the indicated HLA type. Mean and standard deviation are shown for control (black), conjugated (greater than 25% of positive control; gray), and unconjugated (less than 25% of positive control; white). Binding ability was evaluated for six representative supertypes, and in addition, HLA*11:01 was included considering its high prevalence among chronic hepatitis B patient populations. These are each shown in separate graphs (A-G). As negative controls, the inventors included known unconjugated peptides (neg.ct.) and conditions where no peptides were present for each HLA type. Peptides underlined with a solid line are rarely described as epitope sequences in relation to the HLA type being validated. Peptides underlined with a dashed line are epitope sequences that have so far only been described in relation to other HLA types, and the table below summarizes the cross-reactive conjugates among them (H). An asterisk indicates which peptides did not meet the inventors' length and conservation thresholds. [Figure 4] The binding ability of selected predicted HLA-binding peptide sequences against the rank score derived by in silico prediction. As shown in Figure 3, the binding ability of predicted HLA-binding sequences by in vitro HLA-binding assays is plotted against the in silico predicted HLA-binding ability by NetMHCpan, which is expressed as a rank score. A 25% binding cutoff is indicated for the positive control to differentiate between low-binding and non-binding molecules. [Figure 5-1]Immunogenicity of HLA-binding peptide sequences. IFNγ production (after DMSO subtraction) by expanded PBMCs was measured from nine HBV-cleared individuals who responded to incubation with established HLA-binding peptide sequences and well-established c18-27 and p549-557 epitope sequences (Panel B). The gray box indicates the number of responding donors for each HLA-binding peptide sequence as a fraction of the total number of subjects. Epitope sequences underlined with a solid line are rarely described for the validated HLA type. Epitope sequences underlined with a dashed line have so far only been described in relation to other HLA types. An asterisk indicates which HLA-binding peptide sequences did not meet the inventors' length and conservation thresholds. [Figure 5-2] Immunogenicity of HLA-binding peptide sequences. IFNγ production (after DMSO subtraction) by expanded PBMCs was measured from nine HBV-cleared individuals who responded to incubation with established HLA-binding peptide sequences and well-established c18-27 and p549-557 epitope sequences (Panel B). The gray box indicates the number of responding donors for each HLA-binding peptide sequence as a fraction of the total number of subjects. Epitope sequences underlined with a solid line are rarely described for the validated HLA type. Epitope sequences underlined with a dashed line have so far only been described in relation to other HLA types. An asterisk indicates which HLA-binding peptide sequences did not meet the inventors' length and conservation thresholds. [Figure 6]IFNγ response after SLP stimulation, as measured by ELISpot. PBMCs from 15 donors with a history of HBV remission were incubated with 10 μM SLP or equivalent concentration DMSO for 20–24 hours, and subsequent IFNγ production was measured by ELISpot. Cumulative spot-forming units (SFUs) from 4 replicated wells, after subtracting the SFUs from 4 replicated wells under DMSO control conditions, are shown. Each point corresponds to one donor, and all donors are represented by different symbols. Negative SFUs are shown as 0. The number of donors in Figure 6 does not necessarily correspond to the number of donors in Figure 5. [Figure 7] Novel SLPs can boost functional CD8+ and CD4+ T cell responses in vitro in leukocytes from HBV-cleared individuals and chronic HBV patients. PBMCs, rHBV, (A) isolated from the buffy coat of previously HBV-cleared healthy donors, or PBMCs, cHBV, (B) isolated from the whole blood of chronic HBV patients visiting our outpatient clinic, were exposed to an SLP pool containing the indicated SLPs (1, 2, 4, and 6) and expanded for 14 days in the presence of IL-2. After 14 days, the expanded cells were individually re-stimulated with the indicated SLPs for 22 hours, and both the cells and culture supernatants were subsequently evaluated for surface markers (makers) and cytokines, which are indicators of functional T cell activation, respectively. The percentages of CD4+ and CD8+ T cells expressing the activation marker CD69, and the percentage of CD8+ T cells expressing CD107a, an indicator of recent CD8+ T cell cytotoxic activity (subtracting the percentages observed for DMSO-mediated control samples) are shown. The secretion of T cell cytokines IFNγ and TNFα in the supernatant as a result of SLP restimulation is also plotted. Cytokine values ​​were calculated by subtracting twice the standard deviation of the DMSO / unrelated peptide control samples from the mean values ​​observed for those controls. [Figure 8]Novel treatment schedule for elimination of HBV infection. Reduce viral load with nucleoside / nucleotide analog (NA) therapy before initiating therapeutic vaccination (T). Administer therapeutic vaccination once the viral load has stabilized at a low level. Optionally, therapeutic vaccination may be combined with drugs targeting suppressor myeloid cells (MDSCs) (administered before therapeutic vaccination), siRNA (before therapeutic vaccination), drugs modifying T cell metabolism (before or during therapeutic vaccination), or checkpoint blockade (during or after therapeutic vaccination) to further enhance T cell effector function. As a "natural" booster, boost HBV-specific T cells in situ by increasing the presence of viral antigens by discontinuing NA therapy, thereby promoting the elimination of remaining infecting hepatocytes. Preferably, vaccine efficacy should be determined by appropriate monitoring of the viral load, determining follow-up (combination) therapy, and / or discontinuing NA. Lines provide schematic indications of the development of indicated parameters over time. Arrows indicate preferred timing for intervention or monitoring. [Modes for carrying out the invention]

[0019] definition The term "HBV" refers to the hepatitis B virus. Eight distinct genotypes of HBV, designated A through H, have been described. These genotypes share high sequence homology, but there are at least 8% differences in their sequences. Within each genotype, subtypes have been described: they exhibit 4-8% differences in their genomes.

[0020] The terms “HBV polymerase,” or simply “polymerase,” or “Pol” refer to the polymerase encoded by the hepatitis B genome. GenBank NCBI reference NC_003977.2 describes a commonly used reference HBV polymerase sequence, also shown in Sequence ID No. 27 herein. Sequence ID No. 28 shows the consensus sequence of HBV polymerase based on 7489 genotypes. In one embodiment of the present invention, the HBV polymerase fragment present in the peptide of the present invention is identical to the corresponding sequence shown in Sequence ID No. 27 in greater than 85%, greater than 90%, etc., for example, greater than 95%, greater than 98%, etc. In another embodiment of the present invention, the HBV polymerase fragment is identical to the corresponding sequence shown in Sequence ID No. 28 in greater than 85%, greater than 90%, etc., for example, greater than 95%, greater than 98%, etc. In another embodiment, the referenced HBV polymerase is of a genotype selected from the group consisting of A, B, C, D, E, F, G, and H.

[0021] The terms "HBV-X," "HBx," "HBxAg," "HBV-X protein," or "X-protein" refer to the X protein encoded by the hepatitis B genome. GenBank NCBI reference NC_003977.2 describes a commonly used reference HBV-X sequence, which is also shown in Sequence ID No. 29 herein. Sequence ID No. 30 shows the consensus sequence of the HBV-X protein based on 8127 genotypes. In one embodiment of the present invention, the HBV-X fragment present in the peptide of the present invention is identical to the corresponding sequence shown in Sequence ID No. 29 in greater than 85%, greater than 90%, etc., for example, greater than 95%, greater than 98%, etc. In another embodiment of the present invention, the HBV-X fragment is identical to the corresponding sequence shown in Sequence ID No. 30 in greater than 85%, greater than 90%, etc., for example, greater than 95%, greater than 98%, etc. In another embodiment, the referenced HBV-X protein is of a genotype selected from the group consisting of A, B, C, D, E, F, G, and H.

[0022] The numbering of positions within HBV polymerase and HBV-X in this specification is based on the consensus sequences shown in SEQ ID NOs. 28 and 30, respectively. In other words, the numbering of amino acid positions within HBV polymerase and HBV-X proteins corresponds to the numbering in the consensus sequences shown in SEQ ID NOs. 28 and 30, respectively. An amino acid position that "corresponds" an amino acid position in one sequence to an amino acid position in another sequence is one that aligns with the other amino acid when a standard sequence alignment program such as ALIGN or ClustalW is typically used with default settings. Methods for aligning sequences to determine the corresponding position in the consensus sequence for a particular position in the HBV polymerase or HBV-X sequence are well known in the art. For example, alignment may show that, if there is a gap in a given HBV polymerase compared to the consensus sequence, the amino acid fragment from positions 20 to 40 of that given HBV polymerase corresponds to positions 20 to 41 of the consensus sequence. To avoid misunderstanding, while the positional numbering is based on the consensus sequence, the actual amino acid sequences of the fragments may differ from the consensus sequence and can vary depending on the HBV genotype.

[0023] "Sequence identity" is defined herein as the relationship between two or more amino acid sequences as determined by comparing those sequences. Sequence identity can be determined by the alignment of two peptide sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman).

[0024] "Treatment" or "treating" refers to the administration of an effective dose of an immunogenic composition for the purpose of alleviating, improving, inhibiting, eradicating (curing), or preventing a symptom, disorder, or disease state. "Effective dose" refers to the amount and duration of administration that is effective in achieving the desired therapeutic outcome.

[0025] As used herein, the term “immunogenic peptide” means a peptide having the ability to induce or boost an immune response. The immunogenic peptides of the present invention may be unconjugated or unmodified, i.e., simple chains of amino acids linked by peptide bonds, or they may be further modified, such as conjugated, by being covalently bonded to another molecule, such as an adjuvant.

[0026] In the context of the present invention, "20-34 amino acid length" means that the number of amino acid residues is 20-34, i.e., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acid residues. The peptides of the present invention are also called long peptides and exceed the length of the epitope peptide sequences presented by human leukocyte antigens (HLA) class I and class II. Preferably, the long peptides of the present invention are synthetic peptides and are also referred to herein as synthetic long peptides (SLPs).

[0027] In the context of this invention, the term “HBV protein fragment” means a partial sequence of the HBV protein, i.e., an identical amino acid sequence. Therefore, it refers to a continuous sequence of the native HBV protein without insertions, deletions, or substitutions. When a peptide is specified to contain an HBV protein fragment of a particular length, it means that the fragment is neither shorter nor longer than that length. For example, if the fragment is specified to be 20-34 amino acids long, it means that the fragment is between 20 and 34 amino acids long. Therefore, such a peptide will not, for example, contain a continuous sequence of the HBV protein longer than 35 amino acids. However, to avoid misunderstanding, “comprising” has its usual meaning in the art, i.e., “a peptide containing an HBV protein fragment” 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 that are not adjacent to the fragment in the HBV protein.

[0028] In the context of the present invention, the terms "HLA-binding peptide," "HLA conjugate," or "conjugate" refer to a short protein fragment portion of HBV-X or HBV polymerase that can specifically bind to an HLA molecule.

[0029] In the context of the present invention, "epitope" is defined as a short HLA-binding peptide that binds to a designated HLA molecule and, when presented on the surface of a cell, has the ability to elicit a T cell response in an organism.

[0030] The indefinite article "a" or "an" refers to an element, and unless the context explicitly requires that there be one of that element and only one, it does not rule out the possibility that there are more than one of that element. Therefore, the indefinite article "a" or "an" usually means "at least one."

[0031] Further aspects and embodiments of the present invention As described above, in the first main embodiment, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the fragment is 20 to 34 amino acids long, and the fragment is a) At least 10 consecutive amino acids in the HBV-X region from position 57 to 78, preferably, - The amino acid sequence shown in Sequence ID No. 1 (x70-78), and / or - The amino acid sequence shown in Sequence ID No. 2 (x67-75), and / or - The amino acid sequence (x62-73) shown in Sequence ID No. 3, and / or - The amino acid sequence (x58-66) shown in SEQ ID NO: 4, and / or - Amino acid sequence shown in Sequence ID No. 5 (x57-66) things that include, or b) At least 11 consecutive amino acids in the HBV-X region from position 103 to 120, preferably, - The amino acid sequence shown in Sequence ID No. 6 (x103-111), and / or - The amino acid sequence shown in Sequence ID No. 7 (x104-113), and / or - The amino acid sequence shown in Sequence ID No. 8 (x105-113), and / or - Amino acid sequence shown in Sequence ID No. 9 (x110-120) things that include, or c) The amino acid sequence shown in Sequence ID No. 10 (x132-140), or d) The amino acid sequence shown in Sequence ID No. 11 (p124-133), or e) The amino acid sequence shown in Sequence ID No. 12 (p164-173), or f) The amino acid sequence shown in Sequence ID No. 13 (p275-283), or g) At least 10 consecutive amino acids in the HBV polymerase region from position 403 to 415, preferably, - The amino acid sequence shown in Sequence ID No. 14 (p403-412), and / or - The amino acid sequence shown in Sequence ID No. 15 (p404-412), and / or - Amino acid sequence shown in SEQ ID NO: 16 (p407-415) things that include, or h) At least nine consecutive amino acids in the HBV polymerase region from position 509 to 523, preferably, - The amino acid sequence shown in SEQ ID NO: 17 (p509-517), and / or - Amino acid sequence shown in SEQ ID NO: 18 (p515-523) things that include, or i) At least 10 consecutive amino acids in the HBV polymerase region from position 649 to 658, - The amino acid sequence shown in SEQ ID NO: 19 (p649-658), and / or - Amino acid sequence shown in Sequence ID No. 20 (p650-658) things that include, or j) At least 10 consecutive amino acids in the HBV polymerase region from position 693 to 706, preferably, - The amino acid sequence shown in SEQ ID NO: 21 (p693-701), and / or - Amino acid sequence shown in Sequence ID No. 22 (p697-706) things that include, or k) Amino acid sequence shown in Sequence ID No. 23 (p723-731), or l) At least 10 consecutive amino acids in the HBV polymerase region from position 755 to 765, - The amino acid sequence shown in SEQ ID NO: 24 (p755-764), and / or - Amino acid sequence shown in Sequence ID No. 25 (p756-765) things that include, or m) Amino acid sequence shown in Sequence ID No. 26 (p829-837) Includes.

[0032] In one embodiment, the peptide consists of the aforementioned fragment of the HBV protein.

[0033] In another embodiment, the peptide has a length of 20 to 34 amino acids, i.e., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, i.e., 20 to 33 amino acids, for example 20 to 32 amino acids, i.e., 20 to 31 amino acids, for example 20 to 30 amino acids, i.e., 20 to 29 amino acids, for example 20 to 28 amino acids, i.e., 20 to 27 amino acids, for example 20 to 26 or 20 to 25 amino acids.

[0034] In a further embodiment, the fragment has a length of 20 to 33 amino acids, i.e., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 amino acids, for example, 20 to 32 amino acids, i.e., 20 to 31 amino acids, for example, 20 to 30 amino acids, i.e., 20 to 29 amino acids, for example, 20 to 28 amino acids, i.e., 20 to 27 amino acids, for example, 20 to 26 or 20 to 25 amino acids.

[0035] In a further embodiment, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the fragment has a length of 20 to 34 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 amino acids, and the fragment is - The amino acid sequence shown in Sequence ID No. 1 (x70-78), - The amino acid sequence shown in Sequence ID No. 2 (x67-75), - The amino acid sequence shown in Sequence ID No. 3 (x62-73), - The amino acid sequence shown in Sequence ID No. 4 (x58-66), - The amino acid sequence shown in Sequence ID No. 5 (x57-66), - The amino acid sequence shown in Sequence ID No. 6 (x103-111), - The amino acid sequence shown in Sequence ID No. 7 (x104-113), - The amino acid sequence shown in Sequence ID No. 8 (x105-113), - The amino acid sequence shown in Sequence ID No. 9 (x110-120), - The amino acid sequence shown in Sequence ID No. 10 (x132-140), - The amino acid sequence shown in Sequence ID No. 11 (p124-133), - The amino acid sequence shown in Sequence ID No. 12 (p164-173), - The amino acid sequence shown in Sequence ID No. 13 (p275-283), - The amino acid sequence shown in Sequence ID No. 14 (p403-412), - The amino acid sequence shown in Sequence ID No. 15 (p404-412), - The amino acid sequence shown in Sequence ID No. 16 (p407-415), - The amino acid sequence shown in Sequence ID No. 17 (p509-517), - The amino acid sequence shown in SEQ ID NO: 18 (p515-523), - The amino acid sequence shown in SEQ ID NO: 19 (p649-658), - The amino acid sequence shown in Sequence ID No. 20 (p650-658), - The amino acid sequence shown in Sequence ID No. 21 (p693-701), - The amino acid sequence shown in Sequence ID No. 22 (p697-706), - The amino acid sequence shown in Sequence ID No. 23 (p723-731), - The amino acid sequence shown in Sequence ID No. 24 (p755-764), - The amino acid sequence shown in Sequence ID No. 25 (p756-765), and - Amino acid sequence shown in Sequence ID No. 26 (p829-837) It includes one or more sequences selected from the group consisting of, Preferably the fragment is - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 2 (x67-75), or - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 3 (x62-73), or - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 4 (x58-66), or - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 5 (x57-66), or - The amino acid sequence shown in SEQ ID NO: 2 (x67-75) and the amino acid sequence shown in SEQ ID NO: 3 (x62-73), or - The amino acid sequence shown in SEQ ID NO: 2 (x67-75) and the amino acid sequence shown in SEQ ID NO: 4 (x58-66), or - The amino acid sequence shown in Sequence ID No. 2 (x67-75) and the amino acid sequence shown in Sequence ID No. 5 (x57-66), or - The amino acid sequence shown in SEQ ID NO: 3 (x62-73) and the amino acid sequence shown in SEQ ID NO: 4 (x58-66), or - The amino acid sequence shown in SEQ ID NO: 3 (x62-73) and the amino acid sequence shown in SEQ ID NO: 5 (x57-66) or - The amino acid sequence shown in SEQ ID NO: 6 (x103-111) and the amino acid sequence shown in SEQ ID NO: 7 (x104-113), or - The amino acid sequence shown in SEQ ID NO: 6 (x103-111) and the amino acid sequence shown in SEQ ID NO: 8 (x105-113), or - The amino acid sequence shown in SEQ ID NO: 6 (x103-111) and the amino acid sequence shown in SEQ ID NO: 9 (x110-120), or - The amino acid sequence shown in SEQ ID NO: 7 (x104-113) and the amino acid sequence shown in SEQ ID NO: 9 (x110-120), or - The amino acid sequence shown in SEQ ID NO: 8 (x105-113) and the amino acid sequence shown in SEQ ID NO: 9 (x110-120), or - The amino acid sequence shown in SEQ ID NO: 14 (p403-412) and the amino acid sequence shown in SEQ ID NO: 16 (p407-415), or - Amino acid sequences shown in SEQ ID NO: 15 (p404-412) and SEQ ID NO: 16 (p407-415) Includes.

[0036] As described, in one embodiment, the immunogenic peptide comprises a fragment of HBV-X, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in Sequence ID No. 1 (x70-78), and / or - The amino acid sequence shown in Sequence ID No. 2 (x67-75), and / or - The amino acid sequence (x62-73) shown in Sequence ID No. 3, and / or - The amino acid sequence (x58-66) shown in SEQ ID NO: 4, and / or - Amino acid sequence shown in Sequence ID No. 5 (x57-66) It contains at least 10 consecutive amino acids in the HBV-X region from position 57 to 78.

[0037] In a further embodiment, the immunogenic peptide comprises a fragment of HBV-X, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises at least 10 consecutive amino acids from the HBV-X region 57 to 78, and the fragment is - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 2 (x67-75), or - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 3 (x62-73), or - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 4 (x58-66), or - The amino acid sequence shown in SEQ ID NO: 1 (x70-78) and the amino acid sequence shown in SEQ ID NO: 5 (x57-66), or - The amino acid sequence shown in SEQ ID NO: 2 (x67-75) and the amino acid sequence shown in SEQ ID NO: 3 (x62-73), or - The amino acid sequence shown in SEQ ID NO: 2 (x67-75) and the amino acid sequence shown in SEQ ID NO: 4 (x58-66), or - The amino acid sequence shown in Sequence ID No. 2 (x67-75) and the amino acid sequence shown in Sequence ID No. 5 (x57-66), or - The amino acid sequence shown in SEQ ID NO: 3 (x62-73) and the amino acid sequence shown in SEQ ID NO: 4 (x58-66), or - Amino acid sequence shown in SEQ ID NO: 4 (x62-73) and amino acid sequence shown in SEQ ID NO: 5 (x57-66) Includes.

[0038] In a further embodiment, the most N-terminal amino acid of the fragment is the amino acid at position 53 of HBV-X, or the most N-terminal amino acid of the fragment is located at a position closer to the C-terminus than position 53 of HBV-X, i.e., the amino acid at positions 54, 55, 56, etc.

[0039] In another further 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 located at a position further N-terminal than position 91 of HBV-X, i.e., the amino acid at positions 90, 89, 88, etc.

[0040] In further embodiments, - The most N-terminal amino acid of the fragment is the amino acid at position 53 of HBV-X or an amino acid located C-terminal to position 53 of HBV-X, and - The C-terminal amino acid of the fragment is either the amino acid at position 91 of HBV-X or an amino acid located N-terminal to position 91 of HBV-X.

[0041] 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.

[0042] In another embodiment, the immunogenic peptide comprises a fragment of HBV-X, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in Sequence ID No. 6 (x103-111), and / or - The amino acid sequence shown in Sequence ID No. 7 (x104-113), and / or - The amino acid sequence shown in Sequence ID No. 8 (x105-113), and / or - Amino acid sequence shown in Sequence ID No. 9 (x110-120) It contains at least 11 consecutive amino acids in the HBV-X region from position 103 to 120.

[0043] In a further embodiment, the immunogenic peptide comprises a fragment of HBV-X, where the fragment is 20 to 34 amino acid long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises at least 11 consecutive amino acids from the HBV-X region 103 to 120, and the fragment is - The amino acid sequence shown in SEQ ID NO: 6 (x103-111) and the amino acid sequence shown in SEQ ID NO: 7 (x104-113), or - The amino acid sequence shown in SEQ ID NO: 6 (x103-111) and the amino acid sequence shown in SEQ ID NO: 8 (x105-113), or - The amino acid sequence shown in SEQ ID NO: 6 (x103-111) and the amino acid sequence shown in SEQ ID NO: 9 (x110-120), or - The amino acid sequence shown in SEQ ID NO: 7 (x104-113) and the amino acid sequence shown in SEQ ID NO: 9 (x110-120), or - Amino acid sequence shown in SEQ ID NO: 8 (x105-113) and amino acid sequence shown in SEQ ID NO: 9 (x110-120) Includes.

[0044] In a further embodiment, the most N-terminal amino acid of the fragment is the amino acid at position 103 of HBV-X, or the most N-terminal amino acid of the fragment is located at a position C-terminal to 103 of HBV-X, i.e., an amino acid at position 104, 105, 106, etc.

[0045] In another further 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 located at a position N-terminal to 122 of HBV-X, i.e., an amino acid at position 121, 120, 119, etc.

[0046] In further embodiments, the HBV-X fragment contained in the peptide of the present invention does not extend beyond positions 103 and 122 of HBV-X.

[0047] In another embodiment, the immunogenic peptide comprises a fragment of HBV-X, wherein the fragment has a length of 20 to 34 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises the amino acid sequence (x132-140) shown in SEQ ID NO: 10.

[0048] In a further embodiment, the most N-terminal amino acid of the fragment is the amino acid at position 116 of HBV-X, or the most N-terminal amino acid of the fragment is located at a position closer to the C-terminus than 116 of HBV-X, i.e., the amino acids at positions 117, 118, 119, etc.

[0049] In another further 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 located at a position further N-terminal than 140 of HBV-X, i.e., the amino acids at positions 139, 138, 137, etc.

[0050] In further embodiments: - The most N-terminal amino acid of the fragment is the amino acid at position 116 of HBV-X or an amino acid located C-terminal to position 116 of HBV-X, and - The C-terminal amino acid of the fragment is either the amino acid at position 140 of HBV-X or an amino acid located N-terminal to position 140 of HBV-X.

[0051] 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.

[0052] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises the amino acid sequence (p124-133) shown in SEQ ID NO: 11.

[0053] In a further 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 located at a position N-terminal to 155 of the HBV polymerase, i.e., an amino acid at position 154, 153, 152, etc.

[0054] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises the amino acid sequence (p164-173) shown in SEQ ID NO: 12.

[0055] In a further embodiment, the most N-terminal amino acid of the fragment is the amino acid at position 151 of the HBV polymerase, or the most N-terminal amino acid of the fragment is located at a position C-terminal to 151 of the HBV polymerase, i.e., an amino acid at position 152, 153, 154, etc.

[0056] In another further 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 located at a position N-terminal to 174 of the HBV polymerase, i.e., an amino acid at position 173, 172, 171, etc.

[0057] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises the amino acid sequence (p275-283) shown in SEQ ID NO: 13.

[0058] In further embodiments, 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 located C-terminal to position 262 of the HBV polymerase, i.e., the amino acid at positions 263, 264, 265, etc.

[0059] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in Sequence ID No. 14 (p403-412), and / or - The amino acid sequence shown in Sequence ID No. 15 (p404-412), and / or - Amino acid sequence shown in SEQ ID NO: 16 (p407-415) It contains at least 10 consecutive amino acids in the HBV polymerase region from position 403 to 415.

[0060] In a further embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, where the fragment has a length of 20 to 34 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment is - The amino acid sequence shown in SEQ ID NO: 14 (p403-412) and the amino acid sequence shown in SEQ ID NO: 16 (p407-415), or - Amino acid sequences shown in SEQ ID NO: 15 (p404-412) and SEQ ID NO: 16 (p407-415) It contains at least 10 consecutive amino acids in the HBV polymerase region from position 403 to 415.

[0061] In a further 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 located C-terminal to position 390 of the HBV polymerase, i.e., the amino acids at positions 391, 392, 393, etc.

[0062] In another further 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 located at a position N-terminal to 425 of the HBV polymerase, i.e., an amino acid at position 424, 423, 422, etc.

[0063] In further embodiments, - The most N-terminal amino acid of the fragment is the amino acid at position 390 of HBV polymerase or an amino acid located C-terminal to position 390 of HBV polymerase, and - The C-terminal amino acid of the fragment is either the amino acid at position 425 of the HBV polymerase or an amino acid located N-terminal to position 425 of the HBV polymerase.

[0064] Therefore, in this latter embodiment, the HBV polymerase fragment contained in the peptide of the present invention does not extend beyond the 390th and 425th positions of the HBV polymerase.

[0065] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in SEQ ID NO: 17 (p509-517), and / or - Amino acid sequence shown in SEQ ID NO: 18 (p515-523) It contains at least 9 consecutive amino acids in the HBV polymerase region from position 509 to 523.

[0066] In a further 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 located C-terminal to position 503 of the HBV polymerase, i.e., an amino acid at position 504, 505, 506, etc.

[0067] In another further 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 located at a position N-terminal to 532 of the HBV polymerase, i.e., an amino acid at position 531, 530, 529, etc.

[0068] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in SEQ ID NO: 19 (p649-658), and / or - Amino acid sequence shown in Sequence ID No. 20 (p650-658) It contains at least 10 consecutive amino acids in the HBV polymerase region from position 649 to 658.

[0069] In a further 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 located C-terminal to position 624 of the HBV polymerase, i.e., an amino acid at position 625, 626, 627, etc.

[0070] In another further 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 located at a position N-terminal to 658 of the HBV polymerase, i.e., an amino acid at position 657, 656, 655, etc.

[0071] In further embodiments, - The most N-terminal amino acid of the fragment is the amino acid at position 624 of HBV polymerase or an amino acid located C-terminal to position 624 of HBV polymerase, and - The C-terminal amino acid of the fragment is either the amino acid at position 658 of the HBV polymerase or an amino acid located N-terminal to position 658 of the HBV polymerase.

[0072] 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.

[0073] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in SEQ ID NO: 21 (p693-701), and / or - Amino acid sequence shown in Sequence ID No. 22 (p697-706) It contains at least 10 consecutive amino acids in the HBV polymerase region from position 693 to 706.

[0074] In a further 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 located C-terminal to position 672 of the HBV polymerase, i.e., an amino acid at position 671, 670, 696, etc.

[0075] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises the amino acid sequence (p723-731) shown in SEQ ID NO: 23.

[0076] In a further 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 located at a position N-terminal to 751 of the HBV polymerase, i.e., an amino acid at position 750, 749, 748, etc.

[0077] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, where the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, and the fragment is - The amino acid sequence shown in SEQ ID NO: 24 (p755-764), and / or - Amino acid sequence shown in Sequence ID No. 25 (p756-765) It contains at least 10 consecutive amino acids in the HBV polymerase region from position 755 to 765.

[0078] In another embodiment, the immunogenic peptide comprises a fragment of HBV polymerase, wherein the fragment is 20 to 34 amino acids long, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, and the fragment comprises the amino acid sequence (p829-837) shown in SEQ ID NO: 26.

[0079] In a further 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 located C-terminal to position 815 of the HBV polymerase, i.e., the amino acid at positions 816, 817, 818, etc.

[0080] In a further embodiment, the peptide of the present invention is Sequence ID 31: HLSLRGLPVCAFSSAGPCALRFTSA(SLP1), Sequence ID 32: LSAMSTTDLEAYFKDCLFKDWEELG(SLP2), Sequence ID 33: ASSSSSCLHQSAVRKAAYSHLSTSK(SLP3), Sequence ID 34: RKLHLYSHPIILGFRKIPMGVGLSP(SLP4), Sequence ID 35: GFAAPFTQCGYPALMPLYACIQAKQA(SLP5), Sequence ID 36:ARQRPGLCQVFADATPTGWGLAIGH(SLP6) and Sequence ID 37: SPSVPSHLPDRVHFASPLHVAWRPP(SLP7) It includes, or consists of, a sequence selected from the group comprising the following.

[0081] In a further embodiment, the peptide of the present invention is Sequence ID 121: KLHLYSHPIILGFRKIPMGVGLSPFLL(SLP8), Sequence ID 122:GLLGFAAPFTQCGYPALMPLYACIQAKQAFT(SLP9), and Sequence ID 123: ARQRPGLCQVFADATPTGWGLAIGHQRMR(SLP10) It includes, or consists of, a sequence selected from the group comprising the following.

[0082] Sequence IDs 121, 122, and 123 are variants of SLP4, SLP5, and SLP6, respectively. Potential improvements of these variants include the introduction of additional putative epitopes / ligands by N-terminal / C-terminal elongation, or improved manufacturability through N-terminal / C-terminal elongation or shortening without loss of (putative) epitopes / ligands.

[0083] In further embodiments, the peptides of the present invention are the peptides described as Sequence IDs 53, 54, 59, 70, 76, and 79 in International Publication No. 15187009, i.e., Sequence ID 38:VVNEKRRLKLIMPARFYPTHTKYLPLDKGIKPYY (Sequence ID 53 in International Publication No. 15187009), Sequence ID 39:YPTHTKYLPLDKGIKPYYPDQVVNHYFQTRHYL (Sequence ID 54 in International Publication No. 15187009), Sequence ID 40:TAESRLVVDFSQFSRGISRVSWPKFAVPNLQSL (Sequence ID 59 in International Publication No. 15187009), Sequence ID 41: QRMRGTFVAPLPIHTAELLAACFARSRSGAKL (Sequence ID 70 in International Publication No. 15187009), Sequence ID 42:ALPSPSPSAVPADHGAHLSLRGLPVCAFSSAGP (Sequence ID 76 in International Publication No. 15187009), or Sequence ID 43:LEAYFKDCVFKDWEELGEEIRLKVFVLGGCRHKL (Sequence ID 79 in International Publication No. 15187009) It does not include any of the above, nor is it one of them.

[0084] In a further key embodiment, the present invention relates to an immunogenic peptide comprising a fragment of HBV protein, wherein the peptide is 20 to 34 amino acids long, and the fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 26.

[0085] Preferably, the immunogenic peptide of the present invention has the ability to induce a potent combination of antigen-directed CD4+ T helper and CD8+ cytotoxic T cell responses when administered to human subjects. Preferably, the peptide is - Activation or induction of the immune system and / or an increase in antigen-specific activated CD4+ and / or CD8+ T cells in peripheral blood or tissue, as established by ELISpot or ELISA assay or related preferred techniques, or by HLA multimer staining of CD4+ or CD8+ T cells, after at least one week of treatment, or an increase in cytokines produced by these T cells, as established by intracellular cytokine staining of CD4+ and CD8+ T cells or cytokine capture thereto in flow cytometry; and / or - Inhibition of the proliferation of antigen-associated infections or a detectable decrease in antigen-expressing cells or a decrease in the cell viability of antigen-expressing cells; and / or - Induction or increased induction of cell death in antigen-expressing cells; and / or - Inhibition of antigen-expressing cells or prevention of their increase; and / or - A decrease in viral load (e.g., a decrease in serum viral DNA, viral RNA, or viral protein) in the precancerous stage of the disease); and / or - Reduction of hepatocytes containing integrated viral DNA and / or covalent closed circular DNA (cccDNA) - Reduction in reference lesion size; and / or - Reduction in reference tumor size; and / or - Increase in survival rates (e.g., progression-free survival rate, overall survival rate) It can be effectively used for the prevention, partial elimination, and / or treatment or complete elimination of HBV, HBV-related diseases, or conditions in a subject, as preferably detectable by the method.

[0086] In a preferred embodiment, the peptide used in the present invention comprises a CTL epitope as described above and a T helper epitope that exhibits binding affinity, preferably at least moderate, and more preferably high, to an HLA class II molecule encoded by an HLA allele dominant in the human population to be treated.

[0087] In a preferred embodiment, the peptide used in the present invention does not have a cysteine ​​residue at the N-terminus or C-terminus of the peptide.

[0088] Furthermore, in another preferred embodiment, the peptide used in the present invention contains no more than two cysteine ​​residues.

[0089] In another preferred embodiment, the peptide used in the present invention contains no more than three methionine molecules.

[0090] In another preferred embodiment, the peptide used in the present invention does not have glutamine at the N-terminus.

[0091] Preferably, the peptides used in the present invention are isolated peptides, where "isolated" does not reflect the degree of purification of the peptide, but rather indicates that the peptide has been taken out of its natural environment (i.e., subjected to human manipulation), and may be recombinant or synthetically produced peptides.

[0092] Peptides are typically prepared synthetically. This may be done by solid-phase peptide synthesis or by any other preferred method.

[0093] In a further aspect, the present invention relates to a polynucleotide comprising a nucleotide sequence encoding the peptide according to the present invention. As described above, the term “HBV protein fragment” means an amino acid sequence corresponding to a partial sequence of the HBV protein. When a peptide is specified to contain a fragment of the HBV protein of a specific length, it means that the fragment is neither shorter nor longer than that length. For example, when a fragment is specified to be 20 to 34 amino acids long, it means that such a peptide does not contain a continuous sequence of the HBV protein longer than 35 amino acids. As a result, the peptides of the present invention do not contain fragments of the HBV protein longer than 34 amino acids. Therefore, it should be understood that the polynucleotides of the present invention comprising a nucleotide sequence encoding the peptide according to the present invention will also not encode peptides containing fragments of the HBV protein longer than 34 amino acids.

[0094] In a further embodiment, the present invention is - Peptides according to the present invention as described herein or polynucleotides according to the present invention as described herein, and - Pharmacologically acceptable carriers Includes, This invention relates to an immunogenic composition that optionally further contains an adjuvant.

[0095] Suitable methods for polynucleotide-based vaccination are described, for example, in Trimble et al. 2015 Lancet 386:2078; Kranz et al. 2016 Nature 534:396; International Publication No. 2011015656A2; Kratzer, et al. 2018 AASLD, The Liver Meeting 2018, abstract #426; International Publication No. 2017080920; and Boni et al. 2019 Int J Mol Sci 20(11):2754.

[0096] In a further embodiment, the present invention relates to a recombinant virus comprising a polynucleotide according to the present invention.

[0097] The immunogenic compositions used in the present invention are preferably for administration to human subjects and are therefore formulated to be suitable for this purpose. Preferably, administration is parenteral, for example, intravenous, subcutaneous, intramuscular, intradermal, intradermal and / or intratumoral, i.e., by injection.

[0098] The immunogenic composition is preferably chemically stable, i.e., the peptides in the composition do not chemically degrade or decompose. Therefore, preferably, after storing the solution or liquid composition at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the amount of undegraded, undegraded, and / or unreacted peptides in the solution and / or composition is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% by weight compared to the initial amount. Chemical stability can be evaluated using any suitable technique known in the art, for example, by ULC / MS as exemplified herein. When using UPLC / MS, a solution / composition is defined as chemically stable if, after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours, the total percentage peak area on the UV spectrum that does not correspond to the desired peptide product is at most 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% compared to the initial state.

[0099] The immunogenic composition is preferably also physically stable, i.e., the peptides in the composition do not precipitate or redisperse. Physical stability can be evaluated using any suitable technique known in the art, for example, by visual inspection or by particle distribution using a Malvern Mastersizer as illustrated herein (where the average particle size is expressed in units of D(0.5)). When using a Malvern Mastersizer to evaluate physical stability as illustrated herein, the solution / composition is defined as physically stable if the increase in average D(0.5) after storage at room temperature for at least about 0.5, 1, 1.5, 2, or at least 3 hours is at most 50%, 40%, 30%, 20%, 10%, or 5% compared to its initial state (i.e., the freshly prepared solution as is after preparation). Preferably, a solution / composition is defined as physically stable if the increase in the average D(0.5) after storage at room temperature for 3 hours is at most 50%, 40%, 30%, 20%, 10%, or 5%, preferably at most 20%, compared to its initial value.

[0100] In one embodiment, the immunogenic composition comprises or consists of a mixture of dried or lyophilized peptides to be administered together.

[0101] The immunogenic compositions used in the present invention may be prepared by any preferred method. In some embodiments, one or more immunogenic compositions are prepared from dried, preferably lyophilized, peptides.

[0102] For example, this composition, in the following steps: a) A step of providing a vial containing a dried, preferably lyophilized, peptide; b) Preferably a step of thawing the peptide for about 5 to 30 minutes; c) Adding the reconstituted composition to a vial containing the peptide, preferably without rotating the vial; d) Preferably a step of mixing for about 0.5 to 5 minutes; and e) Preferably, the solution is stirred for about 1 to 3 minutes until a clear solution is obtained. It may be prepared by a method including

[0103] Preferably, steps b) to e) are carried out at room temperature.

[0104] Preferably, the vial contains an injection volume as a single dose in a preventive and / or therapeutic method, preferably a therapeutic and / or preventive method as defined herein, i.e., a single drug dose unit, or, if multiple injections are administered to different parts of the body at substantially the same time, a portion thereof.

[0105] In one embodiment, the reconstituted composition of step c) comprises or consists of DMSO and / or water for injection. In another embodiment, the reconstituted composition of step c) of the peptide reconstituted method comprises or consists of an aqueous solution containing an organic acid at about 60-80% v / v, propylene glycol (CAS number 57-55-6) at about 5-10% v / v, a lower alcohol at about 10-20% v / v, and a nonionic hydrophilic surfactant at about 5-10% v / v. In one embodiment, the organic acid is citric acid, which 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 is a. Mono, di, or triglycerides, preferably ethoxylated triglycerides, and / or b. Having a hydrophilic-lipophilic balance (HLB) value of 9 to 14. In further embodiments, the nonionic hydrophilic surfactant is ethoxylated castor oil, preferably polyoxyethylene glycerol trilicinoleic acid 35 (CAS number 61791-12-6).

[0106] In one embodiment, the composition comprises or consists of an aqueous solution containing about 75% v / v about 0.1 M citric acid, about 6.25% v / v propylene glycol (CAS number 57-55-6), about 12.5% ​​v / v ethanol, and about 6.25% v / v polyoxyethylene glycerol trilicinoleic acid 35 (CAS number 61791-12-6).

[0107] 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 peptide reconstituted in step (a) is the total amount of reconstituted peptide in the clear solution obtained after step e), i.e., the amount of reconstituted peptide in the clear solution obtained after step e).

[0108] In one embodiment, the reconstituted composition contains or comprises about 1-2 mg / mL of peptide, 0.038 M citric acid, about 3.13% v / v propylene glycol (CAS No. 57-55-6), about 6.25% v / v ethanol, about 3.13% v / v polyoxyethylene glycerol trilicinoleic acid 35 (CAS No. 61791-12-6), and about 50% oil-based adjuvant, preferably Montanide ISA 51 VG (Seppic), in water.

[0109] The dried peptide may be a peptide without further components, but may also contain buffer components such as trifluoroacetic acid (TFA), and salts such as sodium salts, potassium salts, or phosphates (e.g., NaCl, KCl, and NaPO4). The amount of further components is preferably less than 30%, more preferably less than 25%, of the total weight of the dried peptide to be reconstituted. The dried peptide to be reconstituted may be physically dried, as can be obtained by processes such as rotational evaporation, freeze-drying, and spray-drying, but is not limited to these.

[0110] Adjuvant In one embodiment, the composition of the present invention further comprises an adjuvant, or the treatment or use according to the present invention further comprises the administration of an adjuvant. The term “adjuvant” is used herein to mean a substance that has an immunoenhancing effect and is co-administered with, added to, or co-formulated with an antigen to enhance, induce, elicit, and / or modulate an immune response to the antigen upon administration to a subject. In one embodiment, the adjuvant is physically linked, such as by covalent linkage to one or more peptides to be reconstituted.

[0111] In one embodiment, the adjuvant is an emulsified adjuvant. For example, in one embodiment, the adjuvant is an oil-based adjuvant. Using an oil-based adjuvant, an emulsion (e.g., a water-in-oil or oil-in-water emulsion) can be formed, which in the art has been shown to enhance and induce an immune response. Preferably, the oil-based adjuvant is a mineral oil-based adjuvant. Non-limiting examples of oil-based adjuvants include bio-based oil adjuvants (based on vegetable oil / fish oil, etc.), squalene-based adjuvants (e.g., MF59), and Syntex adjuvant formulations (SAF; Lidgate, Deborah M, Preparation of the Syntex Adjuvant Formulation (SAF, SAF-m, and SAF-1), In: Vaccine Adjuvants, Volume 42 of the series Methods in Molecular Medicine). TMpp. 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. Immunologic adjuvants: general properties, advantages, and limitations. In: Zola, H. (Ed.), Laboratory Methods in Immunology. pp. 39-51), ASO4 (A. Tagliabue, R. Rappuoli Vaccine adjuvants: the dream becomes real Hum. Vaccine, 4(5), 2008, pp. 347-349), purified squalene and Montanide adjuvants based on squalene emulsified with highly purified monooleic acid mannide (e.g., Montanide ISA 25) These are VG, 28 VG, 35 VG, 50 V, 50 V2, 51 VG, 61 VG, 70 VG, 70 M VG, 71 VG, 720 VG, 760 VG, 763 A VG, 775 VG, 780 VG, 201 VG, 206 VG, and 207 VG. More preferably, the oil-based adjuvant is Montanide ISA 51VG(Seppic), which is a mixture of Drakeol VR and mannide monooleate.

[0112] Other suitable adjuvants are those that activate antigen-presenting cells, such as dendritic cells. For example, such adjuvants may be activated via Toll-like receptors and / or via the RIG-I (retinoic acid-inducible gene-1) protein and / or via endothelin receptors. Immunomodulatory compounds capable of activating the innate immune system can be activated particularly well via Toll-like receptors (TLRs), including TLR1-10. Compounds capable of activating TLR receptors, as well as their modifications and derivatives, are well-documented in the art. TLR1 can be activated by bacterial lipoproteins and their acetylated forms, while TLR2 can be activated, in addition, by Gram-positive bacterial glycolipids, LPS, LPA, LTA, pili, outer membrane proteins, bacterial or host-derived heat shock proteins, and mycobacteria lipoarabinomannan. TLR3 can be activated by dsRNA, particularly viral dsRNA, or by the chemical compound poly(I:C). TLR4 can be activated by Gram-negative LPS, LTA, heat shock proteins of host or bacterial origin, viral envelope or covering proteins, taxol or its derivatives, hyaluronan-containing oligosaccharides, and fibronectin. TLR5 can be activated by bacterial flagella or flagellin. TLR6 can be activated by mycobacterial lipoproteins and the heat-labile soluble factor (GBS-F) of group B Streptococcus or modulin of Staphylococcus. TLR7 can be activated by imidazoquinolines such as imiquimod and reximod, and derivatives of imiquimod or reximod (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 containing dsRNA, poly(I:C), poly(I:CLC), unmethylated CpG DNA (which elicits TLR3 and TLR9 receptors), IC31, TLR9 agonists, IMSAVAC, TLR4 agonists, Montanide ISA-51, and Montanide ISA 720 (Seppic, an adjuvant produced in France). It is well known that RIG-I proteins, like TLR3, are activated by ds-RNA (Kato et al, (2005) Immunity, 1:19-28).

[0113] Further particularly preferred TLR ligands are pam3cys and / or derivatives thereof, preferably pam3cys lipopeptides or their variants or derivatives, preferably those described in International Publication No. 2013051936A1, and more preferably U-Pam12, U-Pam14, or AMPLIVANT®. Pam3cys and / or derivatives thereof may optionally be covalently linked to one or more peptide antigens.

[0114] Further preferred adjuvants are cyclic dinucleotides (CDNs), muramyl dipeptides (MDPs), and poly-ICLCs. In preferred embodiments, the adjuvants of the present invention are poly-ICLCs, imidazoquinolines such as pam3cys lipopeptide derivatives as described in International Publication No. 2013051936A1, imiquimod, resikimod or derivatives thereof, CpG oligodeoxynucleotides (CpG-ODNs) having sequences not found in nature, and peptide-based adjuvants, such as muramyl dipeptides (MDPs) or tetanus toxoid peptides containing amino acids not found in nature, and other adjuvants not found in nature.

[0115] Furthermore, 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, ImuFact EV1P321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, Lipoplex, liposomes, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, nanoparticles (including nanoparticles with adjuvants), OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector systems, PLGA microparticles, SRL172, virosoms and other virus-like particles, Pam3Cys-GDPKHPKSF, YF-17D, VEGF Adjuvants selected from the group consisting of trap, R848, β-glucan, Aquila's QS21 stimulon, bajimezan, AsA404 (DMXAA), STING (IFN gene stimulant) agonist (e.g., c-di-GMP VacciGrade®), PCI, NKT (natural killer T cell) agonist (e.g., α-galactosylceramide or α-GalCer), RNAdjuvant® (Curevac), and retinoic acid-inducible protein I ligand (e.g., 3pRNA or 5'-triphosphate RNA) are preferred.

[0116] Treatment and Usage The immunogenic peptides, polynucleotides, recombinant viruses, and immunogenic compositions of the present invention can be used for the treatment of HBV infections, such as chronic HBV infection, and / or HBV-related diseases.

[0117] Examples of diseases treated with the immunogenic peptides, polynucleotides, and immunogenic compositions of the present invention include, without limitation, hepatitis B infections such as chronic hepatitis B infection, hepatitis B-related cirrhosis, and hepatitis B-related hepatocellular carcinoma.

[0118] Accordingly, in a further embodiment, the present invention relates to peptides, polynucleotides, or immunogenic compositions according to the present invention for use as pharmaceuticals.

[0119] In a further embodiment, the present invention relates to peptides, polynucleotides, or immunogenic compositions according to the present invention for use in the treatment or prevention of HBV-related diseases.

[0120] In a further embodiment, the present invention relates to a method for treating or preventing HBV-related disease, comprising the step of administering an immunogenic peptide, a polynucleotide, or an immunogenic composition according to the present invention to a human subject in need thereof.

[0121] Preferably, administration is intravenous, subcutaneous, or intramuscular, but other routes of administration such as mucosal administration or intradermal and / or intradermal administration, for example by injection, can also be considered.

[0122] Preferably, administration of one or more immunogenic compositions induces a cytotoxic CD8+ T cell response to at least one HLA-binding peptide sequence presented by an HLA class I molecule contained in a long peptide. More preferably, administration of one or more immunogenic compositions induces a cytotoxic CD8+ T cell response in conjunction with a helper CD4+ T cell response to an HLA-binding peptide sequence presented by an HLA class II molecule contained in one or more immunogenic compositions. Preferably, administration is - Activation or induction of the immune system and / or an increase in antigen-specific activated CD4+ and / or CD8+ T cells in peripheral blood or tissue, as established by ELISpot or ELISA assay or related preferred techniques, or by HLA multimer staining of CD4+ or CD8+ T cells, after at least one week of treatment, or an increase in cytokines produced by these T cells, as established by intracellular cytokine staining of CD4+ and CD8+ T cells or cytokine capture thereto in flow cytometry; and / or - Inhibition of the proliferation of antigen-associated infections or a detectable decrease in antigen-expressing cells or a decrease in the cell viability of antigen-expressing cells; and / or - Induction or increased induction of cell death in antigen-expressing cells; and / or - Inhibition of antigen-expressing cells or prevention of their increase; and / or - Reduction in viral load (precancerous stage of disease); and / or - Reduction in reference lesion size; and / or - Reduction in reference tumor size; and / or - Increased survival rate (e.g., PFS, OS) The administration is for the prevention, partial elimination and / or treatment or complete elimination of HBV-related infection or disease in a subject, as preferably detectable by [method / tool].

[0123] combination In some embodiments, the treatment involves administering an immunogenic peptide, polynucleotide, or recombinant virus of the present invention in combination with a further immunogenic peptide, polynucleotide, or recombinant virus. For example, the treatment may involve administering two or more immunogenic peptides, such as 3, 4, 5, 6, 7, 8, or more.

[0124] Two or more peptides may all be contained in a single immunogenic composition, or multiple peptides may be divided into two or more compositions. If peptides are divided into two or more compositions, these compositions may be mixed before administration and co-administered, or they may be administered separately. Typically, all compositions, and indeed all peptides in multiple peptides, are administered to the subject within a 24-hour timeframe, preferably within 4, 2, or 1 hours.

[0125] If two or more compositions are administered, the administration may be to the same site, e.g., the same limb, or to two or more different sites. During the course of treatment, one or more compositions may be administered once, or instead, they may be repeated (boosted) two or three times, or more.

[0126] In a preferred embodiment, the therapeutic method comprises a combination of long peptides, wherein the combination of long peptides comprises HLA-binding peptide sequences having the ability to bind to at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of HLA class I molecules encoded by the dominant HLA allele in the human subject population to be treated. Preferred HLA class I HLA-binding peptide sequences in the long peptide according to the present invention are combinations of HLA class I allele supertype classes 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, HLA-A*01 / A*03 and HLA-A*01 / A*24, and their respective subtypes (Sidney et al 2008 BMC Immunology) 9), preferably HLA-A0101;HLA-A0201;HLA-A0206;HLA-A0301;HLA-A1101;HLA-A230 1;HLA-A2402;HLA-A2501;HLA-A2601;HLA-A2902;HLA-A3001;HLA-A3002;HLA-A 3101;HLA-A3201;HLA-A3303;HLA-A6801;HLA-A6802;HLA-A7401;HLA-B0702;H LA-B0801;HLA-B1301;HLA-B1302;HLA-B1402;HLA-B1501;HLA-B1502;HLA-B152 5;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 are HLA-binding peptide sequences.In a preferred embodiment, the therapeutic method comprises a combination of long peptides, the combination of long peptides comprising an HLA-binding peptide sequence having binding ability to at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of HLA class I molecules encoded by a dominant HLA allele in the human subject population to be treated, and an HLA-binding peptide sequence having binding ability to at least 20%, 30%, 40%, 42%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of HLA class II molecules.

[0127] In a preferred embodiment, the long peptide used in the present invention comprises an HLA-binding peptide sequence that exhibits binding affinity, preferably at least moderate, more preferably high, to an HLA class I molecule encoded by a dominant HLA allele in the human target population to be treated, and that elicits a cytotoxic CD8+ T cell response. Preferably, the long peptide used in the present invention comprises an HLA-binding peptide sequence that exhibits binding affinity, preferably at least moderate, more preferably high, to at least one HLA class I molecule from the following group of HLA class I molecules: [ka] [ka] [ka] [ka] [ka] [ka]

[0128] As described, the treatment may include the administration of two or more antigens of the present invention. In one embodiment, the present invention is a method for treating or preventing HBV-related disease in a human subject. - A peptide as described in claim 1a) and a peptide as described in claim 1b), or - A peptide as described in claim 1a) and a peptide as described in claim 1c), or - A peptide as described in claim 1a) and a peptide as described in claim 1d), or - A peptide as described in claim 1a) and a peptide as described in claim 1e), or - A peptide as described in claim 1a) and a peptide as described in claim 1f), or - The peptide as described in claim 1a) and the peptide as described in claim 1g), or - A peptide as described in claim 1a) and a peptide as described in claim 1h), or - A peptide as described in claim 1a) and a peptide as described in claim 1i), or - A peptide as described in claim 1a) and a peptide as described in claim 1j), or - A peptide as described in claim 1a) and a peptide as described in claim 1k), or - A peptide as described in claim 1a) and a peptide as described in claim 1l), or - A peptide as described in claim 1a) and a peptide as described in claim 1m), or - A peptide as described in claim 1b) and a peptide as described in claim 1c), or - A peptide as described in claim 1b) and a peptide as described in claim 1d), or - A peptide as described in claim 1b) and a peptide as described in claim 1e), or - A peptide as described in claim 1b) and a peptide as described in claim 1f), or - The peptide as described in claim 1b) and the peptide as described in claim 1g), or - A peptide as described in claim 1b) and a peptide as described in claim 1h), or - The peptide as described in claim 1b) and the peptide as described in claim 1i), or - A peptide as described in claim 1b) and a peptide as described in claim 1j), or - A peptide as described in claim 1b) and a peptide as described in claim 1k), or - A peptide as described in claim 1b) and a peptide as described in claim 1l), or - A peptide as described in claim 1b) and a peptide as described in claim 1m), or - The peptide as described in claim 1c) and the peptide as described in claim 1d), or - A peptide as described in claim 1c) and a peptide as described in claim 1e), or - A peptide as described in claim 1c) and a peptide as described in claim 1f), or - The peptide as described in claim 1c) and the peptide as described in claim 1g), or - A peptide as described in claim 1c) and a peptide as described in claim 1h), or - The peptide as described in claim 1c) and the peptide as described in claim 1i), or - A peptide as described in claim 1c) and a peptide as described in claim 1j), or - A peptide as described in claim 1c) and a peptide as described in claim 1k), or - A peptide as described in claim 1c) and a peptide as described in claim 1l), or - A peptide as described in claim 1c) and a peptide as described in claim 1m), or - The peptide as described in claim 1d) and the peptide as described in claim 1e), or - The peptide as described in claim 1d) and the peptide as described in claim 1f), or - The peptide as described in claim 1d) and the peptide as described in claim 1g), or - The peptide as described in claim 1d) and the peptide as described in claim 1h), or - The peptide as described in claim 1d) and the peptide as described in claim 1i), or - The peptide as described in claim 1d) and the peptide as described in claim 1j), or - The peptide as described in claim 1d) and the peptide as described in claim 1k), or - A peptide as described in claim 1d) and a peptide as described in claim 1l), or - A peptide as described in claim 1d) and a peptide as described in claim 1m), or - A peptide as described in claim 1e) and a peptide as described in claim 1f), or - The peptide as described in claim 1e) and the peptide as described in claim 1g), or - A peptide as described in claim 1e) and a peptide as described in claim 1h), or - A peptide as described in claim 1e) and a peptide as described in claim 1i), or - A peptide as described in claim 1e) and a peptide as described in claim 1j), or - A peptide as described in claim 1e) and a peptide as described in claim 1k), or - A peptide as described in claim 1e) and a peptide as described in claim 1l), or - A peptide as described in claim 1e) and a peptide as described in claim 1m), or - The peptide as described in claim 1f) and the peptide as described in claim 1g), or - A peptide as described in claim 1f) and a peptide as described in claim 1h), or - A peptide as described in claim 1f) and a peptide as described in claim 1i), or - A peptide as described in claim 1f) and a peptide as described in claim 1j), or - A peptide as described in claim 1f) and a peptide as described in claim 1k), or - A peptide as described in claim 1f) and a peptide as described in claim 1l), or - A peptide as described in claim 1f) and a peptide as described in claim 1m), or - The peptide as described in claim 1g) and the peptide as described in claim 1h), or - The peptide as described in claim 1g) and the peptide as described in claim 1i), or - The peptide as described in claim 1g) and the peptide as described in claim 1j), or - The peptide as described in claim 1g) and the peptide as described in claim 1k), or - The peptide as described in claim 1g) and the peptide as described in claim 1l), or - The peptide as described in claim 1g) and the peptide as described in claim 1m), or - The peptide as described in claim 1h) and the peptide as described in claim 1i), or - A peptide as described in claim 1h) and a peptide as described in claim 1j), or - A peptide as described in claim 1h) and a peptide as described in claim 1k), or - A peptide as described in claim 1h) and a peptide as described in claim 1l), or - A peptide as described in claim 1h) and a peptide as described in claim 1m), or - A peptide as described in claim 1i) and a peptide as described in claim 1j), or - The peptide as described in claim 1i) and the peptide as described in claim 1k), or - The peptide as described in claim 1i) and the peptide as described in claim 1l), or - A peptide as described in claim 1i) and a peptide as described in claim 1m), or - A peptide as described in claim 1j) and a peptide as described in claim 1k), or - A peptide as described in claim 1j) and a peptide as described in claim 1l), or - A peptide as described in claim 1j) and a peptide as described in claim 1m), or - A peptide as described in claim 1k) and a peptide as described in claim 1l), or - A peptide as described in claim 1k) and a peptide as described in claim 1m), or - Peptides as described in claim 1l) and peptides as described in claim 1m) or - One or two polynucleotides containing a sequence encoding any one of the peptide combinations described above. The present invention provides a method including the administration of [a substance].

[0129] Preferably, the immunogenic composition contains or consists of a peptide in an amount constituting a pharmaceutical dose. A pharmaceutical dose is defined herein as the amount of the active ingredient applied to a subject at a given time (i.e., the total amount of peptides in the peptide-based immunogenic composition). The pharmaceutical dose may be applied to a subject in a single volume, i.e., a single injection, or preferably in two, three, four, five or more divided volumes applied to different locations on the body, for example, the right and left limbs. There may be several reasons for applying a single pharmaceutical dose in divided volumes, including avoiding negative side effects, avoiding antigen competition, and / or considerations for compositional analysis.

[0130] The pharmaceutical dose may be an effective dose or a portion thereof. “Effective dose” should be understood herein as the amount or dose of the active ingredient necessary to prevent and / or reduce the symptoms of a disease (e.g., chronic infection, precancerous conditions and / or cancer) compared to an untreated patient. The effective dose of one or more active compounds used in the implementation of the present invention for the prophylactic and / or therapeutic treatment of a disease or condition will vary depending on the method of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and dosage regimen. Such a dose is referred to as the “effective” dose. This effective dose may 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.

[0131] Preferably, the pharmaceutical dose in a single or multiple injection or administration at a specific time point, i.e., the total amount of peptide applied to the target at a given time point, is 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, Contains peptides in any amount between 0.1 micrograms and 20 mg, such as 450 micrograms, 500 micrograms, 650 micrograms, 700 micrograms, 750 micrograms, 800 micrograms, 850 micrograms, 900 micrograms, 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 approximately 20 mg. The preferred dosage ranges are 0.1 micrograms to 20 mg, 1 microgram to 10 mg, 10 micrograms 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.

[0132] In one embodiment, the immunogenic composition used in the present invention is administered in doses of 50 to 150 micrograms of each peptide, such as 1 to 300 micrograms, for example, about 100 micrograms.

[0133] The method of the present invention may be part of a combination therapy with other forms of HBV treatment, the other treatment may be provided as a separate treatment or added to the immunogenic composition of the present invention. The method of the present invention may include drugs that inhibit viral replication (e.g., nucleosides or nucleotide analogs including entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide), and / or drugs that prevent HBV from entering cells (e.g., milkludex), and / or drugs that inhibit viral protein production (e.g., siRNA, shRNA, CRISPR / CAS9-based), and / or drugs that modulate the immune response (e.g., PEG-interferon α), drugs that activate the innate immune response (e.g., αGalCer), and / or vaccines. Hepatitis B immunoglobulin (HBIG) and / or HBV prophylactic vaccines that induce HBsAg-specific antibodies to support the immune response induced by and / or hepatectomy or liver 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)Drugs that stimulate checkpoint molecules (e.g., selected tumor necrosis factor (TNF) receptors), such as 3-dioxygenase, KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), NOX2 (nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2), PD-1 (programmed death 1), PD-L1, TIM-3 (T cell immunoglobulin domain and mucin domain 3), VISTA (V-domain Ig suppressor for T cell activation), SIGLEC7 / CD328 and SIGLEC9 / CD329, NKG2A and / or selected tumor necrosis factor (TNF) receptors. It may be combined with superfamily members (e.g., CD27, CD40, CD122, 4-1BB / CD137, OX-40 / CD134, and GITR (related to the glucocorticoid-inducible 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 further immunomodulators (e.g., compounds known to deplete immunosuppressive Tregs and / or MDSCs).

[0134] In one embodiment, one or more immunogenic peptides or compositions of the present invention are provided in the following steps (i) A step of reducing the viral load by administering antiviral (e.g., NA) therapy to patients infected with HBV, (ii) The step of administering one or more immunogenic peptides or compositions of the present invention (preferably intradermally or subcutaneously) when the viral load has been significantly reduced (for example, to less than 2, 5, or 1 / 10), and (iii) After sufficient time has elapsed for an initial T cell response in the patient (e.g., 2 to 16 weeks after the final dose of the immunogenic composition, i.e., 2 to 12 weeks, e.g., 2 to 8 weeks), an optional immunomonitoring (e.g., characterization of peripheral and intrahepatic T cells, more specifically, determination of the vaccine-specific T cell response to administration of one or more immunogenic peptides of the composition of the present invention by, for example, IFNγ ELISpot analysis or antigen-specific T cell proliferation and / or FACS-based phenotyping of antigen-specific T cells; optionally accompanied by determination of the T cell response to the non-vaccine antigen of interest and unrelated control microbial antigens) may be performed in a therapeutic regimen to evaluate whether a suitable T cell response has been induced by the vaccine (see, for example, Rivino et al. (2018) J Clin Invest 128:668). Next, antiviral therapy is interrupted or discontinued to increase viral antigen presentation and thereby further boost HBV-specific T cells in situ, and to expose infected hepatocytes to the immune system (i.e., by increasing hepatocyte HBV protein expression and antigen presentation) to promote the removal of remaining infected hepatocytes. To further enhance T cell effector function, therapeutic vaccination may be optionally combined with drugs that target suppressor myeloid cells (MDSCs) (administered before therapeutic vaccination), siRNA (before therapeutic vaccination), drugs that modify T cell metabolism (before or during therapeutic vaccination), or checkpoint blockade (during or after therapeutic vaccination). Preferably, the effectiveness of the vaccine is determined by adequate monitoring of the viral load to determine follow-up (combination) therapy and / or discontinuation of NA. Figure 8 provides a non-limiting illustration of a treatment regimen according to this embodiment. This treatment regimen is particularly suitable for patients who are recommended to receive antiviral therapy according to treatment guidelines. Antiviral treatment before vaccination may not always be necessary for patients with a persistent but low viral load.Such patients are eligible to receive therapeutic vaccination at any given time, and optional immune monitoring is performed to assess whether a favorable T-cell response has been induced by the vaccine once sufficient time has elapsed for an initial T-cell response in the patient. To further enhance T-cell effector function, therapeutic vaccination may be optionally combined with drugs that target suppressor myeloid cells (MDSCs) (administered before therapeutic vaccination), siRNA (before therapeutic vaccination), drugs that modify T-cell metabolism (before or during therapeutic vaccination), or checkpoint blockade (during or after therapeutic vaccination). Vaccine efficacy is preferably determined and follow-up (combination) therapy is decided by adequate monitoring of viral load.

[0135] In a further embodiment, the immunogenic peptide or composition may be used in an exovivoimmunization regime. In an exovivoimmunization regime, the peptide or composition may be used to stimulate the creation of antigen-loaded antigen-presenting cells (APCs), such as antigen-loaded activated dendritic cells (DCs), and the subsequent expansion of antigen-specific T cells (e.g., CD4 and CD8-positive circulating T cells, tumor-infiltrating lymphocytes (TILs)). Such antigen-loaded APCs or expanded antigen-specific T cells are subsequently administered to human subjects.

[0136] Accordingly, in a further embodiment, the present invention relates to a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 26, for use in exo vivo stimulation of antigen-loaded activated antigen-presenting cells or expanded antigen-specific T cells.

[0137] Similarly, in another embodiment, the method of the present invention comprises treating a human subject with an antigen-loaded activated antigen-presenting cell (APC) or an expanded population of antigen-specific T cells, wherein the cells are produced ex vivo (i.e., in vitro) using one or more immunogenic compositions described herein. This may be done, for example, by culturing patient PBMCs to produce autologous activated APCs (e.g., DCs), loading them with an immunogenic composition (i.e., antigen-loaded APCs), and subsequently stimulating and expanding T cells or tumor-infiltrating lymphocytes obtained from the PBMCs. Alternatively, antigen-specific T cells may be expanded by incubation with activated APCs cultured from HLA-compatible healthy donor PBMCs loaded with an immunogenic composition. Suitable techniques are described in the art for the purposes of this study, for example, in McCormack et al. (2018) Cytotherapy 20:385; Stevanovic et al. (2015) J Clin Oncol 33:1543; and Stevanovic et al. (2018) Clin Cancer Res, doi:10.1158 / 1078-0432.

[0138] In further embodiments, therapy with activated antigen-presenting cells (APCs) or an expanded population of antigen-specific T cells is combined with direct immunization of a human subject with an immunogenic composition described herein. Such combined protocols may include sequential and / or concurrent administration.

[0139] All patents and references cited herein are incorporated by reference in their entirety. [Examples]

[0140] Example 1: Alignment of HBV-X and HBV polymerase with consensus sequences and functional domains Frequency tables were downloaded from HBVdb V42.0 (Hayer et al, 2013 Nucleic Acid Res 41:566) based on the HBV sequences of all genotypes for HBV-X (n=8127) and HBV polymerase (n=7489). After removing the positions with the highest frequency of gaps (indicated by "-"), the dominant amino acid at each position was determined. The proportion of sequences containing the dominant amino acid was calculated as a conservation score.

[0141] Combining all the dominant amino acids for HBV polymerase gives the consensus sequence (SEQ ID NO: 28): [ka] It leads to this.

[0142] The consensus sequence obtained for HBV-X is (SEQ ID NO: 30): [ka] That was the decision.

[0143] In addition, amino acids previously associated with viral replication loss, either individually or in combination, were aligned with the consensus sequence (Figures 1 and 2). Pol had more functionally relevant amino acids compared to HBV-X. Therefore, an additional threshold of 50% or more loss of viral persistence was introduced for Pol to select the most essential amino acids. Tables 1 and 2 list references regarding functional domains and amino acids.

[0144] [Table 1]

[0145] [Table 2] TIFF0007869419000011.tif212147 TIFF0007869419000012.tif174147

[0146] Example 2: Predicted novel HLA class I binding peptides derived from HBV-x and HBV polymerase The inventors embarked on identifying novel peptides that can bind to at least one of six HLA supertypes (i.e., supertype HLA-A*01; A*02; A*03; A*24; B*07; B*08) that are commonly found in Caucasians, Africans, or Asians, and for which in vitro assays to confirm binding can be optionally used. First, the inventors created a frequency distribution of expected conjugates by predicting conjugates spanning 8 to 14 amino acids for a representative HLA type of the supertype using the established in silico prediction tool NetMHCpan (Nielsen and Andreatta 2016, Genome Med 8:33) (Figures 1 and 2, gray bar charts). The density of all expected conjugates per amino acid was similar between Pol and HBx (mean ± SD Pol 16.36 ± 12.62, HBx 15.60 ± 9.49; Mann-Whitney; p=0.57). Next, since the 9-11mer is most likely to correspond to a relevant epitope (Trolle et al. 2016 J Immunol 196:1480), the predicted conjugates spanning 9-11 amino acids were aligned with the inventors' map illustrating the preservation and function outlined in Example 1 (Figures 1 and 2).

[0147] This prediction resulted in a total of 251 novel HLA conjugate candidates for HBx and 1655 for Pol. Of these, the inventors selected the most promising peptides to validate binding using an in vitro HLA binding assay based on UV-induced peptide exchange (Toebes et al. 2006 Nat Med 12:246). For practical and economic reasons, the inventors aimed to test the binding of 96 unique peptide sequences across various HLA types for both proteins. The inventors placed the binding ability of the inventors' newly identified conjugates in context, including two well-described epitopes (cores 18-27 and Pol 549-557). These 96 conjugate candidates were selected based on peptide length (preferably 9mer), expected HLA binding strength, conservation of included amino acids, and functional importance. Not all of the 96 conjugate candidates always met all criteria. For HLA-A*01 and HLA-A*24, the number of expected conjugates to HBx was unsatisfactory in terms of maintaining the inventors' stringent thresholds for conservation and peptide length. Therefore, for these conditions, the inventors also included peptides with somewhat lower conservation or peptides spanning 8 to 12 amino acids (see Example 3, indicated by an asterisk in Figure 3). In addition, the inventors selected several peptides that have rarely been reported as epitopes in the literature (once or twice) and are therefore considered unestablished (see Example 3, underlined with a solid line in Figure 3). This included c123-130, as it was the only HBV-derived epitope registered in Hepitopes in relation to HLA-B*08. Furthermore, throughout this selection procedure, prioritizing peptides expected to bind to several HLA types, a total of 113 pairs of conjugate candidates were tested. Overall, this selection procedure yielded 45 candidate conjugates for HBx and 68 for Pol for validation in in vitro conjugation assays. The vast majority of these mapped to highly conserved regions with established functional importance.The median conservation rates among the selected peptides were over 93% for HBx and over 96% for Pol.

[0148] Example 3: In vitro binding ability of selected peptides derived from HBx and polymerase 3.1 Method In the in vitro binding assay as previously described (Karimzadeh et al. 2018 J Virol 92:e01891), a synthetic peptide of the selected HLA conjugate candidate (Peptide 2.0 Inc.) was used. Specifically, the peptide exchange reaction was carried out by exposing a conditional peptide-HLA complex (pHLA) (0.53 μM) to long-wavelength ultraviolet light using a 366 nm UV lamp (Camag) for 30 minutes, either in the presence or absence of the indicated peptide (50 μM). Subsequently, the peptide exchange efficiency was analyzed using an HLA class I enzyme-linked immunosorbent assay (ELISA) to detect β-2 microglobulin of the peptide-stabilized HLA class I complex in the exchange reaction mixture. For this purpose, streptavidin (2 μg / ml) was conjugated to polystyrene microtiter wells (Nunc MaxiSorp). After washing and blocking, HLA complexes or controls present in the exchange reaction mixture were captured by streptavidin on a microtiter plate using their biotinylated heavy chains (incubation at 37°C for 1 hour). Unbound material was removed by washing. Subsequently, horseradish peroxidase (HRP) conjugate antibody against human β-2-microglobulin (0.6 μg / ml; Sanquin Reagents BV) was added (incubation at 37°C for 1 hour). After removing the unbound HRP conjugate by washing, ABTS[2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt](Sanquin Reagents BV) substrate solution was added to the wells. After 8 minutes (incubation at room temperature), the reaction was stopped by adding 2% (w / v) oxalic acid dihydrate stop solution (Sanquin Reagents BV), and the readings were taken at 414 nm using a Thermo Electron Multiskan Ascent ELISA reader. Each peptide was independently exchanged twice. In this HLA class I ELISA, all exchange mixtures were measured in duplicate. The absorbance of all peptides was normalized to the absorbance of a known HLA allele-specific ligand with high affinity for each corresponding allele (corresponding to 100%; Table 3).Negative controls included ultraviolet irradiation of the conditional HLA class I complex in the absence of HLA allele-specific non-conjugates and rescue peptides.

[0149]

Table 3

[0150] 3.2 Results In the plate-based in vitro binding assay as described in Section 3.1, the binding ability of the selected peptides was tested. Peptides were classified as HLA binders when their binding ability was higher than 25% of that of a known high-affinity peptide. Since many HBV-infected patients are Asian and the prevalence of HLA-A*11:01 is high in this population compared to HLA-A*03:01, a representative example of a supertype with a high prevalence in Caucasians (Chang et al 2013 Eur J Immunol 43:1109), both HLA-A*11:01 and HLA-A*03:01 were tested as members of the HLA-A*03 supertype.

[0151] The inventors identified 13 conjugates for HBx and 33 conjugates for Pol across various HLA supertypes (grey bars in Figure 3). This included novel conjugates previously described in the context of other HLA types (dashed underlines in Figures 3A - 3G and Figure 3H). Notably, for each HLA supertype tested, both HBx-derived and Pol-derived conjugates were identified. For HLA-A*02, the well-established epitopes c18-27 and p549-557 had better scores than the positive control (Figure 3B). In contrast, the binding of epitopes that have been rarely reported (solid underlines in Figure 3) was not always confirmable.

[0152] In general, this binding prediction proved to be quite poor, as effectively only one-third of the predicted conjugates exhibited binding ability above the threshold. Many peptides that were predicted to bind strongly (low rank scores in the in silico prediction) did not show above-threshold binding in the in vitro assay (Figure 4).

[0153] Table 4 compares the predicted scores (ranks) of the peptides tested for each HLA type with the assay results (binding percentage).

[0154] [Table 4] TIFF0007869419000015.tif185147 TIFF0007869419000016.tif173147 TIFF0007869419000017.tif173147 TIFF0007869419000018.tif172147 TIFF0007869419000019.tif179147 TIFF0007869419000020.tif172147

[0155] Example 4: Immunogenicity of selected HLA-binding peptides 4.1 Method Immunogenicity was evaluated for peptides with binding scores higher than 25% in in vitro HLA binding assays. In short, PBMCs were isolated from buffy coats of nine donors who had previously been HBV-cleared by Ficoll (GE Healthcare) density centrifugation. The buffy coats were provided by local blood banks along with their corresponding two-digit HLA types. Four-digit HLA typing was performed in seven of the nine donors using the Global Screening Array (GSA) (Illumina through Human Genomics Facility Erasmus MC Rotterdam) (Table 5).

[0156] [Table 5]

[0157] All donors submitted written informed consent. PBMCs were cultured in IMDM (Lonza) + 2% human serum (Sanquin) + 50 IU / ml hIL-2 (Miltenyi) in the presence of a peptide pool of up to five target peptides based on HLA matching at 10 μg / ml / peptide. After 14 days, 200,000 cells were restimulated in triplicates with the target peptides at 10 μg / ml / peptide at 37°C for 48 hours. The supernatant from the restimulation was then used in an hIFNγ ELISA (BioLegend) according to the manufacturer's instructions. Plates were read at 450 nm wavelength using an Infinite 200Pro ELISA reader. hIFNγ levels were calculated from the OD values ​​(mean of triplicates) after background subtraction, using supernatants derived from previously successful restimulations quantified in a separate ELISA using the hIFNγ standard provided by the manufacturer (c18-27). HLA conjugates with an average OD of at least the average of the DMSO control + 2 × SD were quantified.

[0158] 4.2 Results Next, we tested whether the immunogenicity of HBx-derived and Pol-derived conjugates could be confirmed. PBMCs from blood donors who had previously cleared HBV infection were expanded for two 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 was detected in response to the well-established epitopes c18-27 and p549-557 (Figure 5B). There was considerable variability in IFNγ production, and generally, some donors appeared to respond better than others (Figure 5). Overall, we observed responses to five completely novel HBx-derived peptides and 17 novel Pol-derived peptides. In addition, we observed IFNγ production in response to one rarely described HBx-derived epitope and three Pol-derived epitopes, but the responses to these were all not very high (Figure 5; underlined with solid line). Importantly, four additional Pol-derived epitopes elicited responses in donors that were negative for the HLA types previously described for those epitopes (Figure 5; underlined with dashed line and Table 3), suggesting that these peptide sequences are epitopes for multiple HLA types. No measurable response was observed for the six Pol-derived and seven HBx-derived HLA conjugates in any of the tested donors (Figure 5; gray box), which may be due to the small number of donors. Therefore, further testing of donors in the future may confirm the immunogenicity of these HLA conjugates.

[0159] Example 5: 5.1 Method To evaluate whether the claimed peptide fragments are immunogenic in a human setting, seven SLPs containing one or more epitope peptide sequences were designed, manufactured, and tested using PBMC samples from 15 different donors who had previously cleared HBV infection.

[0160] The design of the seven SLPs was based on the following criteria: 1) the innate HBV-X HBV polymerase genotype sequence; 2) high manufacturability expected using an in silico machine learning algorithm trained on a large-scale real-world peptide synthesis yield set, based on common synthetic principles, particularly those described herein above; and 3) the inclusion of one or more epitope peptide sequences (see Table 6). The preferred length for the SLPs was set at 25AA. Where deemed necessary, manufacturability was improved by including adjacent regions along the corresponding HBV-X or HBV polymerase sequence, taking into account the expected poor manufacturability based on established peptide synthesis experience. Six SLPs of length 25AA and one SLP of length 26AA were obtained.

[0161] According to established methods, individual SLPs (Table 6) are subjected to solid-phase Fmoc / t The peptides were synthesized using Bu chemistry, treated with a cleavage cocktail, purified by HPLC, and analyzed by UPLC-MS. All reagents and solvents for solid-phase peptide synthesis (SPPS) were purchased from Merck, Sigma Aldrich, Actu-All, Bachem, Biosolve, and GL Biochem, and used as received. Peptide synthesis was performed using a Tetras peptide synthesizer (Advanced ChemTech). The resin was dried, cooled, and treated with a trifluoroacetic acid (TFA)-based cleavage cocktail. After filtering the resin, the reaction mixture was shaken at room temperature. Subsequently, the peptides were 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 pellet was dissolved in an acetonitrile (ACN) and TFA-containing or acetic acid-containing H2O-based mixture and freeze-dried overnight. After purification by HPLC based on solvent systems of TFA in H2O and TFA in ACN, or TFA in H2O and TFA in tert-butanol-containing ACN, the selected purified fractions were pooled and freeze-dried overnight. The identity and purity of the high-purity peptides were determined by UPLC-MS. Before use, the SLP was reconstituted in 10% DMSO and 90% H2O to a concentration of 2 mM.

[0162] Using the IFNγ ELISpot assay, the ability of SLP to induce IFNγ production by PBMC after 24 hours of stimulation was tested. Briefly, PBMC were separated from buffy coats of 15 HLA-typed donors (Sanquin Blood Bank) who had previously cleared HBV infection by density gradient centrifugation. PBMC were cultured in PVDF plates (MSIPS4510, Millipore) coated with IFNγ capture antibody (5 μg / ml, Mab-1-D1K, Mabtech) in the presence of 10 μM SLP or equivalent DMSO control. Cells were seeded at a density of 200,000 cells per well in IMDM + 8% human serum in 4 replicate wells. After incubation for 20 - 24 hours, IFNγ detection antibody (0.3 μg / ml, Mab-7-B6-1-biotin, Mabtech) was added, followed by streptavidin-ALP (1 μg / ml, Mabtech). Spot development was performed by adding BCIP / NBT-plus substrate (100 μl / well, Mabtech), and spots were counted using a CTL Immunospot S6 Ultimate analyzer (Immunospot). The number of spot-forming units (SFU) from 4 replicate wells was added together, and the cumulative spot number of 4 replicate DMSO control wells was subtracted.

[0163] 5.2 Results To test the ability of SLP to induce an IFNγ production response, an IFNγ ELISpot assay was performed on PBMC from 15 HBV resolvers. These donors had previously cleared HBV infection and thus were thought to have HBV-specific T cell responses. All SLP derived from both polymerase and HBx had the ability to induce an IFNγ response (Figure 6).

[0164] [Table 6]

[0165] Example 6. Novel SLP can boost the functional CD8+ and CD4+ T cell response of leukocytes in HBV-resolved individuals and chronic HBV patients in vitro. 6.1 Method The functional booster capacity of novel SLPs was tested in an expanded-scale experiment. Specifically, PBMCs were isolated from either buffy coats derived from healthy donors who had previously undergone HBV removal (n=6) or from the blood of chronic HBV patients who visited the outpatient clinic at Erasmus Medical Center Rotterdam (n=5). PBMCs were cultured for 14 days in IMDM (Lonza) + 2% human serum (Sanquin) in the presence of an SLP pool (3 μM of each SLP). After 2 days, 50 IU / ml IL-2 was added to the culture, and this was repeated three times a week until day 14. After 14 days, 200,000 cells in each well were re-stimulated in 4 replicates with individual SLPs (10 μM of each SLP) or DMSO as a medium control. After 22 hours, the supernatant was collected and subjected to cytokine analysis, and the cells were used for flow cytometry analysis. Cells were pooled and stained with the following panels: 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, under dark conditions at 4°C for 30 minutes. Cells were captured using a BD FACSCanto instrument and analyzed using a FlowJo v10 (BD). Marker expression rates were determined by subtracting the expression rates observed in the DMSO control for the corresponding markers. Secreted cytokines in the culture supernatant were determined using Luminex technology. Cytokines were analyzed on ThermoFisher custom Procarta plates and analyzed using a MAGPIX instrument (Merck Millipore). The amount of secreted cytokines was calculated using standards. Background subtraction was performed by subtracting the mean (DMSO and unrelated peptides) + 2 × SD (DMSO and unrelated peptides) from the calculated values.

[0166] 6.2 Results In vitro expansion experiments mimicking vaccination showed that all four novel SLPs (SLP1, SLP2, SLP4, and SLP6) were capable of boosting functional CD8+ and CD4+ T cell responses in vitro in leukocytes from HBV-cleared individuals (rHBV1-6) and chronic HBV patients (cHBV1-5), with each of these SLPs eliciting a response in at least one donor. T cell activation was demonstrated by the increased presence of CD69 in response to the SLPs in both cell types (Figure 7). The fact that CD8+ T cells presented CD107a in response to the SLPs, indicating the new secretion of cytotoxic agents, and the presence of type I T cell cytokines IFNγ and TNFα, essential for T cell effector function, demonstrated that this expansion and activation of donor T cells also generated functional T cells.

Claims

1. An immunogenic peptide comprising the amino acid sequence shown in Sequence ID No.

31.

2. An immunogenic composition, - The peptide according to claim 1, and - Pharmaceutically acceptable carriers Includes, An immunogenic composition further comprising an adjuvant, optionally.

3. The immunogenic composition according to claim 2 for the treatment or prevention of HBV-related diseases in human subjects.

4. The immunogenic composition according to claim 2 for ex vivo stimulation of antigen-loaded activated antigen-presenting cells or expanded antigen-specific T cells.

5. An immunogenic composition according to claim 2 for the treatment of HBV-related disease in human subjects, wherein the treatment comprises the following steps: (i) A step of reducing the viral load by administering antiviral therapy to HBV-infected patients, (ii) The step of administering the immunogenic composition when the viral load has decreased significantly, and (iii) The step of interrupting or discontinuing antiviral therapy after sufficient time has elapsed for an initial T cell response to occur in the patient. An immunogenic composition containing [the specified substance].

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