WO2015187009A1 - Synthetic long peptides (slp) for therapeutic vaccination against hepatitis b virus infection - Google Patents

Synthetic long peptides (slp) for therapeutic vaccination against hepatitis b virus infection Download PDF

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Publication number
WO2015187009A1
WO2015187009A1 PCT/NL2015/050390 NL2015050390W WO2015187009A1 WO 2015187009 A1 WO2015187009 A1 WO 2015187009A1 NL 2015050390 W NL2015050390 W NL 2015050390W WO 2015187009 A1 WO2015187009 A1 WO 2015187009A1
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Prior art keywords
peptide
hbv
slp
hla
protein
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PCT/NL2015/050390
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French (fr)
Inventor
Wilhelmus Johannes Theodorus Alexander KREBBER
Johan Herman Kessler
Cornelis Joseph Maria Melief
Kitty Michelle Corinne KWAPPENBERG
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Isa Pharmaceuticals B.V.
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Priority to KR1020167036642A priority Critical patent/KR102569204B1/en
Priority to CA2950863A priority patent/CA2950863C/en
Priority to JP2016570861A priority patent/JP6780852B2/en
Priority to US15/315,526 priority patent/US10376576B2/en
Priority to ES15732967T priority patent/ES2979072T3/en
Priority to EP15732967.3A priority patent/EP3148566B1/en
Priority to CN201580041909.4A priority patent/CN106573960B/en
Publication of WO2015187009A1 publication Critical patent/WO2015187009A1/en
Priority to US16/458,894 priority patent/US10898567B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Synthetic Long Peptides for therapeutic vaccination against Hepatitis B virus infection
  • the present invention relates to the fields of medicine and immunology.
  • it relates to novel peptides that may be used in the treatment and/or prevention of a Hepatitis B viral infection and/or an Hepatitis B related disease or condition.
  • HBV hepatitis B virus
  • HBV human immunodeficiency virus
  • Infection with HBV can develop as an acute viral hepatitis, an illness that begins with general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, and then progresses to development of jaundice. The illness lasts for a few weeks and then gradually improves in most affected adults. A few people may have more severe liver disease (fulminant hepatic failure), and may die as a result. The infection may be entirely asymptomatic and may go unrecognized. Chronic infection with hepatitis B virus either may be asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of many years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (liver cancer), also with a latency of many years.
  • antiviral drugs such as nucleoside/nucleotide analogues (e.g. Entecavir and Tenofovir) or interferon (IFN)a efficiently decreases serum viral loads.
  • antiviral therapy rarely leads to a sustained virological response and drug resistance occurs (Zoulim et al, 2012).
  • the great majority of HBV carriers remains untreated.
  • HCC liver cirrhosis and associated liver failure or hepatocellular carcinoma
  • the present invention is meant to enable efficacious therapeutic vaccination against chronic HBV infection.
  • Therapeutic vaccination constitutes a promising strategy to treat chronic hepatitis B (Michel et al., 2011).
  • T cell responses are crucial in HBV clearance and control.
  • HBV-infected chimpanzees have shown the essential role of HBV-specific CD8 + T cells as effector cells in this process (Thimme et al, 2003).
  • the T cell responses are usually very weak, focused on only a few epitopes and functionally impaired (Michel et al, 2011).
  • the goal of therapeutic vaccination is to install vigorous and robust multivalent CTL and T-helper cell responses directed to many HBV antigens, thereby pursuing viral clearance, hepatitis control and cure.
  • the inventors identified a selection of HBV antigens to be used for efficacious therapeutic vaccination. Based on HLA class I and class II binding capacities of HBV protein-derived peptides and analysis of the generation of these HLA class I binding peptides by cleavages made by the proteasome, the most immunogenic regions, covering a very high percentage of all possible T cell epitopes in the global hepatitis B patient population, have been uncovered in the HBV polymerase protein, core protein, X protein and large surface protein.
  • These regions contain high numbers of T cell epitopes and when administered to the hepatitis B patient - either as chemically synthesized long peptide or via genetic approaches - such a vaccination is envisioned to induce a vigorous T cell response, resolving the HBV infection.
  • peptides are highly preferred for medical purposes as these can be efficiently synthesized in vitro, which is not possible or uneconomical for native proteins larger than approximately 100, i.e. 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 amino acids.
  • Chemical synthesis of peptides is routine practice and various suitable methods are known to the skilled person. Chemical synthesis of peptides also overcomes the problems associated with recombinant production of intact proteins, which is difficult to standardize and requires extensive purification and quality control measures.
  • Peptides with a length that exceeds the length of human leukocyte antigen (HLA) class I and class II epitopes e.g.
  • APC professional antigen presenting cells
  • DC Dendritic cell
  • HLA human leukocyte antigen
  • the present invention relates to novel peptides of about 15 to about 100 amino acids in length, also denominated herein as long peptides, that each exceed the length of human leukocyte antigen (HLA) class I and class II presented epitopes and that induce a combined CD4 + and CD8 + T cell response that is therapeutically successful and inducing cure in a high percentage of patients.
  • the long peptides of the invention are synthetic peptides, denominated herein as synthetic long peptides (SLPs).
  • SLPs synthetic long peptides
  • the long peptide and/or SLP sequences with the highest coverage of HLA class I-restricted cytotoxic T lymphocyte epitopes and HLA class II-restricted T helper epitopes were identified.
  • the selections as described herein identify the long peptide and/or SLP sequences that incorporate HBV- derived T cell epitopes that are presented on all predominantly expressed HLA class I and class II alleles.
  • the long peptide and/or SLP vaccine composition can be used in all HBV infected individuals.
  • the present invention describes the identification and selection of HBV-derived long peptides, preferably SLPs, that are highly immunogenic and capable of inducing a potent combined HB V-directed CD4 + T helper and CD8 + cytotoxic T cell response, when administered as a vaccine composition to patients.
  • HBV-derived long peptides preferably SLPs
  • Such highly immunogenic long peptides from HBV have not been disclosed in the prior art.
  • the HBV-derived long peptides of the invention were identified based on a putative immunogenicity score developed and validated by the inventors and as disclosed herein. The putative immunogenicity is quantified herein using the T cell Regional Immunogenicity Assessment (TRIA) score.
  • TAA T cell Regional Immunogenicity Assessment
  • the TRIA score is based on the cumulative Class I-BCI score of said peptide, which is indicative for their immunogenic CTL activating capacity, and the cumulative Class II-B score of said peptide, which is indicative for their immunogenic Th-cell activating capacity. Calculation of the cumulative Class I-BCI score and the cumulative Class II-B score is described in detail herein in the Examples section.
  • the TRIA score is calculated as the sum of the cumulative Class I-BCI score and the cumulative Class II-B score. A strong correlation was found between this TRIA score and the T cell responses found in PBMC of HBV-immune donors. Therefore, the TRIA score enables the selection of optimal immunogenic long peptides.
  • the present invention provides a peptide derived from an HBV protein.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471, more preferably the peptide of the invention comprises or consist of a peptide selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 76, 77, 1142 and 1469, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 77, 1142 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, even more preferably selected from the group consisting
  • a peptide of the invention that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51, 60, 63, 64, 68, 71, 74-77. Also preferred is a peptide of the invention that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 63, 71 and 75.
  • the peptide of the invention comprises at least about 70 predicted T-cell epitopes. More preferably, the peptide of the invention comprises at least about 70 predicted T-cell epitopes and at least about 3 proteasomal cleavage sites.
  • the peptide if the invention comprises at least about 70 predicted HLA class I-restricted CD8 + cytotoxic T-cell epitopes, at least about 1 predicted HLA class II-restricted CD4 + T-helper epitope. More preferably, the peptide if the invention comprises at least about 70 predicted HLA class I-restricted CD8 + cytotoxic T-cell epitopes, at least about 1 predicted HLA class II-restricted CD4 + T-helper epitope and at least about 3 proteasomal cleavage sites.
  • HLA class I-restricted CD8 + cytotoxic T-cell epitope is also denominated herein as CTL epitope and HLA class II-restricted CD4 + T-helper epitope is also denominated herein as Th-cell epitope.
  • the peptide of the invention comprises at least about 70 predicted CTL epitopes, at least about 15 predicted Th-cell epitopes. More preferably, the peptide of the invention comprises at least about 70 predicted CTL epitopes, at least about 15 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites.
  • the peptide of the invention comprises at least about 95 predicted CTL epitopes, at least about 25 predicted Th-cell epitopes. More preferably, the peptide of the invention comprises at least about 95 predicted CTL epitopes, at least about 25 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites.
  • the peptide of the invention comprises at least about 125 predicted CTL epitopes, at least about 50 predicted Th-cell epitopes. More preferably, a peptide of the invention comprises at least about 125 predicted CTL epitopes, at least about 50 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites.
  • a peptide of the invention has a TRIA score of at least about 6300, at least about 8000, at least about 9000, at least about 10000, or preferably at least about 14000.
  • a peptide of the invention can advantageously be used in the prevention and/or treatment of an HBV related disease or condition in a subject, preferably a mammal, more preferably a human.
  • the peptide of the invention comprises or consists of an amino acid sequence, preferably a contiguous amino acid sequence, of any of the proteins selected from the group consisting of HBV protein polymerase, HBV core protein, HBV X-protein and HBV large surface protein.
  • said peptide comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471.
  • a peptide of this group is characterized in that it has a TRIA score of at least 6300, indicating the high immunogenic capacity for CD4+ and CD8+ T cell activation. Furthermore, a peptide of this group is characterized in that it comprises at least 70 predicted HLA class I-restricted CD8 + cytotoxic T-cell epitopes, at least 1 predicted HLA class II-restricted CD4 + T-helper epitope. . Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55-57, 60-66, 68-78, 1142- 1145 and 1468-1471.
  • a peptide of this group is characterized in that it has a TRIA score of at least 8000.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55-57, 60-66, 68, 69, 71-79, 1142-1145 and 1468-1471.
  • a peptide of this group is characterized in that it comprises at least 70 predicted CTL epitopes, at least 15 predicted Th-cell epitopes.
  • a peptide of this group comprises at least 3 proteasomal cleavage sites.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55, 57, 60, 63, 64, 66, 68, 71, 72,74-78, 1142, 1145, 1468-1471.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 53, 55-57, 60-66, 68, 69, 71, 73-78, 1142-1145, 1468-1471.
  • a peptide of this group is characterized in that it has a TRIA score of at least 9000.
  • the peptide of the invention is a peptide that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 55-57, 60-65, 68, 69, 71, 74, 75, 77, 78, 1142-1145, 1468, 1469 and 1471.
  • a peptide of this group is characterized in that it has a TRIA score of at least 10000.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 52, 53, 55, 57, 60, 61, 63, 64, 68, 69, 71, 72, 75, 77, 78, 1142-1145, 1468, 1469 and 1471.
  • a peptide of this group is characterized in that it comprises at least 95 predicted CTL epitopes, at least 25 predicted Th-cell epitopes.
  • a peptide of this group comprises at least 3 proteasomal cleavage sites.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 75, 77, 1142, 1469.
  • the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 63, 75, 1143-1145, 1468 and 1469.
  • a peptide of this group is characterized in that it has a TRIA score of at least 14000.
  • a peptide of this group is characterized in that it comprises at least 125 predicted CTL epitopes, at least 50 predicted Th-cell epitopes.
  • a peptide of this group comprises at least 3 proteasomal cleavage sites.
  • T-cell epitope is defined herein as a linear fragment of a polypeptide antigen, which is recognized and bound by a T-cell receptor, preferably a human T-cell receptor, after being made accessible to a T-cell receptor by intracellular proteolytic processing of the polypeptide antigen and subsequent presentation by an HLA class I or HLA class II molecule on the cell surface of an antigen-presenting cell.
  • a "predicted T- cell epitope” is to be understood herein as a linear fragment of a polypeptide antigen for which liberation from the source protein or peptide by proteolytic cleavage and T-cell receptor recognition and/or binding has been predicted using bioinformatics analyses based on algorithms that predict HLA class I and II peptide binding and C-terminal generation by the proteasome of all possible HLA class I binding peptides (with a length of a HLA class I ligand; 8 - 12 aa) contained in the HBV proteins.
  • a "confirmed T-cell epitope” is to be understood herein as a linear fragment of a polypeptide antigen for which liberation from the source protein or source polypeptide by proteolytic cleavage and T-cell receptor recognition and/or binding, and more preferably CD4 + or CD8 + T cell activation capability, have been established experimentally as disclosed herein.
  • a “linear fragment” is understood herein to be a contiguous amino acid sequence of a polypeptide antigen, said polypeptide antigen preferably being an HBV protein, more preferably a protein selected from the group consisting of HBV protein polymerase, HBV core protein, HBV X-protein and HBV large surface protein.
  • An identical linear fragment of a polypeptide antigen showing binding affinity to a second or further type of HLA class I or HLA class II molecule is to be understood herein as a second or further T-cell epitope.
  • a specific linear fragment of a polypeptide antigen being capable to bind to two types of HLA molecules is understood herein to be two separate or distinct T-cell epitopes, and is scored twice within the cumulative BCI Class I- and/or Class II-B score.
  • a T-cell epitope typically comprises or consists of at least 8 amino acids and up to 20 or (exceptionally) even more amino acids.
  • a T-cell epitope can be an HLA class I-restricted CD8 + cytotoxic T cell (CTL) epitope or an HLA class Il-restricted CD4 + T-helper (Th-)cell epitope.
  • CTL cytotoxic T cell
  • Th- T-helper
  • HLA class I restricted epitopes also referred to as CTL epitopes
  • HLA class-II molecules are typically loaded with linear fragments in the late endosomal compartment.
  • a peptide according to the invention comprises T-cell epitopes that are selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466 (see Tables 4-7).
  • a preferred peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a more preferred peptide according to the invention comprises at least 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • An even more preferred peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466.
  • An even more preferred peptide according to the invention comprises at least 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466.
  • the predicted T-cell epitopes of the present invention are confirmed experimentally as disclosed herein.
  • a preferred peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a more preferred peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • An even more preferred peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466.
  • An even more preferred peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466.
  • proteasomal cleavage site is understood herein as a site in a protein or polypeptide that is cleaved by the proteasome, preferably a human proteasome/proteasome naturally present in a human cell.
  • a specific proteasomal cleavage site liberating the C-terminus of the epitope is preferably present exactly after the C-terminus of the epitope amino acid sequence, in order to allow the epitope's C- terminal residue to be liberated from the larger peptide and to be presented by the HLA class I molecule.
  • the first important event that defines an HLA class I-restricted CD8 + cytotoxic T-cell (CTL) epitope is the release of the epitope (or the epitope-precursor) from its flanking protein regions through enzymatic cleavage by cytosolic peptidases.
  • the multicatalytic proteasome is the primary enzyme complex required for the generation of the exact C-terminus of the vast majority of CTL epitopes (Rock et al, 2004).
  • Proteasomes are multicatalytic enzyme complexes abundantly present intracellularly and are considered responsible for the generation of the C terminus of the vast majority of CTL epitopes (Craiu et al, 1997; Stoltze et al., 1998; Mo et al., 1999).
  • the generation of the amino-terminus of a CTL epitope is much more flexible because several amino-terminal exo-peptidases (like ERAPl, puromycin sensitive aminopeptidase, bleomycin hydrolase and others) reside in the cytosol and endoplasmic reticulum and these trimming enzymes have the capacity to shorten an N-terminal elongated epitope-precursor to its precise length.
  • proteasome-mediated cleavage sites in a protein or in a polypeptide, like a peptide of the invention can be used as an important identifier of almost every CTL epitope, because the proteasomal cleavages determine and enable C-terminal epitope generation (Kessler et al, 2001; Kessler and Melief, 2007).
  • the assessment of proteasomal cleavage sites in the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein identifies the C-termini of intracellularly produced HBV peptide fragments, specifically for HLA class I presented peptide fragments.
  • T-cell epitope requirements have been used in the present invention to localize and design long peptides derived from the full length sequences of an HBV protein which comprises preferred CD8 + cytotoxic T cell (CTL) and CD4 + T- helper (Th-)cell epitopes and/or combinations thereof and are thus highly immunogenic and therefore suitable peptides for synthesis and (therapeutic) vaccination purposes.
  • CTL cytotoxic T cell
  • Th- T- helper
  • Proteasome mediated proteolytic cleavages can be predicted in silico using a prediction algorithm. Cleavage as performed by the proteasome can be verified in a proteasome mediated cleavage assay as disclosed herein, which measures the C- terminal liberation of the epitope from its flanking regions (Kessler et al, 2001; Kessler and Melief, 2007).
  • a cell free proteasome cleavage assay identifying and quantitatively measuring the amino acid (aa) positions and the abundancy of cleavages by the proteasome in a polypeptide can be used to determine which peptides are generated from the source protein (or source polypeptide), thereby establishing the peptide pool available for epitope generation.
  • the cell free proteasome cleavage assay involves the co-incubation of a polypeptide (preferably having a length of 28-40 aa, more preferably having a length of 30-39 aa) with a preparation of purified proteasomes in an appropriate buffer solution.
  • a polypeptide preferably having a length of 28-40 aa, more preferably having a length of 30-39 aa
  • proteasomes Two main forms of proteasomes exist, the immunoproteasomes, which are mainly expressed in professional antigen presenting cells, like e.g. Dendritic Cells, and the constitutive proteasomes, which are expressed mainly in other cell types. These types contain variant catalytic subunits with slightly different catalytic activity.
  • proteasome- mediated cleavage assays may be performed separately with these two proteasome types.
  • a constitutive 20S-proteasome or immune 20S-proteasome is used as disclosed herein.
  • the reaction mixture comprising the peptides to be cleaved and either of the two proteasome types (purified proteasome preparations) is incubated at 37°C and samples are drawn at 1 h, 3 h, 6 h and 24 h time points as detailed in the Examples herein.
  • a cleavage site can be confirmed by detection of fragments containing as COOH terminus the residue H2-terminal from the cleavage site together with the (possible) complementary fragment(s), as calculated from the intensities of the fragment peaks in the mass spectra (preferably present for >1%, more preferably present for >7%, at 24 h incubation), in both the digestion with constitutive proteasomes and the digestion with immunoproteasomes.
  • a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, preferably at least 3, proteasomal cleavage site as defined herein.
  • a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, preferably at least 3, proteasomal cleavage site as assessed and verified in a proteasomal cleavage assay as described above.
  • T-cell epitopes are HLA class I-restricted CD8 + cytotoxic T cell (CTL) epitopes and HLA class Il-restricted CD4 + T-helper (Th-)cell epitopes.
  • CTL epitope is understood herein as a linear fragment of a polypeptide antigen that is liberated from the source protein by proteasome mediated proteolytic cleavage and subsequently presented by an HLA class I molecule on the cell surface of an antigen presenting cell (APC), preferably a human antigen presenting cell.
  • APC antigen presenting cell
  • a "predicted CTL epitope” is understood herein as a linear fragment of a polypeptide antigen for which liberation from its source protein by proteolytic cleavages and HLA class I molecule binding have been predicted using bioinformatics analyses based on algorithms that predict HLA class I peptide binding and C-terminal generation by the proteasome of all HLA class I binding short peptides (with a length of a CTL epitope; 8 - 12 aa) contained in the HBV proteins.
  • a predicted CTL epitope of the present invention is confirmed experimentally as disclosed herein.
  • a CTL epitope of the invention is preferably capable of activating a CD8 + T cell response.
  • a “confirmed CTL epitope” is understood herein as a linear fragment of a polypeptide antigen for which liberation by proteolytic cleavages and HLA class I molecule binding, more preferably CD8 + T cell activation, have been established experimentally as disclosed herein.
  • a CTL epitope of the invention is preferably capable of activating a CD8 + T cell response.
  • a CTL epitope typically comprises at least 8 up to 12, or exceptionally up to 13 or 14 amino acids.
  • Preferably a CTL epitope consists of 8-14 amino acids, i.e. has a length of at least 8 up to 14 amino acids.
  • a CTL epitope is defined by two important intracellular events being (i) proteasome mediated proteolytic cleavage and (ii) binding to an HLA class I molecule, which takes place in the endoplasmic reticulum (ER).
  • proteasome mediated proteolytic cleavage and (ii) binding to an HLA class I molecule, which takes place in the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • HLA class I molecule specific binding to an HLA class I molecule is predicted using an in silico prediction algorithm and established by using an HLA class I peptide binding assay as known by the person skilled in the art (Kessler and Melief, 2007; Kessler et al., 2003).
  • the HLA class I-restricted epitope in a long peptide according to the invention is predicted to be generated at its C-terminus by the proteasome and preferably has a predicted high affinity binding capacity for the HLA class I molecule using an assay as described in van der Burg et al, 1995 and Kessler et al, 2003; e.g.
  • IC50 ⁇ about 5 ⁇ may be considered high affinity binding
  • about 5 ⁇ ⁇ IC 5 o ⁇ about 15 ⁇ may be considered intermediate affinity binding
  • about 15 ⁇ ⁇ IC 5 o ⁇ 100 ⁇ may be considered low affinity binding
  • IC50 > about 100 ⁇ may be considered as no binding.
  • various HLA class I binding assays are available.
  • the assays can be divided into cell-free assays (using soluble HLA) versus cellular assays (using HLA class I molecules on the cell surface), and competitive assays (resulting in semi-quantitative data) versus assays that do not use a labeled reference peptide and are therefore quantitative (Kessler and Melief, 2007; Viatte et al., 2006).
  • the assays have in common that the HLA class I peptide binding affinity is reliably assessed.
  • the biochemical approach involves the biochemical purification of HLA-epitope complexes from cells expressing the HBV antigen of the invention together with the presenting HLA class I molecule (geno)type, followed by the mass spectrometric search for the epitope in the eluted HLA class I-bound CTL receptor ligands as known by the person skilled in the art (Schirle et al., 2000; Schirle et al., 2001).
  • the functional approach involves a CTL line or clone that is specifically recognizing the HLA-epitope, which is used as a tool to demonstrate the natural processing and actual presentation of the epitope by HLA class I molecules.
  • a CTL induction assay as known in the art, either the synthetically generated minimal (i.e. exact length) epitope or the peptide sequence of interest encompassing the epitope, for instance a peptide, long peptides and/or SLP as defined herein, is used to stimulate and select HLA-epitope-specific cytotoxic T cells.
  • a multivalent CD8 + T cell population or a multivalent mixed CD8 + and CD4 + T cell population, is stimulated with autologous target cells of which the HLA class I molecules on the cell surface are either exogenously loaded with the precise synthetic epitope or endogenously loaded with intracellularly generated CTL epitopes derived from the exogenously loaded long peptide of the invention after its uptake by the antigen presenting target cells.
  • autologous target cells are loaded with a peptide, e.g.
  • the epitope is generated after cellular uptake of the peptide and its intracellular processing by the proteasome together with other N-terminal trimming peptidases.
  • the HLA-epitope- specific CTL is used to demonstrate the intracellular generation and natural presentation of the epitope of the invention by HLA class I molecules on the surface of HBV-infected cells.
  • Specific recognition of an HLA class I restricted epitope by a CTL demonstrates the cell surface expression of the epitope and reveals its immunogenicity, i.e. the presence of epitope-specific T cells in the (T-cell receptor) repertoire of a selected donor.
  • the CD8 + T cell activating capability has been demonstrated ex vivo and/or in vivo, in T cells from human healthy control individuals or even more preferably in T cells from a human patient with an HBV related disease or condition and/or from a healthy control.
  • the activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HBV related disease.
  • a CTL epitope for which liberation by proteolytic cleavage and HLA class I molecules presentation, or preferably CD8+ T cell activating capability, has been demonstrated experimentally is denominated herein as a confirmed CTL epitope.
  • a peptide of the invention preferably comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes as defined herein.
  • a peptide of the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes as defined herein.
  • a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes from the HBV core protein, HBV polymerase, HBV X protein or HBV large surface protein.
  • a peptide according to the invention comprises or consists of a contiguous amino acid sequence of any of the proteins selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and HBV large surface protein, wherein said contiguous amino acid sequence comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes.
  • a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 CTL predicted epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 (see Tables 4a, 5a, 6a and 7a).
  • a peptide of the invention preferably comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, or preferably at least 95 confirmed CTL epitopes as defined herein and verified using a biochemical or functional assay as described above.
  • a peptide of the invention that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or preferably at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes as defined herein and verified using a functional assay as described above.
  • a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or preferably at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846- 923, 959-1090, and 1146-1395.
  • Th-cell epitope is understood herein to be a linear peptide fragment that is recognized by an HLA class II molecule.
  • a "predicted Th-cell epitope” is understood herein as a linear fragment of a polypeptide antigen for which HLA class II molecule recognition has been predicted using sophisticated bioinformatics analyses that are experimentally underpinned.
  • a predicted Th-cell epitope of the present invention is confirmed experimentally as disclosed herein.
  • a Th-cell epitope of the invention is preferably capable of inducing a CD4 + T cell response.
  • a "confirmed Th- cell epitope” is understood herein as a linear fragment of a polypeptide antigen for which HLA class II molecule recognition has been established experimentally as known by the person skilled in the art and further detailed herein.
  • An HLA class II-restricted CD4 + T-helper cell (Th-cell) epitope typically comprises 15 up to 20, or exceptionally even more, amino acids.
  • an HLA class II-restricted T-helper cell epitope comprises or 10 - 20 or 10 - 15 amino acids.
  • Specific recognition of a predicted HBV-derived Th-cell epitope can be tested and/or verified in a Th-cell induction assay.
  • long peptide and/or SLP sequence of interest comprising the predicted Th-cell epitope is exogenously loaded on the surface of target cells and subsequently these peptide-loaded target cells are co-incubated with a multivalent autologous T helper cell population.
  • epitope-specific T helper cells can be selected and can be back-tested for the recognition of the T helper cell epitope contained in the peptide or SLP thereby proving its natural cell surface presentation.
  • an HLA class II-restricted CD4 + T-helper cell epitope comprised in a peptide according to the invention is capable of inducing or activating a CD4 + T-helper cell in a human patient with an HBV related disease or condition.
  • the induction or activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HBV related disease.
  • the HLA class Il-restricted epitope is capable of activating a CD4 + T-helper memory and/or CD4 + T-helper effector response, i.e. activation of a CD45RO-positive CD4 + T-helper cell.
  • a CD45RO-positive CD4 + T-helper cell This will lead, by virtue of the 'license to kill' signal through CD40-triggering of DC (Lanzavecchia, 1998) to a more robust CD8 + effector and memory cytotoxic T cell response.
  • the activated CD4 + T-helper cells may activate non-HLA restricted killer cells of the immune system.
  • a Th-cell epitope for which recognition by an HLA class II molecule, or preferably CD4+ activating capability, has been demonstrated experimentally is denominated herein as a confirmed Th-cell epitope.
  • a peptide according to the invention comprises at least one predicted
  • Th-cell epitope from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10 or preferably at least 15 predicted Th-cell epitope(s) as defined herein. Preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s) from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a peptide according to the invention comprises or consists of a contiguous amino acid sequence of any of the proteins selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and HBV large surface protein, wherein said contiguous amino acid sequence comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s).
  • a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s) selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466 (see Tables 4b, 5b, 6b, and 7b). More preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8,
  • a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7,
  • a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466.
  • a peptide according to the invention comprises both at least 70 predicted CTL epitopes and at least one predicted Th-cell epitope from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a peptide according to the invention is a peptide derived from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, preferably is a fragment of the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, that comprises at least 70 predicted CTL epitopes, at least one predicted Th-cell epitope and at least 3 proteasomal cleavage sites.
  • Activated DCs display upregulated costimulatory molecules and secrete CTL-promoting cytokines. This allows both a more robust CD8 + CTL response induced by such an activated DC that presents HLA class I restricted epitopes and a much more robust CTL memory response (Ridge et al,. 1998; Schoenberger et al., 1998; Sun et al, 2004).
  • the need for CD40 expression on DC for robust CD8 + CTL responses following vaccination with synthetic long peptides (length of 35 aa.) has been demonstrated in Zwaveling et al. (2002).
  • a preferred peptide according to the invention comprises at least 70 predicted CTL epitopes and at least one predicted Th-cell epitope; preferably at least 70 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one predicted Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466.
  • a more preferred peptide according to the invention comprises at least 70 predicted CTL epitopes and at least 15 predicted Th-cell epitopes; preferably at least 70 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 15 predicted Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-95, 1091-1140, and 1396-1466.
  • a peptide according to the invention comprises at least 71, 72, 73, 74, 75, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 up to 175 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67
  • a peptide according to the invention comprises at least 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 up to 175 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638- 685; 846-923, 959-1090, and 1 146-1395 and at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
  • a preferred peptide according to the invention comprises at least 5 confirmed CTL epitopes and at least one confirmed Th-cell epitope; preferably at least 5 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80- 276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146 - 1395 and at least one confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466.
  • a more preferred peptide according to the invention comprises at least 15 confirmed CTL epitopes and at least one confirmed Th-cell epitope; preferably at least 15 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-95, 1091-1140, and 1396-1466.
  • a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146 - 1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 confirmed Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466.
  • a peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146 - 1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 confirmed Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466.
  • a peptide according to the invention comprises at least 15 confirmed CTL epitopes as defined herein and at least 5 confirmed Th-cell epitopes as defined herein.
  • the HLA class I epitopes in the peptides according to the invention are preferably capable of being presented on HLA molecules being encoded by HLA alleles that are predominant in the population of human subjects to be treated.
  • Preferred HLA class I epitopes in peptides according to the invention are epitopes capable of binding to: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101; HLA- A2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001; HLA- A3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA- A7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA- B1501; HLA-
  • a peptide of the invention covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the HLA class I molecules that are encoded by HLA alleles predominant in the population of human subjects to be treated, wherein "Cover an HLA class I molecule" is understood herein as comprising a CTL epitope that shows binding affinity, preferably intermediate binding affinity, more preferably high binding affinity to said HLA class I molecule.
  • a peptide of the invention covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of group of HLA class I molecules consisting of: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101 ; HLA-A2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001 ; HLA- A3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA-A7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA-B1501; HLA-B1502; HLA-B1525; HLA-B1801; HLA-B2702; HLA-B2705; HLA
  • the HBV genome (SEQ ID NO: 3; see Table 1) consists of a partially double- stranded circular DNA molecule having four overlapping open reading frames (ORFs) that are responsible for the transcription and expression of seven different hepatitis B proteins through the use of multiple in-frame start codons.
  • the HBV proteins are the core protein and the e antigen (HBeAg) encoded by the C gene, the HBV polymerase encoded by the P gene, the viral surface proteins (small (S), middle (M), and large (L)) encoded by the S gene, and X protein encoded by the X gene.
  • the outer surface coat surrounds an inner protein shell, composed of HBc protein. This inner shell is referred to as the core particle or capsid.
  • the core particle surrounds the viral DNA and the enzyme DNA polymerase.
  • the HBV core protein is the major component of the viral nucleocapsid.
  • the amino acid sequences of the HBV polymerase, HBV core protein, HBV X protein and HBV large surface protein are represented by SEQ ID NO: 1, 4, 45 and 1141 respectively (see Table 1).
  • a preferred amino acid sequence of a human HBV polymerase protein is a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1;
  • a preferred coding sequence is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 2.
  • a preferred amino acid sequence of an HBV core protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 4;
  • a preferred coding sequence is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 5.
  • a preferred amino acid sequence of an HBV large surface protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1141
  • a preferred coding sequence is a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1467.
  • the full length consensus amino acid sequence of the X protein was obtained by deducing the optimal sequence from the 39 published and reviewed full length (154 amino acid) HBV X protein amino acid sequences in the UniProt database (at: www.uniprot.org). These 39 sequences were first aligned and subsequently for each aa position the most frequently occurring aa was selected for that position in the consensus sequence.
  • a preferred consensus amino acid sequence of a human HBV X protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 45.
  • the consensus amino acid sequence may be encoded by any coding sequence known or designed; the person skilled in the art knows how to design a coding sequence from a known amino acid sequence; such coding sequence may be a codon-optimized sequence.
  • the terms "HBV X protein” and "consensus HBV X protein” are used interchangeably herein.
  • Percentage of identity is herein determined by calculating the ratio of the number of identical nucleotides/amino acids in the sequence divided by the length of the total nucleotides/amino acids of said sequence, minus the lengths of any gaps.
  • Identity with a given SEQ ID NO means identity based on the full length of said sequence (i.e. over its whole length or as a whole).
  • a peptide derived from an HBV protein means that the peptide comprises at least 15 and at most 100 consecutive amino acids originating from the HBV core protein, HBV polymerase, HBV X consensus protein and/or HBV large surface protein.
  • a peptide derived from the HBV polymerase protein comprises at most 100 consecutive amino acids of SEQ ID NO: 1
  • a peptide derived from the HBV core protein comprises at most 100 consecutive amino acids of SEQ ID NO: 4
  • a peptide derived from the HBV X consensus protein comprises at most 100 consecutive amino acids of SEQ ID NO: 45
  • a peptide derived from the HBV large surface protein comprises at most 100 consecutive amino acids of SEQ ID NO: 1 141.
  • a peptide derived from the HBV polymerase protein consists of at most 100 consecutive amino acids of SEQ ID NO: 1
  • a peptide derived from the HBV core protein consists of at most 100 consecutive amino acids of SEQ ID NO: 4
  • a peptide derived from the HBV X consensus protein consists of at most 100 consecutive amino acids of SEQ ID NO: 45
  • a peptide derived from the HBV large surface protein consists of at most 100 consecutive amino acids of SEQ ID NO: 1141. Therefore, by definition, a peptide according to the invention is distinct from a full length HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, as these full length proteins are all longer than 100 amino acids.
  • the peptide of the present invention is from about 15 to about 100 amino acids in length. More preferably the length of the peptide is from 15 up to 100 length indicated herein as the length of the peptide is 15-100 amino acids, or preferably the length of the peptide is 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15-65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids.
  • a peptide which comprises at most 100 amino acids derived from an HBV protein preferably means that the number of consecutive amino acids originating from an HBV protein, preferably being a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81,
  • a peptide which comprises at least 15 amino acids derived from an HBV protein preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids.
  • a peptide which comprises 15-100 amino acids derived from an HBV protein preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and no more than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82,
  • a peptide which comprises 15-100 amino acids derived from an HBV protein preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of SEQ ID NO: 1, 4, 45 and 1141 and present in a peptide as defined herein, is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and no more than about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59
  • the length of the contiguous amino acid sequence from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein comprised within the peptide is 15-100 amino acids, or preferably 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15- 65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids.
  • the length of the contiguous amino acid sequence from the sequences selected from the group consisting of SEQ ID NO: 1, 4, 45 and 1141 comprised within the peptide is 15-100 amino acids, or preferably 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15- 65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids.
  • a peptide according to the invention may comprise additional amino acids than the ones originating from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein or may entirely be made of or consist of an amino acid sequence originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a peptide according to the invention may comprise several parts of non-contiguous amino acid sequences from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, wherein it is to be understood that said peptide has a length, a TRIA score and/or amount and type of T cell epitopes as defined herein.
  • a peptide according to the invention consists of any of the contiguous amino acid sequence from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein as defined herein and indicated by its representing SEQ ID NO, whereby it is understood that no amino acids are appended to either end of the said peptide.
  • the peptide according to the invention comprises any of the contiguous amino acid sequences from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein as defined herein and indicated by its representing SEQ ID NO and further may comprise a modified amino acid and/or a covalently linked functional group such as a fluorinated group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chains or glycan, a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1, CpG oligodeoxynucleotides (CpG-ODNs), Cyclic dinucleotides (CDNs), 2-aminoisobutyric acid (Abu), Muramyl dipeptide (MDP), a DC pulse cassette, a tet
  • the peptide of the invention comprises or consists of a non- naturally occurring sequence as a result of the synthesis of non-natural lengths or as a result of comprising additional amino acids not originating from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein or as a result of comprising non-contiguous amino acid sequences from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, and/or as a result of comprising a modified amino acid and/or a non-naturally occurring amino acid and/or a covalently linked functional group such as a fluorinated group, a fluorcarbon group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chains or glycan, a pam3cys and/or derivative thereof preferably such as described in WO2013051936A1, CpG oligodeoxynu
  • the peptide of the invention may comprise 2-aminoisobutyric acid (Abu, an isostereomer of cysteine).
  • a cysteine of the peptide of the invention may be replaced by Abu.
  • Encompassed within the present invention is a peptide of SEQ ID NO: 77, wherein the N-terminal cysteine has been replaced by Abu.
  • a peptide of the invention is an isolated peptide, wherein "isolated” does not reflect the extent to which the peptide is purified, but indicates that the peptide has been removed from its natural milieu (i.e., that has been subject to human manupilation), and may be a recombinantly produced peptide or a synthetically produced peptide.
  • the invention relates to a peptide that can be effectively used in the prevention, partial clearance and/or treatment or full clearance of a HBV related disease or condition in a subject, preferably as detectable by:
  • a subject is preferably a mammal, more preferably a human.
  • a subject may be an animal model, preferably a non- human mammalian model with humanized HLA class I and class II molecules, or a mammalian, preferably human, organ, such as a liver.
  • HBV related disease or condition is preferably defined as acute HBV infection, chronic HBV infection and other conditions where the hepatitis virus is found in the blood or body fluids containing blood of a subject, such as liver cirrhosis and liver cancer, or optionally of an asymptomatic subject that is characterized by the presence of the virus in the body of said subject.
  • a patient may survive and may be considered as being disease free as a consequence of treatment according to the invention.
  • the disease or condition may have been stopped or regressed (i.e. cleared or partially cleared infection).
  • a significant increase of HBV-specific activated CD4 + or CD8 + cells in peripheral blood at least one week after vaccination is preferably at least a 5%, 10%, 20%, 30% increase or more.
  • An inhibition of the proliferation of HBV infected cells is preferably at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% inhibition or more.
  • An induction of HBV infected cell death may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more.
  • HBV infection may be inhibited at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% induction, or more.
  • HBV infected cells may be reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%.
  • the assay may be carried out by comparison to a subject not treated or to the same subject before treatment.
  • Acute and chronic HBV infection can be treated using the present invention.
  • a peptide according to the invention comprising epitopes which are to be presented to T- cell receptors of CD8 + cytotoxic T cells and/or CD4 + T-helper cells preferably fulfill a number of structural requirements as defined herein.
  • In vitro and ex vivo T cell experiments are preferably used to confirm the capability of peptides according to the invention to induce substantial CD4 + T-helper and CD8 + cytotoxic T cell responses.
  • the peptides of the present invention thereby provide a marked improvement in the selection of relatively short peptides that may be chemically synthesized, comprising the most potent and most widely applicable HLA class I and/or class II presented T cell epitopes derived from HBV.
  • a peptide is distinct from a contiguous sequence of amino acids of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a peptide according to the invention comprising a T-cell epitope from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C-terminus with additional amino acids or functional groups, which may improve bio-availability, targeting to T-cells, or comprise or release immune modulating substances that provide adjuvant or (co) stimulatory functions.
  • the optional additional amino acids at the N- and/or C-terminus are preferably not present in the corresponding positions in the native amino acid sequence of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • a peptide according to the invention comprising a T-cell epitope is obtainable by chemical synthesis and subsequent purification according to methods well-known in the art. (see e.g. Atherton et al., 1989; Barany et al., 1979; Fields et al, 1997).
  • a peptide according to the invention is preferably soluble in physiologically acceptable watery solutions (e.g. PBS) comprising no more than 35, 20, 10, 5 or 0% DMSO. In such a solution the peptide according to the invention is preferably soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml.
  • a mixture of more than one different peptide according to the invention is soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml in such solutions.
  • the peptides according to the invention may be easily synthesized and are large enough to be taken up by professional antigen presenting cells, in particular dendritic cells (DC), processed by the proteasome and/or the endosomal/lysosomal degradation and antigen processing system and preferably have sufficient length to contain at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 to preferably up to 175 CTL epitopes and/or at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to preferably up to 96
  • a peptide according to the invention is a peptide comprising a peptide with an amino acid sequence selected from the group consisting of:
  • the length of the preferably contiguous amino acid sequence is preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids and/or preferably no more than 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 amino acids, most preferably a length of 30-39 amino acids.
  • the invention provides a polynucleotide encoding a peptide according to the invention, preferably a peptide as defined herein above.
  • a polynucleotide may be any polynucleotide comprising e.g. RNA, DNA, and/or cDNA; a polynucleotide may be single stranded or double stranded and may comprise nucleotide analogues and/or nucleotide equivalents such as a peptide nucleic acid (PNA) and a morpholino nucleotide analogue.
  • a polynucleotide may be codon optimized for a host of choice to facilitate expression of the encoded subject matter.
  • the polynucleotide according to the invention does not encode a wild-type full length HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, but rather encode a peptide according to the invention as such, or flanked by amino acid sequence that are not contiguous with a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
  • flanking amino acids may be from proteins other than a wild-type HBV and/or they may be from other locations within a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein that are not contiguous with the peptide they flank.
  • the polynucleotide encodes two or more peptides according to the invention arranged as beads-on-string, whereby the peptides according to the invention (the beads) are linked directly together and/or are linked through linker sequences that are from proteins other than a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, and/or from other locations within a wild- type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, that are not contiguous with the peptide they flank.
  • the amino acid sequences flanking or linking the peptides may comprise proteolytic cleavage sites.
  • a polynucleotide according to the invention may be applied to deliver a peptide according to the invention in various ways.
  • a polynucleotide according to the invention may e.g. be used in the production of recombinant protein or peptide in a suitable host cell (e.g. a bacterial host cell such as E. coli, a suitable yeast host cell such as S. cerevisiae, a suitable filamentous fungal such as an Aspergillus or mammalian host cell) from which the recombinant protein or peptide may be purified.
  • a suitable host cell e.g. a bacterial host cell such as E. coli, a suitable yeast host cell such as S. cerevisiae, a suitable filamentous fungal such as an Aspergillus or mammalian host cell
  • the polynucleotide may be operably linked to expression regulatory sequences (promoters and the like) and incorporated in an expression construct for human cells.
  • Such (autologous) cells may be transfected or transduced ex vivo to be (re)-administered to a subject in need thereof.
  • expression construct according to the invention may be incorporated into a suitable gene therapy vector.
  • Viral vectors (based on a defective virus) are more efficient agents for gene transfer as compared to non- viral agents.
  • Suitable viral expression constructs include e.g. vectors that are based on adenovirus, adeno-associated virus (AAV), retroviruses or modified vaccinia Ankara (MVA).
  • the polynucleotide according to the invention may also be operably linked to a sequence encoding and adjuvant such as a Toll-like receptor (TLR) ligand, a NOD ligand, or a RIG-I ligand.
  • TLR Toll-like receptor
  • NOD NOD
  • RIG-I RIG-I
  • the present invention provides a cell comprising the polynucleotide according to the second aspect of the invention.
  • Such cell can be used for e.g. production of a peptide according to the invention or for medical purposes such as prevention and/or treatment of an HBV related disease as defined elsewhere herein.
  • Said cell may be any host cell.
  • the host cell may be a prokaryote or may be a eukaryote.
  • a preferred prokaryote cell is E. coli.
  • the cell is a eukaryote, the cell preferably is a mammalian, insect, plant, fungal, or algal cell.
  • Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6 cells, and antigen presenting cells such as dendritic cells.
  • Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof.
  • Preferred fungal cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia and filamentous fungal cells.
  • the eukaryotic cell is a human antigen presenting cell, preferably a dendritic cell.
  • polynucleotide may be provided with proper control sequences such as a promoter and terminator sequence and may be inserted into a proper vector such as a plasmid or a method described in the second aspect of the invention may be used.
  • the present invention also provides for an antigen presenting cell such as a dendritic cell as defined earlier herein that has been contacted and/or loaded with a peptide according to the invention, preferably a peptide according to the first aspect of the present invention.
  • an antigen presenting cell such as a dendritic cell as defined earlier herein that has been contacted and/or loaded with a peptide according to the invention, preferably a peptide according to the first aspect of the present invention.
  • a dendritic cell may be used for immune therapeutic treatment of a subject in need thereof.
  • Such dendritic cell can be isolated from the subject, loaded with at least one peptide according to the invention and used for treatment.
  • the present invention provides a method for the preparation of an HBV specific T-cell, said method comprising contacting a T-cell with an antigen presenting cell expressing a polynucleotide according to the invention and/or contacting a T-cell with an antigen presenting cell loaded with a peptide according to the invention; and, optionally, culturing said T-cell.
  • the T-cell is preferably a CD8 + cytotoxic T-cell or a CD4 + T-helper cell.
  • a polynucleotide according to the invention is introduced into the antigen presenting cell (APC), preferably a dendritic cell, using transfection.
  • APC antigen presenting cell
  • the polynucleotide according to the invention may be provided with proper control sequences, or be comprised into a proper vector such as described elsewhere herein.
  • a T-cell with a peptide according to the invention can be performed by any method known to the person skilled in the art.
  • a peptide or an epitope comprised in a peptide is presented to the CD8 + cytotoxic T-cell or CD4 + T- helper cell by an HLA class I or an HLA class II molecule on the surface of an antigen presenting cell, preferably a dendritic cell.
  • an antigen presenting cell preferably a dendritic cell.
  • Culturing said T-cell may be performed using any method known by the person skilled in the art. Maintaining a T-cell under conditions to keep the cell alive is herein also to be construed to be culturing.
  • the T-cell according to this aspect of the invention is contacted with a peptide according to the invention as defined in the first aspect of the invention.
  • the present invention provides a T-cell obtainable by the method depicted in the fourth aspect of the present invention.
  • such T-cell is a T-cell that is obtained by the method according to the fourth aspect of the invention.
  • the T-cell is preferably a CD8 cytotoxic T-cell or a CD4 + T-helper cell.
  • the T-cell according to this aspect of the invention has been contacted with a peptide according to the invention as defined in the first aspect of the invention.
  • the present invention provides a composition useful for the prevention and/or treatment of an HBV related disease or related condition, comprising a peptide according to the invention and/or a polynucleotide according to the invention and/or a cell, preferably a T-cell, according to the invention and/or a cell, preferably a T-cell, obtained by the method according to the fourth aspect of the invention and a pharmaceutically acceptable carrier.
  • the composition according to the invention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different peptides according to the invention.
  • a composition according to the invention comprises a peptide according to the invention as defined in the first aspect of the invention.
  • a composition of the invention comprises a combination of peptides wherein said combination of peptides covers at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%,98%, 99% or 100% of the HLA class I molecules that are encoded by HLA alleles predominant in the population of human subjects to be treated as defined herein above.
  • the composition according to the invention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different polynucleotides according to the invention.
  • a composition according to the invention comprises a polynucleotide according to the invention as defined in the second aspect of the invention.
  • the composition according to the invention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different cells, preferably T-cells that have been contacted with a peptide according to the invention.
  • said T-cells have been contacted with a peptide according to the invention as defined in the first aspect of the invention.
  • the T-cell is preferably a CD8 + cytotoxic T-cell or a CD4 + T-helper cell.
  • composition of the invention comprises at least
  • composition that comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different peptides of the peptides consisting of or comprising of a peptide selected from the group consisting of SEQ ID NO: 51, 60, 63, 64, 68, 71, 74-77, more preferably selected from the group consisting of SEQ ID NO: 63, 71 and 75.
  • the composition of the invention comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63 and peptide that comprises or consists of a peptide of SEQ ID NO: 1143.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1143 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 71 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 71 and peptide that comprises or consists of a peptide of SEQ ID NO: 63.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 63. Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 1143.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, a peptide that comprises or consists of a peptide of SEQ ID NO: 1143, and a peptide that comprises or consists of a peptide of SEQ ID NO: 75.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, a peptide that comprises or consists of a peptide of SEQ ID NO: 1143, a peptide that comprises or consists of a peptide of SEQ ID NO: 75, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1144.
  • compositions that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 75, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1469. Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1469.
  • composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 75, a peptide that comprises or consists of a peptide of SEQ ID NO: 1469, and a peptide that comprises or consists of a peptide of SEQ ID NO: 63.
  • said composition further comprising a peptide that comprises or consists of a peptide of SEQ ID NO: 60 and/or that comprises or consists of a peptide of SEQ ID NO: 71, and/or that comprises or consists of a peptide of SEQ ID NO: 74.
  • a preferred composition of the invention comprises a peptide that consists of or comprises a peptide of SEQ ID NO: 75, a peptide that consists of or comprises a peptide of SEQ ID NO: 63, and a peptide that consists of or comprises a peptide of SEQ ID NO: 1469
  • a preferred composition of the invention comprises a peptide that consists of or comprises a peptide of SEQ ID NO: 75, a peptide that consists of or comprises a peptide of SEQ ID NO: 63, and a peptide that consists of or comprises a peptide of SEQ ID NO: 71.
  • a pharmaceutically acceptable carrier can be any such carrier known to the person skilled in the art, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).
  • composition according to the present invention further comprises at least one adjuvant.
  • adjuvant may be any adjuvant known to the person skilled in the art. Preferred adjuvants are defined later herein.
  • a preferred use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention is the use as a medicament.
  • a specific preferred use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention is for the treatment and/or prevention of an HBV related disease or condition.
  • the invention provides for the use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention for the manufacturing of a medicament for the treatment and/or prevention of an HBV related disease.
  • the invention further provides a method for the prevention and/or treatment of an HBV related disease or condition comprising administering to a subject an effective amount of a peptide, polynucleotide, composition, cell or T-cell according to the invention and/or a T-cell obtainable or obtained by a method according to the invention.
  • compositions and medicaments according to the invention are preferably formulated to be suitable for intravenous or subcutaneous, or intramuscular administration, although other administration routes can be envisaged, such as mucosal administration or intradermal and/or intracutaneous administration, e.g. by injection.
  • Intradermal administration is preferred herein.
  • Advantages and/or preferred embodiments that are specifically associated with intradermal administration are later on defined in a separate section entitled "intradermal administration”.
  • the administration of a peptide, a polynucleotide, a composition and/or a cell according to the invention and/or a cell obtainable or obtained by a method according to the invention with an appropriate pharmaceutical excipient such as an adjuvant and/or a carrier may be carried out as a single administration.
  • the administration may be repeated if needed and/or distinct peptides, polynucleotides, compositions and/or cells according to the invention and/or cells obtainable or obtained by a method according to the invention with appropriate pharmaceutical excipients such as adjuvants and/or carriers, may be sequentially administered.
  • the peptide, polynucleotide, composition and/or cell according to the invention and/or cell obtainable or obtained by a method according to the invention may preferably comprise at least one immune response stimulating compound or adjuvant.
  • the medicaments according to the invention may additionally comprise one or more synthetic adjuvants.
  • Such adjuvant may be admixed to the medicament according to the invention or may be administered separately to the subject, mammal or human, to be treated.
  • Particularly preferred are those adjuvants that are known to act via the Toll-like receptors and/or via a RIG-I (Retinoic acid- Inducible Gene-1) protein and/or via an endothelin receptor.
  • TLRs Toll like receptors
  • TLR1 may be activated by bacterial lipoproteins and acetylated forms thereof
  • TLR2 may in addition be activated by Gram positive bacterial glycolipids, LPS, LP A, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and Mycobacterial lipoarabinomannans.
  • TLR3 may be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(LC).
  • TLR4 may be activated by Gram negative LPS, LTA, Heat shock proteins from the host or from bacterial origin, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides and fibronectins.
  • TLR5 may be activated with bacterial flagellae or flagellin.
  • TLR6 may be activated by mycobacterial lipoproteins and group B Streptococcus heat labile soluble factor (GBS- F) or Staphylococcus modulins.
  • TLR7 may be activated by imidazoquinolines, such as imiquimod, resiquimod and derivatives imiquimod or resiquimod.
  • TLR9 may be activated by unmethylated CpG DNA or chromatin - IgG complexes.
  • TLR3, TLR7 and TLR9 play an important role in mediating an innate immune response against viral infections, and compounds capable of activating these receptors are particularly preferred for use in the methods of treatment and in the compositions or medicaments according to the invention.
  • Particularly preferred adjuvants comprise, but are not limited to, synthetically produced compounds comprising dsRNA, poly(LC), unmethylated CpG DNA which trigger TLR3 and TLR9 receptors, IC31, a TLR 9 agonist, IMSAVAC, a TLR 4 agonist, Montanide ISA-51, Montanide ISA 720 (an adjuvant produced by Seppic 7, France).
  • RIG-I protein is known to be activated by dsRNA just like TLR3 (Kato et al, 2005).
  • a particularly preferred TLR ligand is a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1.
  • Further preferred adjuvants are Cyclic dinucleotides (CDNs), Muramyl dipeptide (MDP) and poly-ICLC.
  • the adjuvants of the invention are non-naturally occurring adjuvants such as the pam3cys lipopeptide derivative as described in WO2013051936A1, Poly-ICLC, imidazoquinoline such as imiquimod, resiquimod or derivatives thereof, CpG oligodeoxynucleotides (CpG-ODNs) having a non-naturally occurring sequence, and peptide-based adjuvants, such as muramyl dipeptide (MDP) or tetanus toxoid peptide, comprising non-naturally occurring amino acids.
  • non-naturally occurring adjuvants such as the pam3cys lipopeptide derivative as described in WO2013051936A1, Poly-ICLC, imidazoquinoline such as imiquimod, resiquimod or derivatives thereof, CpG oligodeoxynucleotides (CpG-ODNs) having a non-naturally occurring sequence
  • the synthetic adjuvant compounds are physically linked to the peptides of the invention.
  • Physical linkage of adjuvants and costimulatory compounds or functional groups to the ULA class I and ULA class II epitope comprising peptides provides an enhanced immune response by improved targeting to antigen-presenting cells, in particular dendritic cells, that internalize, metabolize and display antigen and by simultaneously stimulating such cells to up- regulate expression of a variety of co-stimulatory molecules, thereby becoming efficient T cell response inducing and enhancing cells.
  • Another preferred immune modifying compound is an inhibitor of an endothelin receptor such as BQ-788 (Buckanovich RJ et al, 2008; Ishikawa K, 1994).
  • BQ-788 is N-cis-2,6- dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D - 1 - methoxycarbonyltryptophanyl-D-norleucine.
  • Another preferred immune response stimulating compound or adjuvant is Interferon alpha (IFNa), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately to the subject as an immuno-modulatory agent.
  • an immune response stimulating compound when admixed to the medicament according to the invention, it is depicted as an adjuvant; when administered separately, it is depicted as an immuno-modulatory agent, or an immuno-modulator, which terms are used herein interchangeably.
  • an immuno-modulatory agent when administered separately, it is depicted as an immuno-modulatory agent, or an immuno-modulator, which terms are used herein interchangeably.
  • antigen presenting cell (co)stimulatory molecules as set out in WO99/61065 and in WO03/084999, in combination with the peptides and compositions of the invention is preferred.
  • a preferred embodiment comprises delivery of the medicaments according to the invention, with or without additional immune stimulants such as TLR ligands and/or anti CD40/anti-4-lBB/ anti-OX-40 or anti-CD27 antibodies in a slow release vehicle such as mineral oil (e.g. Montanide ISA 51) or PLGA.
  • the medicament according to the invention may be delivered intradermally, e.g. by injection, with or without immune stimulants (adjuvants and/or immuno-modulators).
  • the medicaments according to the invention are administered in a composition consisting of the medicaments and one or more immunologically inert pharmaceutically acceptable carriers, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).
  • a medicament according to the invention as defined herein is formulated to be suitable for intradermal administration or application.
  • Intradermal is known to the skilled person. In the context of the invention, intradermal is synonymous with intradermal and is distinct from subcutaneous. A most superficial application of a substance is epicutaneous (on the skin), then would come an intradermal application (in or into the skin), then a subcutaneous application (in the tissues just under the skin), then an intramuscular application (into the body of the muscle). An intradermal application is usually given by injection. An intradermal injection of a substance can be done to test a possible reaction, allergy and/or cellular immunity to it, but can also be performed to elicit a specific antibody or T cell immune response. A subcutaneous application is usually also given by injection: a needle is injected in the tissues under the skin.
  • intradermal administration is that the formulation procedure can be simplified and be made more robust. Furthermore, intradermal vaccine delivery has been repeatedly shown to allow significant dose sparing for eliciting vaccine-induced antibody or T cell responses when compared to conventional administration methods such as intramuscular and subcutaneous administration. This effect is attributed to the relatively dense network of immune cells present in the skin. This was also shown with the HPV16 synthetic long peptides in a human study published by Van der Burg et al. (2007). In this study it was shown that intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of HPV16-specific T cells circulating in the blood.
  • a medicament according to the invention does not comprise any adjuvant such as Montanide ISA-51, and specifically Montanide ISA-51.
  • the formulation of the medicament is more simple: an oil-water based emulsion is preferably also not present in a medicament according to the invention.
  • a medicament according to the invention preferably does not comprise an adjuvant such as Montanide ISA-51 and specifically Montanide ISA-51 and/or does not comprise an oil-in-water based emulsion; more preferably a medicament according to the invention comprises neither of these to adjuvant and even more preferably comprises no adjuvant at all.
  • the medicament according to the invention is a, preferably buffered, aqueous solution, preferably at physiological ionic strength and/or osmolality, such as e.g. PBS (Phosphate Buffer Saline) or water for injection (WFI), comprising or consisting of one or more medicaments as defined earlier herein.
  • physiological ionic strength and/or osmolality such as e.g. PBS (Phosphate Buffer Saline) or water for injection (WFI), comprising or consisting of one or more medicaments as defined earlier herein.
  • PBS Phosphate Buffer Saline
  • WFI water for injection
  • a medicament according to the invention has another advantage, which is that by intradermally administering low amounts of a medicament, preferably a peptide as earlier herein defined, an immunogenic effect may still be achieved.
  • the amount of each peptide used is preferably ranged from 1 and 1000 ⁇ g, more preferably from 5 and 500 ⁇ g, even more preferably from 10 and 100 ⁇ g.
  • the medicament according to the invention comprises a peptide as earlier defined herein and at least one adjuvant, said adjuvant being not formulated in an oil-in water based emulsion and/or not being of an oil-in-water emulsion type as earlier defined herein.
  • This type of medicament according to the invention may be administered as a single administration.
  • the administration of a peptide as earlier herein defined and/or an adjuvant may be repeated if needed and/or distinct peptides and/or distinct adjuvants may be sequentially administered.
  • a peptide according to the invention is administered intradermally whereas an adjuvant as defined herein is sequentially administered.
  • the adjuvant may be intradermally administered.
  • a preferred immune response stimulating compound (immuno-modulator) or adjuvant for intradermal administration is Interferon alpha (IFNa), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately, for example by subcutaneous injection to the subject.
  • IFNa Interferon alpha
  • pegylated Interferon alpha which may be admixed to the medicament according to the invention, or may be administered separately, for example by subcutaneous injection to the subject.
  • the Interferon alpha is preferably also administered subcutaneously and is preferably administered at a dose of 1 microgram/kilogram of body weight within 10 cm proximity to the site where the medicament according to the invention is administered, such described in Zeestraten et al, 2013.
  • Another typical advantage of the medicaments according to the invention is that relatively low amounts of peptides may be used, in one single shot, in a simple formulation and without any adjuvant known to give undesired side-effects as Montanide IS A- 51.
  • the medicament for intradermal administration may be any medicament according to the invention as defined herein.
  • a medicament according to the invention used for subcutaneous administration may be the same as the one used for intradermal administration and may thus be any medicament according to the invention as defined herein.
  • the skilled person knows how to formulate a medicament suited for subcutaneous administration.
  • a medicament according to the invention for subcutaneous administration comprises a peptide as already herein defined in combination with an adjuvant.
  • Preferred adjuvants or immune modulators have already been mentioned herein.
  • Other preferred adjuvants are of the type of an oil-in water emulsions such as incomplete Freund's adjuvant or IF A, Montanide ISA-51 or Montanide ISA 720 (Seppic France).
  • a medicament according to the invention suited for subcutaneous administration comprises one or more peptides according to the invention, an adjuvant or immune modulator as earlier defined herein and an inert pharmaceutically acceptable carrier and/or excipients all as earlier defined herein.
  • Interferon alpha Interferon alpha
  • pegylated Interferon alpha which may be admixed to the medicament according to the invention, or may be administered separately to the subject.
  • the Interferon alpha is preferably also administered subcutaneously and is preferably administered at a dose of 1 microgram/kilogram of body weight within 10 cm proximity to the site where the medicament according to the invention is administered, such described in Zeestraten et al, 2013.
  • the medicament according to the invention suited for intradermal administration may be simultaneously administered with a medicament according to the invention suited for subcutaneous administration.
  • both medicaments may be sequentially intradermally and subsequently subcutaneously administered or vice versa (first subcutaneous administration followed by intradermal administration).
  • the intradermal and/or subcutaneous administration of a medicament according to the invention, preferably a peptide according to the invention, and/or of an adjuvant may be repeated if needed and/or of distinct medicament, preferably peptides and/or of distinct adjuvants may be sequentially intradermally and/or subcutaneously administered.
  • a medicament according to the invention preferably a peptide is administered intradermally and/or subcutaneously whereas an adjuvant as defined herein is sequentially administered as immune-modulator.
  • the adjuvant or immune- modulator may be intradermally and/or subcutaneously administered. However any other way of administration may be used for the adjuvant or immune-modulator.
  • an intradermal and a subcutaneous administration of a medicament according to the invention is advantageous.
  • DC in the epidermis are clearly different from DC in the dermis and in the subcutis.
  • the intracutaneous (intradermal) immunization will cause antigen processing and activation of epidermal DC (Langerin-positive Langerhans cells) that through their dendritic network are in close contact with the keratinocytes. This will also optimally activate inflammatory pathways in the interactions between Langerhans cell and keratinocytes, followed by trafficking of antigen loaded and activated Langerhans cell to the skin-draining lymph nodes.
  • the subcutaneous administration will activate other DC subsets, that will also become loaded with antigen and travel independently to the skin- draining lymph nodes.
  • the use of a medicament which may be administered both intradermally and subcutaneously may lead to a synergistic stimulation of T-cells in these draining nodes by the different DC subsets.
  • a medicament according to the present invention and the methods of treatment described herein using a medicament according to the invention may advantageously be combined with other medicaments and methods of treatment.
  • a medicament according to the invention or a method of treatment according to the invention may be combined with e.g. therapy, and/or antibody therapy against an HBV related disease or may be combined with e.g. immunotherapy and/or antibody therapy against another than HBV related disease, or may be combined with immunotherapy against another antigen than HBV to treat an HBV related disease.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • sequence errors the sequence of the HBV core, HBV polymerase and HBV large surface protein polypeptides obtainable by expression of the gene present in SEQ ID NO: 5, 2 and 1467 containing the respective nucleic acid sequence coding for the polypeptides should prevail.
  • Figure 1 Proteasome-mediated cleavage pattern of long vaccine peptides as predicted in silico and observed in experimental in vitro digestions. For each SLP, the observed cleavage sites are indicated by arrows (major and minor cleavage sites are indicated by bold and thin arrows, respectively, as further detailed herein).
  • the predicted C-score is indicated in the first row
  • the confirmed predicted cleavage sites are indicated in the second row (indicated as '+')
  • the amino acid position within the source protein is indicated in the third row
  • the respective amino acids in the sequence are indicated in the fourth row
  • the amino acid number within the SLP is indicated in the fifth row.
  • C* indicates the cysteine replacement by Abu.
  • Figure 2 Overview of responding naive donors after T cell induction with HBV- derived long peptides. Combined data of IFNy production as measured by ELISA ('ELISA'); hatched box SI (Stimulation Index) > 1.5, and T cell proliferation ('Prolif ) as measured by 3H thymidine incorporation; hatched box SI > 1.5. N.t : not tested
  • FIG 3 Overview of responding HBV-immune donors after stimulation with HBV- derived long peptides. Data represent IFNy-ELISpot results, with hatched boxes indicating a positive response with SI (Stimulation Index)>3, white boxes SI ⁇ 3 .
  • Figure 4 The average Stimulation Index (SI) for each peptide, as measured in an IFNy ELISpot assay, plotted against the predicted TRIA score of the corresponding peptide. Abu-SLP27 is indicated ( ⁇ ).
  • T cell inducing vaccine composition consisting of synthetic long peptides to treat chronically HBV-infected patients.
  • a selection of 37 long peptides (Table 3; sequences ranging from 30 to 39 amino acids) was made encompassing the regions of the HBV Polymerase, Core protein, Surface Antigen and X-protein with the highest putative T cell immunity inducing capacity.
  • HLA class I restricted cytotoxic T cell (CTL) epitopes and putative HLA class II restricted T helper epitopes were identified in these proteins using algorithms that predict HLA class I and II peptide binding and C- terminal generation by the proteasome of all HLA class I binding short peptides (with a length of a CTL epitope; 8 - 12 aa) contained in the HBV proteins. Numerical values were assigned to all putative CTL epitopes and T helper epitopes reflecting their immunogenicity quality.
  • Class I-BCI score Class I-Binding-Cleavage-Immunogenicity score
  • Putative T helper epitopes were assessed using the so-called Class II- B score (Class II-Binding score) reflecting their binding capacity and thus predicted immunogenic quality.
  • Class II-Binding score Class II-Binding score
  • the TRIA score enabled the assessment of the total T cell immunogenicity of any possible long peptide (length 30 - 39 aa) in the HBV proteins.
  • the 37 HBV-derived long peptides with the highest TRIA scores were selected.
  • PBMC peripheral blood mononuclear cells
  • Peptides were synthesized using solid phase Fmoc/tBu chemistry on a PTI Prelude peptide synthesizer and purified on a Gilson preparative HPLC system to > 95% purity. The identity and purity of the peptides were confirmed with UPLC-MS on a Waters Acquity UPLC/TQD system.
  • the putative CTL immunity inducing capacity per SLP was predicted by calculating the cumulative Class I-BCI score per SLP.
  • the cumulative BCI score is based on the Class I-B score, which is a measure for HLA class I peptide binding, and the C-score, which is a measure for proteosomal epitope liberation.
  • the putative T helper cell immunity inducing capacity per SLP was predicted by calculating the cumulative Class II-B score per SLP. As detailed below, the cumulative Class II-B score is based on the Class II-B score, which is a measure for HLA class II peptide binding.
  • the total putative immunity inducing capacity per SLP was predicted by summation of the cumulative Class I-BCI score and the cumulative Class II-B score, which value is denominated herein as the TRIA score.
  • HBV polymerase- derived peptides HBV Core protein-derived peptides
  • HBV Surface Antigen-derived peptides HBV X protein-derived peptides
  • the upper 1.5 th percentile of the predicted binding peptides to each HLA class I molecule was selected.
  • the 'Class I-Binding score (Class I-B score)' is derived from the ranking of the predicted binding affinity of the peptides. Briefly, the ranking was first reversed and subsequently normalized to 100, so that the peptide predicted to bind best has a score of 100. Example: five peptides were selected (5 within the 1.5 th percentile).
  • C-score Cleavage score
  • Class I-BCI Class I- Binding-Cleavage-Immunogenicity
  • Cumulative Class I-BCI score The cumulative Class I-BCI score for each long peptide according to the invention was used as (one of two) selection criterion to identify the peptides of the invention.
  • the cumulative Class I-BCI score is a quantitative reflection of both the total number of CD8 + cytotoxic T cell epitopes that are contained in a long peptide according to the invention and their predicted quality, in terms of binding capacity and likelihood of intracellular generation by the proteasome, and is as such indicative for the CD8 + cytotoxic T cell-inducing power of each peptide according to the invention (its CD8 + T cell immunogenicity).
  • a relatively high cumulative Class I-BCI score of a peptide according to the invention indicates a high CD8 + T cell immunogenicity.
  • HBV polymerase-derived peptides was assessed in silico using a proprietary algorithm.
  • the 'Class II-Binding score' (Class II-B score) is derived from the ranking of the predicted binding affinity of the peptides. Briefly, the ranking was first reversed and subsequently normalized to 100, so that the peptide predicted to bind best has a score of 100. To reduce the number of peptides in the list, all length variants of peptides predicted to bind to a particular HLA class II molecule with a lower predicted binding (lower Class II-B score) are discarded in the list.
  • the cumulative Class II-B score for each long peptide according to the invention was used as the second selection criterion to identify the peptides of the invention.
  • the cumulative Class II-B score is a quantitative reflection of both the total number of CD4 + T-helper cell epitopes that are contained in a long peptide according to the invention and their predicted quality, in terms of binding capacity, and is as such indicative for the CD4 + T cell-inducing power of each peptide according to the invention (its CD4 + T cell immunogenicity).
  • a relatively high cumulative Class II-B score of a peptide according to the invention indicates a high CD4 + T cell immunogenicity
  • T cell Regional Immunogenicity Assessment The TRIA score for a particular peptide of the invention (SLP) is the sum of cumulative Class I-BCI score and the cumulative Class II-B score of that particular long peptide of the invention (SLP). Proteasomal digestion
  • Dithiotreitol (DTT; Sigma- Aldrich) was freshly dissolved in UPLC-grade water and added to a 2X concentrated proteasome digestion buffer (60 mM Trizma-base; pH 7.5; Sigma-Aldrich, 20 mM KC1; Sigma-Aldrich, 10 mM MgCl 2 ; Sigma-Aldrich, 10 mM NaCl; Sigma-Aldrich) to an end concentration DTT of 2 mM. Then, 130 ⁇ UPLC- grade water was added to 3 reaction vials, along with 150 ⁇ of the 2X concentrated proteasome digestion buffer containing 2 mM DTT, and 10 ⁇ of the peptide to be tested (stock concentration 300 nmol/ml).
  • a 2X concentrated proteasome digestion buffer 60 mM Trizma-base; pH 7.5; Sigma-Aldrich, 20 mM KC1; Sigma-Aldrich, 10 mM MgCl 2 ; Sigma-
  • a Q-TOF1 mass spectrometer (Waters) equipped with an online nanoelectrospray interface with an approximate flow rate of 250 nl/min was used for electrospray ionization-mass spectrometry. Peptide-digestion samples were trapped on a precolumn
  • PBMC Peripheral blood mononuclear cells
  • DCs dendritic cells
  • approximately 50* 10 6 PBMC were brought to a concentration of 3* 10 6 cells/ ml complete medium (EVIDM, Lonza, supplemented with 8% HS, Seralab; penicillin/streptomycin, Lonza; L-glutamin, Lonza) and seeded 3 ml /well in a 6-wells plate (Corning). After incubation for 1.5 hours at 37°C, non-adherent cells were washed away in three washing steps using complete medium (day 0).
  • the adherent cells were cultured for three days at 37°C in 2 ml/well of complete medium containing 800 U/ml GM-CSF and 500 U/ml IL-4 (Peprotech). On day 3, 1 ml of complete medium containing 2400 U/ml GM-CSF and 1500 U/ml IL-4 was added to each well and cultured for another three days at 37°C. Induction of T cells
  • Pool 1 comprises SLP 26 (SEQ ID NO: 76), SLP 24 (SEQ ID NO: 74), SLP 1 (SEQ ID NO: 51) and SLP 30 (SEQ ID NO: 1142);
  • Pool 2 comprises Abu-SLP 27 (SEQ ID NO: 77, wherein the cysteine on amino acid position 1 is replaced by Abu), SLP 25 (SEQ ID NO: 75), SLP 10 (SEQ ID NO: 60) and SLP 34 (SEQ ID NO: 1469); and Pool 3 comprises SLP 5 (SEQ ID NO: 55), SLP 13 (SEQ ID NO: 63), SLP 14 (SEQ ID NO: 64) and SLP 21 (SEQ ID NO: 21).
  • DC and autologous PBMC were co- cultured in a 1 : 10 ratio for 10 days at 37°C in the presence of 10 ng/ml IL-7 and 100 pg/ml IL-12p70.
  • the T cell lines generated by this process were checked every 2-3 days and split when necessary.
  • a second batch of autologous DC was differentiated and loaded with peptide pools as described above.
  • the peptide-loaded DC were washed twice with complete medium and added to the T cells in a 1 : 10 (DC:T cell) ratio in the presence of 10 ng/ml IL-7 and 100 pg/ml IL-12p70.
  • the cells were co-cultured for 7 days.
  • a new batch of DC was differentiated and loaded with peptide pools as described above.
  • DC and T cells were co-cultured in a 1 : 10 ratio.
  • the PBMC were first pre-stimulated with 3 nmol/ml of the indicated peptide for 72 hours at 37°C. During this stimulation, ELISpot PVDF plates (Mabtech) were coated with 5 ug/ml anti-human IFNy mAb 1-DlK coating antibody (Mabtech) in PBS and incubated overnight at 4°C. After stimulation of the PBMC, the coating antibody was aspirated from the plate, and washed 4 times with PBS. To block aspecific binding, 100 ⁇ of IMDM containing 8% FCS was added to all wells and incubated at 37°C for 1 hour.
  • PBMC peripheral blood mononuclear cells
  • the biotinylated anti-human IFNy mAb 7-B6-1 (Mabtech) was added to all wells (100 ⁇ /well) at a 0.3 ⁇ g/ml concentration in PBS with 1% FBS, and incubated for 2 hours at RT. Next, plates were washed 6 times using PBS/Tween20 0.05% and 1 ⁇ g/ml Extravidin- Alkaline phosphatase (ALP) (Sigma-Aldrich) was added to all wells (100 ⁇ /well) in PBS with 1% FBS. The plates were incubated for 1 hour at RT.
  • ALP Extravidin- Alkaline phosphatase
  • ALP substrate solution BCIP/NBT-plus (Mabtech) was prepared and 100 ⁇ /well was added to all wells after the plates were washed 4 times with PBS/Tween20 0.05%. To terminate the colorimetric reaction (after 1-20 minutes), tap water was used to wash the plates extensively. After drying, the formed spots were measured on a Biosys Bioreader 5000.
  • a high quality CD8 + T cell epitope is defined as a peptide that possesses both a predicted high affinity for the HLA class I molecule to which it binds and is also predicted to be generated at its C-terminus by a proteolytic cleavage of the proteasome.
  • Peptides according to the present invention were selected in HBV protein regions that contain optimally high numbers of high quality CD8 + and CD4 + T cell epitopes. To this end, first the HLA class I binding and C-terminal generation of all possible CD8 + T cell epitopes was assessed using a proprietary HLA class I peptide binding algorithm and two proprietary algorithms predicting the cleavages by the proteasome.
  • binding-cleavage- immunogenicity (BCI) Class I-score that for each short peptide (8 - 13 amino acids) incorporates both its predicted binding affinity for the HLA class I molecule to which it binds and the likelihood that the peptide is generated by the proteasome in the cells.
  • the Class I-BCI score is calculated from (1) the binding Class I-score (Class I-B score), which is derived from the results of the in silico prediction of HLA class I peptide binding using the aforementioned algorithm, and (2) the cleavage score (C- score), which is derived from the results of the in silico prediction of the proteasome- mediated C-terminal generation of the peptide by the proteasome using the aforementioned algorithms.
  • Tables 4a, 5a, 6a, and 7a present the Class I-BCI score for all possible CD8 + T cell epitopes of these Polymerase, Core protein, Surface Antigen or X-protein derived SLPs, respectively, together with the cumulative BCI Class I score.
  • Tables 4b, 5b, 6b and 7b present the Class II-B score for all possible CD4 + T cell epitopes of these Polymerase, Core protein, Surface Antigen or X-protein derived SLPs, respectively, together with the cumulative Class II-B score. Together the cumulative Class I-BCI score (for CTL epitopes) and the cumulative B Class II-score (for T helper epitopes) resulted in one quantitative value, the so-called Total Regional Immunogenicity Assessment (TRIA) score, reflecting the overall T cell immunogenicity of a long vaccine peptide (Table 3). Based on the highest TRIA scores, 37 SLPs derived from HBV Polymerase, Core protein, Surface Antigen or X-protein were selected for further evaluation (Table 3).
  • TRIA Total Regional Immunogenicity Assessment
  • Digestion experiments were performed separately with 20S constitutive proteasomes and 20S immuno proteasome preparations.
  • the combined analysis of cleavages produced by both types of proteasomes allows the assessment of the C- terminal generation of CTL epitopes that are expressed both on the surface of antigen presenting cells (mainly dendritic cells), containing immunoproteasomes, and on the surface of cancer cells, especially from solid tumors, which mainly express constitutive proteasomes.
  • antigen presenting cells mainly dendritic cells
  • these epitopes are preferred because vaccination with such epitopes will allow the induction of CTL by vaccination and the subsequent eradication of cancer cells by these CTL after recognition of the epitopes on the surface of cancer cells.
  • 10 long peptides of the invention (length 30 - 39 aa) were co-incubated in an appropriate buffer with the proteasome preparations at 37°C for 0, 1, 3, 6 and 24 h. After incubation for the indicated interval reactions were stopped and digestion mixtures, containing the digestion fragments, were measured by mass spectrometry as described herein. The mass spectra were (semi-quantitatively) analyzed to assess the position and abundance of the cleavage sites. The results of 24 h digestion are shown in Figure 1.
  • Major cleavage site fragments containing as COOH terminus the residue MTi-terminal from the cleavage site together with the (possible) complementary fragment(s) are present for >7% at 24 h incubation, as calculated from the intensities of the fragment peaks in the mass spectra.
  • Minor cleavage site fragments containing as COOH terminus the residue MTi-terminal from the cleavage site together with the (possible) complementary fragment(s) are present for ⁇ 7% at 24 h incubation. Cleavage sites with a cumulative fragment abundance of ⁇ 1% are not shown.
  • Figure 1 also indicates the C-score of proteasomal cleavage prediction. This score indicates the likelihood of cleavage C-terminal of the residue directly under the C- score. If the C-score > 1 the cleavage site is considered predicted to be cleaved by both the constitutive proteasome and the immunoproteasome. As described herein, the C- score is a summation of the predictions by two in silico algorithms separately predicting the proteasome-mediated cleavages by constitutive proteasomes and the cleavages by immunoproteasomes. Each separate prediction can attain a maximal value of 1. Accordingly, the maximal value of the C-score is 2. C-scores > 1 are together counted as the total number of predicted cleavage sites (the C-terminus of the long substrate peptide is not taken into account, because cleavage after this residue cannot be tested).
  • Figure 1 further indicates the confirmed cleavages (indicated by '+'), which are those predicted cleavage sites (C-score > 1) that are confirmed to be cleaved after 24 h in the proteasome-mediated digestion assay.
  • PBMC peripheral blood mononuclear cells
  • T cell responses against the 13 selected HBV peptides in PBMC derived from six HBV-immune donors After isolation of the PBMC, cells were stimulated with each of the 13 peptides and an IFNy-ELISpot was performed to detect T cell responses.
  • PBMC samples from 3 out of 6 tested donors showed a positive IFNy response against one or more of the 13 peptides (SI > 3).
  • the average SI of the IFNy-responses was calculated; 1.21 for SLPl, 2.1 1 for SLP5, 2.40 for SLP10, 2.44 for SLP13, 1.97 for SLP 14, 1.86 for SLP 18, 2.87 for SLP21, 2.54 for SLP24, 2.66 for SLP25, 1.56 for SLP26, 1,66 for Abu-SLP27, 1.49 for SLP30 and 3.02 for SLP34.
  • HBV-derived long vaccine peptides of the present invention encompass the HBV protein regions with the highest number of high quality HLA class I and HLA class II binding epitopes in an outbred population.
  • a preferred combination of the vaccine peptides will be incorporated in a novel HBV SLP vaccine composition to treat chronically HBV-infected patients.
  • the TRIA score is a quantitative representation of the quality and quantity of all putative HLA class I restricted CD8 + cytotoxic T cell epitopes and HLA class II restricted CD4 + T helper epitopes contained in a long peptide.
  • the TRIA score was calculated for all possible HBV peptides with a length of 30 - 39 aa, which is the optimal peptide length for vaccination purposes, enabling the rational selection of a set of highly immunogenic long vaccine peptides.
  • C TRIA score is the sum of the Cumulative Class I-BCI score and the Cumulative Class II-B score.
  • Table 4a Predicted HLA class I-restricted CD8 + cytotoxic T cell epitopes contained in SLP sequences from HBV polymerase protein.
  • HLA class I binding peptide of HBV polymerase is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
  • Predicted HLA class Il-restricted CD4 + T cell epitopes contained sequences from HB V polymerase protein.
  • SLP Peptide of invention

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Abstract

The present invention relates to the fields of medicine and immunology. In particular, it relates to novel peptides that may be used in the treatment and/or prevention of a Hepatitis B viral infection and/or an Hepatitis B related disease or condition.

Description

Synthetic Long Peptides (SLP) for therapeutic vaccination against Hepatitis B virus infection
FIELD OF THE INVENTION
The present invention relates to the fields of medicine and immunology. In particular, it relates to novel peptides that may be used in the treatment and/or prevention of a Hepatitis B viral infection and/or an Hepatitis B related disease or condition. BACKGROUND OF THE INVENTION
Chronic infection with the hepatitis B virus (HBV) is a major global health problem. HBV is the prototype member of the Hepadnaviridae family, which have a strong preference for infecting liver cells (Ganem et al, 2004).
Despite the availability since three decades of an efficacious preventive vaccine for the protection against hepatitis B, an estimated two billion people have nevertheless been infected with HBV and more than 240 million currently have chronic (long-term) hepatitis B infection, with a geographical predominance in regions outside Western Europe and North America (World Health Organization, July 2013).
Transmission of the virus between people occurs by direct blood-to-blood contact or via semen or vaginal fluid of an infected person. In endemic areas, the infection occurs characteristically by perinatal transmission from mother to child. Thus, although HBV is not transmitted casually, the virus - via similar modes of entry as human immunodeficiency virus (HIV) but being at least 50 times more infectious - can be easily transmitted by perinatal, percutaneous or sexual exposure. Frequent person-to- person contact with infected individuals accordingly poses a serious risk to groups like health workers.
Infection with HBV can develop as an acute viral hepatitis, an illness that begins with general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, and then progresses to development of jaundice. The illness lasts for a few weeks and then gradually improves in most affected adults. A few people may have more severe liver disease (fulminant hepatic failure), and may die as a result. The infection may be entirely asymptomatic and may go unrecognized. Chronic infection with hepatitis B virus either may be asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), leading to cirrhosis over a period of many years. This type of infection dramatically increases the incidence of hepatocellular carcinoma (liver cancer), also with a latency of many years.
Treatment of chronically HBV-infected individuals with antiviral drugs such as nucleoside/nucleotide analogues (e.g. Entecavir and Tenofovir) or interferon (IFN)a efficiently decreases serum viral loads. However, antiviral therapy rarely leads to a sustained virological response and drug resistance occurs (Zoulim et al, 2012). Moreover, the great majority of HBV carriers remains untreated.
Approximately 15-40% of chronic HBV carriers will develop clinically significant liver diseases in their lifetime with a high risk of death from liver cirrhosis and associated liver failure or hepatocellular carcinoma (HCC) (Lok, 2002; and Huang et al, 2011). Yearly up to one million people die worldwide due to the acute or chronic consequences of hepatitis B (Michel et al, 2001 ; Grimm et al, 2013). Due to the failure of antiviral drugs to eradicate infection, and consequently the need for long-term if not lifelong antiviral therapy with its drawbacks such as toxic side-effects and high costs, there is an urgent need for novel therapeutic approaches (Grimm et al., 2013).
The present invention is meant to enable efficacious therapeutic vaccination against chronic HBV infection. Therapeutic vaccination constitutes a promising strategy to treat chronic hepatitis B (Michel et al., 2011).
Next to the humoral immune response against HBV, which is predominantly involved in the protection against HBV infection by the current prophylactic vaccines (Lok, 2002), the cellular immune response is unequivocally involved in the natural resistance against HBV infection.
Perinatal transmission of HBV from mothers to neonates and infections during the first years of life result in persistent infection in more than 90% of children. By contrast, infection during adulthood clears spontaneously in more than 90% of cases and results in lifelong protective immunity (Rehermann et al., 2005).
In acute, self-limited hepatitis B virus infection, vigorous polyclonal and multispecific CD8+ cytotoxic T cell (CTL) and CD4+ T-helper (Th) cell responses to many HBV antigens are readily demonstrable in the peripheral blood (Michel et al, 2011).
These T cell responses are crucial in HBV clearance and control. Experiments in HBV-infected chimpanzees have shown the essential role of HBV-specific CD8+ T cells as effector cells in this process (Thimme et al, 2003). In contrast to the response in patients with resolved HBV infections, in patients with chronic hepatitis B the T cell responses are usually very weak, focused on only a few epitopes and functionally impaired (Michel et al, 2011). The goal of therapeutic vaccination is to install vigorous and robust multivalent CTL and T-helper cell responses directed to many HBV antigens, thereby pursuing viral clearance, hepatitis control and cure.
Despite the fact that great progress has been made in understanding the etiology and epidemiology of the disease, there is still a need for an effective therapeutic HBV vaccine.
DETAILED DESCRIPTION OF THE INVENTION
The inventors identified a selection of HBV antigens to be used for efficacious therapeutic vaccination. Based on HLA class I and class II binding capacities of HBV protein-derived peptides and analysis of the generation of these HLA class I binding peptides by cleavages made by the proteasome, the most immunogenic regions, covering a very high percentage of all possible T cell epitopes in the global hepatitis B patient population, have been uncovered in the HBV polymerase protein, core protein, X protein and large surface protein. These regions contain high numbers of T cell epitopes and when administered to the hepatitis B patient - either as chemically synthesized long peptide or via genetic approaches - such a vaccination is envisioned to induce a vigorous T cell response, resolving the HBV infection.
The use of relatively short peptides is highly preferred for medical purposes as these can be efficiently synthesized in vitro, which is not possible or uneconomical for native proteins larger than approximately 100, i.e. 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 amino acids. Chemical synthesis of peptides is routine practice and various suitable methods are known to the skilled person. Chemical synthesis of peptides also overcomes the problems associated with recombinant production of intact proteins, which is difficult to standardize and requires extensive purification and quality control measures. Peptides with a length that exceeds the length of human leukocyte antigen (HLA) class I and class II epitopes (e.g. having a length as indicated below herein) are particularly advantageous for use as vaccine component because they are large enough to be taken up by professional antigen presenting cells (APC), in particular Dendritic cell (DC), as explained in WO02/070006, and processed in the DC before cell surface presentation of the contained HLA class I-presented and HLA class Il-presented epitopes takes place. Therefore, the disadvantageous induction of T cell tolerance by the systemic presentation of minimal HLA class I-presented epitopes on non-antigen presenting cells (as shown in Toes et al, 1996a, and Toes et al, 1996b), is prevented by the application of peptides exceeding the length of human leukocyte antigen (HLA) class I and class II epitopes (as shown in Zwaveling et al., 2002).
The present invention relates to novel peptides of about 15 to about 100 amino acids in length, also denominated herein as long peptides, that each exceed the length of human leukocyte antigen (HLA) class I and class II presented epitopes and that induce a combined CD4+ and CD8+ T cell response that is therapeutically successful and inducing cure in a high percentage of patients. Preferably, the long peptides of the invention are synthetic peptides, denominated herein as synthetic long peptides (SLPs). As compared to vaccination with the peptides of the present invention, therapeutic vaccination with full length HBV proteins is likely to be less potent (Rosalia et al, 2013). From the viewpoint of manufacture and administration of peptides to patients, immunization with the complete set of overlapping long peptides or SLPs spanning the full length HBV polymerase, HBV core protein, HBV X protein and HBV large surface protein is not feasible. To narrow the number of peptides in a vaccine, it is needed to incorporate the most immunogenic SLPs that are recognized by the highest percentage of patients. The present invention provides for peptides and peptide vaccines to meet this need. Using a stepwise sophisticated selection procedure based on bioinformatics analyses that are experimentally underpinned, the long peptide and/or SLP sequences with the highest coverage of HLA class I-restricted cytotoxic T lymphocyte epitopes and HLA class II-restricted T helper epitopes were identified. The selections as described herein identify the long peptide and/or SLP sequences that incorporate HBV- derived T cell epitopes that are presented on all predominantly expressed HLA class I and class II alleles. By covering the vast majority of worldwide expressed HLA haplotypes (Bui et al, 2006), the long peptide and/or SLP vaccine composition can be used in all HBV infected individuals. The present invention describes the identification and selection of HBV-derived long peptides, preferably SLPs, that are highly immunogenic and capable of inducing a potent combined HB V-directed CD4+ T helper and CD8+ cytotoxic T cell response, when administered as a vaccine composition to patients. Such highly immunogenic long peptides from HBV have not been disclosed in the prior art. The HBV-derived long peptides of the invention were identified based on a putative immunogenicity score developed and validated by the inventors and as disclosed herein. The putative immunogenicity is quantified herein using the T cell Regional Immunogenicity Assessment (TRIA) score. The TRIA score is based on the cumulative Class I-BCI score of said peptide, which is indicative for their immunogenic CTL activating capacity, and the cumulative Class II-B score of said peptide, which is indicative for their immunogenic Th-cell activating capacity. Calculation of the cumulative Class I-BCI score and the cumulative Class II-B score is described in detail herein in the Examples section. The TRIA score is calculated as the sum of the cumulative Class I-BCI score and the cumulative Class II-B score. A strong correlation was found between this TRIA score and the T cell responses found in PBMC of HBV-immune donors. Therefore, the TRIA score enables the selection of optimal immunogenic long peptides. In a first aspect, the present invention provides a peptide derived from an HBV protein. Preferably the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471, more preferably the peptide of the invention comprises or consist of a peptide selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 76, 77, 1142 and 1469, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 77, 1142 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 60, 63, 71, 74, 75 and 1469, most preferably selected from the group of SEQ ID NO: 75, 1469 and 63. Further preferred is a peptide of the invention that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51, 60, 63, 64, 68, 71, 74-77. Also preferred is a peptide of the invention that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 63, 71 and 75. Preferably, the peptide of the invention comprises at least about 70 predicted T-cell epitopes. More preferably, the peptide of the invention comprises at least about 70 predicted T-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, the peptide if the invention comprises at least about 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes, at least about 1 predicted HLA class II-restricted CD4+ T-helper epitope. More preferably, the peptide if the invention comprises at least about 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes, at least about 1 predicted HLA class II-restricted CD4+ T-helper epitope and at least about 3 proteasomal cleavage sites. HLA class I-restricted CD8+ cytotoxic T-cell epitope is also denominated herein as CTL epitope and HLA class II-restricted CD4+ T-helper epitope is also denominated herein as Th-cell epitope. Preferably, the peptide of the invention comprises at least about 70 predicted CTL epitopes, at least about 15 predicted Th-cell epitopes. More preferably, the peptide of the invention comprises at least about 70 predicted CTL epitopes, at least about 15 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises at least about 95 predicted CTL epitopes, at least about 25 predicted Th-cell epitopes. More preferably, the peptide of the invention comprises at least about 95 predicted CTL epitopes, at least about 25 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises at least about 125 predicted CTL epitopes, at least about 50 predicted Th-cell epitopes. More preferably, a peptide of the invention comprises at least about 125 predicted CTL epitopes, at least about 50 predicted Th-cell epitopes and at least about 3 proteasomal cleavage sites. Preferably, a peptide of the invention has a TRIA score of at least about 6300, at least about 8000, at least about 9000, at least about 10000, or preferably at least about 14000.
A peptide of the invention can advantageously be used in the prevention and/or treatment of an HBV related disease or condition in a subject, preferably a mammal, more preferably a human. Preferably, the peptide of the invention comprises or consists of an amino acid sequence, preferably a contiguous amino acid sequence, of any of the proteins selected from the group consisting of HBV protein polymerase, HBV core protein, HBV X-protein and HBV large surface protein. Preferably, said peptide comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471. A peptide of this group is characterized in that it has a TRIA score of at least 6300, indicating the high immunogenic capacity for CD4+ and CD8+ T cell activation. Furthermore, a peptide of this group is characterized in that it comprises at least 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes, at least 1 predicted HLA class II-restricted CD4+ T-helper epitope. . Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites.
More preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55-57, 60-66, 68-78, 1142- 1145 and 1468-1471. A peptide of this group is characterized in that it has a TRIA score of at least 8000.
More preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55-57, 60-66, 68, 69, 71-79, 1142-1145 and 1468-1471. A peptide of this group is characterized in that it comprises at least 70 predicted CTL epitopes, at least 15 predicted Th-cell epitopes. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 51-53, 55, 57, 60, 63, 64, 66, 68, 71, 72,74-78, 1142, 1145, 1468-1471.
More preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 53, 55-57, 60-66, 68, 69, 71, 73-78, 1142-1145, 1468-1471. A peptide of this group is characterized in that it has a TRIA score of at least 9000.
Even more preferably, the peptide of the invention is a peptide that comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 55-57, 60-65, 68, 69, 71, 74, 75, 77, 78, 1142-1145, 1468, 1469 and 1471. A peptide of this group is characterized in that it has a TRIA score of at least 10000.
Even more preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 52, 53, 55, 57, 60, 61, 63, 64, 68, 69, 71, 72, 75, 77, 78, 1142-1145, 1468, 1469 and 1471. A peptide of this group is characterized in that it comprises at least 95 predicted CTL epitopes, at least 25 predicted Th-cell epitopes. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites. Preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 75, 77, 1142, 1469.
Most preferably, the peptide of the invention comprises or consists of a peptide selected from the group consisting of SEQ ID NO: 63, 75, 1143-1145, 1468 and 1469. A peptide of this group is characterized in that it has a TRIA score of at least 14000. Furthermore, a peptide of this group is characterized in that it comprises at least 125 predicted CTL epitopes, at least 50 predicted Th-cell epitopes. Preferably, a peptide of this group comprises at least 3 proteasomal cleavage sites.
A "T-cell epitope" is defined herein as a linear fragment of a polypeptide antigen, which is recognized and bound by a T-cell receptor, preferably a human T-cell receptor, after being made accessible to a T-cell receptor by intracellular proteolytic processing of the polypeptide antigen and subsequent presentation by an HLA class I or HLA class II molecule on the cell surface of an antigen-presenting cell. A "predicted T- cell epitope" is to be understood herein as a linear fragment of a polypeptide antigen for which liberation from the source protein or peptide by proteolytic cleavage and T-cell receptor recognition and/or binding has been predicted using bioinformatics analyses based on algorithms that predict HLA class I and II peptide binding and C-terminal generation by the proteasome of all possible HLA class I binding peptides (with a length of a HLA class I ligand; 8 - 12 aa) contained in the HBV proteins. A "confirmed T-cell epitope" is to be understood herein as a linear fragment of a polypeptide antigen for which liberation from the source protein or source polypeptide by proteolytic cleavage and T-cell receptor recognition and/or binding, and more preferably CD4+ or CD8+ T cell activation capability, have been established experimentally as disclosed herein. A "linear fragment" is understood herein to be a contiguous amino acid sequence of a polypeptide antigen, said polypeptide antigen preferably being an HBV protein, more preferably a protein selected from the group consisting of HBV protein polymerase, HBV core protein, HBV X-protein and HBV large surface protein. An identical linear fragment of a polypeptide antigen showing binding affinity to a second or further type of HLA class I or HLA class II molecule is to be understood herein as a second or further T-cell epitope. In other words, a specific linear fragment of a polypeptide antigen being capable to bind to two types of HLA molecules is understood herein to be two separate or distinct T-cell epitopes, and is scored twice within the cumulative BCI Class I- and/or Class II-B score. A T-cell epitope typically comprises or consists of at least 8 amino acids and up to 20 or (exceptionally) even more amino acids. A T-cell epitope can be an HLA class I-restricted CD8+ cytotoxic T cell (CTL) epitope or an HLA class Il-restricted CD4+ T-helper (Th-)cell epitope. HLA class I restricted epitopes (also referred to as CTL epitopes) are typically presented via the classical proteasome dependent HLA class I processing route, whereas HLA class-II molecules are typically loaded with linear fragments in the late endosomal compartment. Preferably, a peptide according to the invention comprises T-cell epitopes that are selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466 (see Tables 4-7). A preferred peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. A more preferred peptide according to the invention comprises at least 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. An even more preferred peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466. An even more preferred peptide according to the invention comprises at least 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 or from about 230 to about 233 predicted T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466. Preferably, the predicted T-cell epitopes of the present invention are confirmed experimentally as disclosed herein. A preferred peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. A more preferred peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed T-cell epitopes from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. An even more preferred peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466. An even more preferred peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed T-cell epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-1140, and 1146 - 1466.
A "proteasomal cleavage site" is understood herein as a site in a protein or polypeptide that is cleaved by the proteasome, preferably a human proteasome/proteasome naturally present in a human cell. A specific proteasomal cleavage site liberating the C-terminus of the epitope is preferably present exactly after the C-terminus of the epitope amino acid sequence, in order to allow the epitope's C- terminal residue to be liberated from the larger peptide and to be presented by the HLA class I molecule. The first important event that defines an HLA class I-restricted CD8+ cytotoxic T-cell (CTL) epitope is the release of the epitope (or the epitope-precursor) from its flanking protein regions through enzymatic cleavage by cytosolic peptidases. The multicatalytic proteasome is the primary enzyme complex required for the generation of the exact C-terminus of the vast majority of CTL epitopes (Rock et al, 2004). Proteasomes are multicatalytic enzyme complexes abundantly present intracellularly and are considered responsible for the generation of the C terminus of the vast majority of CTL epitopes (Craiu et al, 1997; Stoltze et al., 1998; Mo et al., 1999). The generation of the amino-terminus of a CTL epitope, on the other hand, is much more flexible because several amino-terminal exo-peptidases (like ERAPl, puromycin sensitive aminopeptidase, bleomycin hydrolase and others) reside in the cytosol and endoplasmic reticulum and these trimming enzymes have the capacity to shorten an N-terminal elongated epitope-precursor to its precise length. In contrast, C- terminal trimming has not been reported. Therefore the identification of the proteasome-mediated cleavage sites in a protein or in a polypeptide, like a peptide of the invention, can be used as an important identifier of almost every CTL epitope, because the proteasomal cleavages determine and enable C-terminal epitope generation (Kessler et al, 2001; Kessler and Melief, 2007). The assessment of proteasomal cleavage sites in the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein identifies the C-termini of intracellularly produced HBV peptide fragments, specifically for HLA class I presented peptide fragments. Length requirements are much less strict for loading of HLA class II with peptide fragments. Therefore, precise enzymatic generation of the HLA class II binding peptide fragment is not needed. These T-cell epitope requirements have been used in the present invention to localize and design long peptides derived from the full length sequences of an HBV protein which comprises preferred CD8+ cytotoxic T cell (CTL) and CD4+ T- helper (Th-)cell epitopes and/or combinations thereof and are thus highly immunogenic and therefore suitable peptides for synthesis and (therapeutic) vaccination purposes.
Proteasome mediated proteolytic cleavages can be predicted in silico using a prediction algorithm. Cleavage as performed by the proteasome can be verified in a proteasome mediated cleavage assay as disclosed herein, which measures the C- terminal liberation of the epitope from its flanking regions (Kessler et al, 2001; Kessler and Melief, 2007). A cell free proteasome cleavage assay identifying and quantitatively measuring the amino acid (aa) positions and the abundancy of cleavages by the proteasome in a polypeptide can be used to determine which peptides are generated from the source protein (or source polypeptide), thereby establishing the peptide pool available for epitope generation. The cell free proteasome cleavage assay involves the co-incubation of a polypeptide (preferably having a length of 28-40 aa, more preferably having a length of 30-39 aa) with a preparation of purified proteasomes in an appropriate buffer solution. Two main forms of proteasomes exist, the immunoproteasomes, which are mainly expressed in professional antigen presenting cells, like e.g. Dendritic Cells, and the constitutive proteasomes, which are expressed mainly in other cell types. These types contain variant catalytic subunits with slightly different catalytic activity. Although most epitopes are liberated by both types of proteasomes, sometimes differential epitope generation occurs dependent on proteasome type (Morel et al, 2000; Chapiro et al, 2006). Accordingly, proteasome- mediated cleavage assays may be performed separately with these two proteasome types. Preferably, a constitutive 20S-proteasome or immune 20S-proteasome is used as disclosed herein. The reaction mixture comprising the peptides to be cleaved and either of the two proteasome types (purified proteasome preparations) is incubated at 37°C and samples are drawn at 1 h, 3 h, 6 h and 24 h time points as detailed in the Examples herein. Subsequently, generated peptide cleavage fragments and the remaining source polypeptide are identified and quantified by mass spectrometry (Kessler et al, 2001). This assay reveals both the positions in the polypeptide (and thus in the source protein) where the proteasome cleaves and the cleavage efficiency (abundancy) at these positions. A cleavage site can be confirmed by detection of fragments containing as COOH terminus the residue H2-terminal from the cleavage site together with the (possible) complementary fragment(s), as calculated from the intensities of the fragment peaks in the mass spectra (preferably present for >1%, more preferably present for >7%, at 24 h incubation), in both the digestion with constitutive proteasomes and the digestion with immunoproteasomes. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, preferably at least 3, proteasomal cleavage site as defined herein. More preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, preferably at least 3, proteasomal cleavage site as assessed and verified in a proteasomal cleavage assay as described above.
As indicated above, examples of T-cell epitopes are HLA class I-restricted CD8+ cytotoxic T cell (CTL) epitopes and HLA class Il-restricted CD4+ T-helper (Th-)cell epitopes. A "CTL epitope" is understood herein as a linear fragment of a polypeptide antigen that is liberated from the source protein by proteasome mediated proteolytic cleavage and subsequently presented by an HLA class I molecule on the cell surface of an antigen presenting cell (APC), preferably a human antigen presenting cell. A "predicted CTL epitope" is understood herein as a linear fragment of a polypeptide antigen for which liberation from its source protein by proteolytic cleavages and HLA class I molecule binding have been predicted using bioinformatics analyses based on algorithms that predict HLA class I peptide binding and C-terminal generation by the proteasome of all HLA class I binding short peptides (with a length of a CTL epitope; 8 - 12 aa) contained in the HBV proteins. Preferably, a predicted CTL epitope of the present invention is confirmed experimentally as disclosed herein. A CTL epitope of the invention is preferably capable of activating a CD8+ T cell response. A "confirmed CTL epitope" is understood herein as a linear fragment of a polypeptide antigen for which liberation by proteolytic cleavages and HLA class I molecule binding, more preferably CD8+ T cell activation, have been established experimentally as disclosed herein. A CTL epitope of the invention is preferably capable of activating a CD8+ T cell response. A CTL epitope typically comprises at least 8 up to 12, or exceptionally up to 13 or 14 amino acids. Preferably a CTL epitope consists of 8-14 amino acids, i.e. has a length of at least 8 up to 14 amino acids. A CTL epitope is defined by two important intracellular events being (i) proteasome mediated proteolytic cleavage and (ii) binding to an HLA class I molecule, which takes place in the endoplasmic reticulum (ER). The stronger a linear peptide fragment binds and the slower the off-rate, the more likely that this linear peptide fragment will become a cell surface presented immunogenic CTL epitope (Van der Burg et al., 1996). Analysis of proteasome mediated proteolytic cleavages can be performed as indicated above. Preferably, specific binding to an HLA class I molecule is predicted using an in silico prediction algorithm and established by using an HLA class I peptide binding assay as known by the person skilled in the art (Kessler and Melief, 2007; Kessler et al., 2003). Preferably, the HLA class I-restricted epitope in a long peptide according to the invention is predicted to be generated at its C-terminus by the proteasome and preferably has a predicted high affinity binding capacity for the HLA class I molecule using an assay as described in van der Burg et al, 1995 and Kessler et al, 2003; e.g. IC50≤ about 5 μΜ may be considered high affinity binding, about 5 μΜ < IC5o≤ about 15 μΜ may be considered intermediate affinity binding, about 15 μΜ < IC5o < 100 μΜ may be considered low affinity binding and IC50 > about 100 μΜ may be considered as no binding. To measure class I binding affinity of a peptide or fragments thereof, various HLA class I binding assays are available. The assays can be divided into cell-free assays (using soluble HLA) versus cellular assays (using HLA class I molecules on the cell surface), and competitive assays (resulting in semi-quantitative data) versus assays that do not use a labeled reference peptide and are therefore quantitative (Kessler and Melief, 2007; Viatte et al., 2006). The assays have in common that the HLA class I peptide binding affinity is reliably assessed.
The actual presentation of a CTL epitope on the cell surface, i.e. the net result of both proteasomal cleavage, possible other proteolytic events like N-terminal trimming, and binding and presentation by an HLA class I molecule, which events together define a CTL epitope as indicated above, can be demonstrated by a biochemical approach or by a functional approach using cytotoxic T cells with a T-cell receptor specific for the epitope and HLA class I molecule (geno)type, as known by the person skilled in the art (Kessler and Melief, 2007).
The biochemical approach involves the biochemical purification of HLA-epitope complexes from cells expressing the HBV antigen of the invention together with the presenting HLA class I molecule (geno)type, followed by the mass spectrometric search for the epitope in the eluted HLA class I-bound CTL receptor ligands as known by the person skilled in the art (Schirle et al., 2000; Schirle et al., 2001).
The functional approach involves a CTL line or clone that is specifically recognizing the HLA-epitope, which is used as a tool to demonstrate the natural processing and actual presentation of the epitope by HLA class I molecules. In this methodology, using a CTL induction assay as known in the art, either the synthetically generated minimal (i.e. exact length) epitope or the peptide sequence of interest encompassing the epitope, for instance a peptide, long peptides and/or SLP as defined herein, is used to stimulate and select HLA-epitope-specific cytotoxic T cells. To that end, briefly, a multivalent CD8+ T cell population, or a multivalent mixed CD8+ and CD4+ T cell population, is stimulated with autologous target cells of which the HLA class I molecules on the cell surface are either exogenously loaded with the precise synthetic epitope or endogenously loaded with intracellularly generated CTL epitopes derived from the exogenously loaded long peptide of the invention after its uptake by the antigen presenting target cells. In case the autologous target cells are loaded with a peptide, e.g. the synthetic long peptide of the invention, or fragments thereof, encompassing the epitope, the epitope is generated after cellular uptake of the peptide and its intracellular processing by the proteasome together with other N-terminal trimming peptidases. Subsequently, using a T cell recognition assay, the HLA-epitope- specific CTL is used to demonstrate the intracellular generation and natural presentation of the epitope of the invention by HLA class I molecules on the surface of HBV-infected cells. Specific recognition of an HLA class I restricted epitope by a CTL demonstrates the cell surface expression of the epitope and reveals its immunogenicity, i.e. the presence of epitope-specific T cells in the (T-cell receptor) repertoire of a selected donor. Preferably, the CD8+ T cell activating capability has been demonstrated ex vivo and/or in vivo, in T cells from human healthy control individuals or even more preferably in T cells from a human patient with an HBV related disease or condition and/or from a healthy control. The activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HBV related disease. A CTL epitope for which liberation by proteolytic cleavage and HLA class I molecules presentation, or preferably CD8+ T cell activating capability, has been demonstrated experimentally is denominated herein as a confirmed CTL epitope. A peptide of the invention preferably comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes as defined herein. Preferably, a peptide of the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes as defined herein. Preferably, a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. More preferably a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes from the HBV core protein, HBV polymerase, HBV X protein or HBV large surface protein. Even more preferably, a peptide according to the invention comprises or consists of a contiguous amino acid sequence of any of the proteins selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and HBV large surface protein, wherein said contiguous amino acid sequence comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 predicted CTL epitopes. Preferably, a peptide according to the invention comprises at least 70, 71, 72, 73, 74, 75, 79, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 and to up to 233 CTL predicted epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 (see Tables 4a, 5a, 6a and 7a). Even more preferably, a peptide of the invention preferably comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, or preferably at least 95 confirmed CTL epitopes as defined herein and verified using a biochemical or functional assay as described above. Most preferred is a peptide of the invention that comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or preferably at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes as defined herein and verified using a functional assay as described above. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or preferably at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846- 923, 959-1090, and 1146-1395.
A "Th-cell epitope" is understood herein to be a linear peptide fragment that is recognized by an HLA class II molecule. A "predicted Th-cell epitope" is understood herein as a linear fragment of a polypeptide antigen for which HLA class II molecule recognition has been predicted using sophisticated bioinformatics analyses that are experimentally underpinned. Preferably, a predicted Th-cell epitope of the present invention is confirmed experimentally as disclosed herein. A Th-cell epitope of the invention is preferably capable of inducing a CD4+ T cell response. A "confirmed Th- cell epitope" is understood herein as a linear fragment of a polypeptide antigen for which HLA class II molecule recognition has been established experimentally as known by the person skilled in the art and further detailed herein.
An HLA class II-restricted CD4+ T-helper cell (Th-cell) epitope typically comprises 15 up to 20, or exceptionally even more, amino acids. Preferably, an HLA class II-restricted T-helper cell epitope comprises or 10 - 20 or 10 - 15 amino acids. Specific recognition of a predicted HBV-derived Th-cell epitope can be tested and/or verified in a Th-cell induction assay. To this end the peptide or fragment thereof, long peptide and/or SLP sequence of interest comprising the predicted Th-cell epitope is exogenously loaded on the surface of target cells and subsequently these peptide-loaded target cells are co-incubated with a multivalent autologous T helper cell population. After several rounds of stimulation, epitope-specific T helper cells can be selected and can be back-tested for the recognition of the T helper cell epitope contained in the peptide or SLP thereby proving its natural cell surface presentation. Preferably, an HLA class II-restricted CD4+ T-helper cell epitope comprised in a peptide according to the invention is capable of inducing or activating a CD4+ T-helper cell in a human patient with an HBV related disease or condition. The induction or activation is preferably assessed ex vivo or in vivo, more preferably in a human patient with an HBV related disease. Most preferably, the HLA class Il-restricted epitope is capable of activating a CD4+ T-helper memory and/or CD4+ T-helper effector response, i.e. activation of a CD45RO-positive CD4+ T-helper cell. This will lead, by virtue of the 'license to kill' signal through CD40-triggering of DC (Lanzavecchia, 1998) to a more robust CD8+ effector and memory cytotoxic T cell response. In another setting the activated CD4+ T-helper cells may activate non-HLA restricted killer cells of the immune system. A Th-cell epitope for which recognition by an HLA class II molecule, or preferably CD4+ activating capability, has been demonstrated experimentally is denominated herein as a confirmed Th-cell epitope.
Preferably, a peptide according to the invention comprises at least one predicted
Th-cell epitope from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10 or preferably at least 15 predicted Th-cell epitope(s) as defined herein. Preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s) from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. Even more preferably, a peptide according to the invention comprises or consists of a contiguous amino acid sequence of any of the proteins selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and HBV large surface protein, wherein said contiguous amino acid sequence comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s). Preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 predicted Th-cell epitope(s) selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466 (see Tables 4b, 5b, 6b, and 7b). More preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8,
7, 8, 9, 10, or preferably at least 15 confirmed Th-cell epitope(s) as defined herein. Even more preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7,
8, 9, 10, or preferably at least 15 confirmed Th-cell epitope(s) as defined herein and verified using an T helper cell induction assay as described above. Preferably, a peptide of the invention comprises at least 1, 2, 3, 4, 5, 6, 8, 7, 8, 9, 10, or preferably at least 15 confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466. Preferably, a peptide according to the invention comprises both at least 70 predicted CTL epitopes and at least one predicted Th-cell epitope from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. More preferably, a peptide according to the invention is a peptide derived from the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, preferably is a fragment of the HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, that comprises at least 70 predicted CTL epitopes, at least one predicted Th-cell epitope and at least 3 proteasomal cleavage sites. The presence of at least 3 proteasomal cleavage sites, at least 70 predicted CTL epitopes and at least 1 Th epitope within a single peptide according to the invention, being a continuous amino acids fragment of an antigen protein of interest, has been observed to be particularly advantageous due to synergy between the Th response and the CTL response in mounting and maintaining an effective CD8+ cytotoxic T cell response. Several published studies have demonstrated that CD4+ T-helper cells upon interaction with HLA class II epitope presenting dendritic cells (DC) upregulate CD40 ligand. The interaction of the Th-cell by its CD40 ligand with the CD40 molecule on the DC leads to activation of the DC. Activated DCs display upregulated costimulatory molecules and secrete CTL-promoting cytokines. This allows both a more robust CD8+ CTL response induced by such an activated DC that presents HLA class I restricted epitopes and a much more robust CTL memory response (Ridge et al,. 1998; Schoenberger et al., 1998; Sun et al, 2004). The need for CD40 expression on DC for robust CD8+ CTL responses following vaccination with synthetic long peptides (length of 35 aa.) has been demonstrated in Zwaveling et al. (2002).
Accordingly, a preferred peptide according to the invention comprises at least 70 predicted CTL epitopes and at least one predicted Th-cell epitope; preferably at least 70 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one predicted Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466. A more preferred peptide according to the invention comprises at least 70 predicted CTL epitopes and at least 15 predicted Th-cell epitopes; preferably at least 70 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 15 predicted Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-95, 1091-1140, and 1396-1466. More preferably, a peptide according to the invention comprises at least 71, 72, 73, 74, 75, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 up to 175 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 up to 96 predicted Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Even more preferably, a peptide according to the invention comprises at least 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 up to 175 predicted CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638- 685; 846-923, 959-1090, and 1 146-1395 and at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 up to 96 predicted Th- cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Preferably, a peptide according to the invention comprises at least 95 predicted CTL epitopes as defined herein and at least 25 predicted Th-cell epitopes as defined herein.
More preferably, a preferred peptide according to the invention comprises at least 5 confirmed CTL epitopes and at least one confirmed Th-cell epitope; preferably at least 5 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80- 276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146 - 1395 and at least one confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. A more preferred peptide according to the invention comprises at least 15 confirmed CTL epitopes and at least one confirmed Th-cell epitope; preferably at least 15 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146-1395 and at least one confirmed Th-cell epitope selected from the group consisting of SEQ ID NO: 686-845; 924-95, 1091-1140, and 1396-1466. More preferably, a peptide according to the invention comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146 - 1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 confirmed Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396-1466. Even more preferably, a peptide according to the invention comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 confirmed CTL epitopes selected from the group consisting of SEQ ID NO: 80-276, 278-314, 316-429, 432-483, 486-545, 548-636, 638-685; 846-923, 959-1090, and 1146 - 1395 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 confirmed Th-cell epitopes selected from the group consisting of SEQ ID NO: 686-845; 924-958, 1091-1140, and 1396 - 1466. Preferably, a peptide according to the invention comprises at least 15 confirmed CTL epitopes as defined herein and at least 5 confirmed Th-cell epitopes as defined herein.
The HLA class I epitopes in the peptides according to the invention are preferably capable of being presented on HLA molecules being encoded by HLA alleles that are predominant in the population of human subjects to be treated. Preferred HLA class I epitopes in peptides according to the invention are epitopes capable of binding to: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101; HLA- A2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001; HLA- A3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA- A7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA- B1501; HLA-B1502; HLA-B1525; HLA-B1801; HLA-B2702; HLA-B2705; HLA- B3501; HLA-B3503; HLA-B3701; HLA-B3801; HLA-B3901; HLA-B4001; HLA- B4002; HLA-B4402; HLA-B4403; HLA-B4601; HLA-B4801; HLA-B4901; HLA- B5001; HLA-B5101; HLA-B5201; HLA-B5301; HLA-B5501; HLA-B5601; HLA- B5701; HLA-B5801 and HLA-B5802. In a preferred embodiment, a peptide of the invention, covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the HLA class I molecules that are encoded by HLA alleles predominant in the population of human subjects to be treated, wherein "Cover an HLA class I molecule" is understood herein as comprising a CTL epitope that shows binding affinity, preferably intermediate binding affinity, more preferably high binding affinity to said HLA class I molecule. Preferably, a peptide of the invention covers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of group of HLA class I molecules consisting of: HLA-A0101; HLA-A0201; HLA-A0206; HLA-A0301; HLA-A1101 ; HLA-A2301; HLA-A2402; HLA-A2501; HLA-A2601; HLA-A2902; HLA-A3001 ; HLA- A3002; HLA-A3101; HLA-A3201; HLA-A3303; HLA-A6801; HLA-A6802; HLA-A7401; HLA-B0702; HLA-B0801; HLA-B1301; HLA-B1302; HLA-B1402; HLA-B1501; HLA-B1502; HLA-B1525; HLA-B1801; HLA-B2702; HLA-B2705; HLA-B3501; HLA-B3503; HLA-B3701; HLA-B3801; HLA-B3901; HLA-B4001; HLA-B4002; HLA-B4402; HLA-B4403; HLA-B4601; HLA-B4801; HLA-B4901; HLA-B5001; HLA-B5101; HLA-B5201; HLA-B5301; HLA-B5501; HLA-B5601; HLA-B5701; HLA-B5801 and HLA-B5802.
The HBV genome (SEQ ID NO: 3; see Table 1) consists of a partially double- stranded circular DNA molecule having four overlapping open reading frames (ORFs) that are responsible for the transcription and expression of seven different hepatitis B proteins through the use of multiple in-frame start codons. The HBV proteins are the core protein and the e antigen (HBeAg) encoded by the C gene, the HBV polymerase encoded by the P gene, the viral surface proteins (small (S), middle (M), and large (L)) encoded by the S gene, and X protein encoded by the X gene. There is an outer shell (or envelope) composed of several proteins known collectively as HBs or surface Proteins. This outer shell is frequently referred to as the surface coat. The outer surface coat surrounds an inner protein shell, composed of HBc protein. This inner shell is referred to as the core particle or capsid. Finally the core particle surrounds the viral DNA and the enzyme DNA polymerase.
The HBV core protein is the major component of the viral nucleocapsid. The amino acid sequences of the HBV polymerase, HBV core protein, HBV X protein and HBV large surface protein are represented by SEQ ID NO: 1, 4, 45 and 1141 respectively (see Table 1).
A preferred amino acid sequence of a human HBV polymerase protein is a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1; a preferred coding sequence is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 2. A preferred amino acid sequence of an HBV core protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 4; a preferred coding sequence is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 5. A preferred amino acid sequence of an HBV large surface protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1141, a preferred coding sequence is a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 1467.
The full length consensus amino acid sequence of the X protein was obtained by deducing the optimal sequence from the 39 published and reviewed full length (154 amino acid) HBV X protein amino acid sequences in the UniProt database (at: www.uniprot.org). These 39 sequences were first aligned and subsequently for each aa position the most frequently occurring aa was selected for that position in the consensus sequence. The 39 sequences with the following entries were included in the analysis: P69713; P03165; P0C686; P69714; Q8JMY5; Q69604; Q05499; 091531; Q9PX75; P20976; P20975; P20977; P24026; Q9PXA2; Q67923; P0C685; P0C678; 093195; Q9E6S8; P12936; Q91C38; Q913A9; Q8JMY3; Q8JN06; Q8JMZ5; Q69607; Q9IBI5; Q80IU5; Q4R1 S9; Q4R1 S1; Q9YZR6; P0C687; Q9QMI3; P0C681; Q80IU8; Q99HR6; P17102; Q67877; and Q69027 (see Table 2). A preferred consensus amino acid sequence of a human HBV X protein is a sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with the sequence depicted in SEQ ID NO: 45. The consensus amino acid sequence may be encoded by any coding sequence known or designed; the person skilled in the art knows how to design a coding sequence from a known amino acid sequence; such coding sequence may be a codon-optimized sequence. The terms "HBV X protein" and "consensus HBV X protein" are used interchangeably herein.
Percentage of identity is herein determined by calculating the ratio of the number of identical nucleotides/amino acids in the sequence divided by the length of the total nucleotides/amino acids of said sequence, minus the lengths of any gaps. Identity with a given SEQ ID NO means identity based on the full length of said sequence (i.e. over its whole length or as a whole).
Within the context of the present invention, "a peptide derived from an HBV protein" means that the peptide comprises at least 15 and at most 100 consecutive amino acids originating from the HBV core protein, HBV polymerase, HBV X consensus protein and/or HBV large surface protein. In other words, "a peptide derived from the HBV polymerase protein" comprises at most 100 consecutive amino acids of SEQ ID NO: 1, "a peptide derived from the HBV core protein" comprises at most 100 consecutive amino acids of SEQ ID NO: 4, "a peptide derived from the HBV X consensus protein" comprises at most 100 consecutive amino acids of SEQ ID NO: 45, and "a peptide derived from the HBV large surface protein" comprises at most 100 consecutive amino acids of SEQ ID NO: 1 141. Preferably, "a peptide derived from the HBV polymerase protein" consists of at most 100 consecutive amino acids of SEQ ID NO: 1, "a peptide derived from the HBV core protein" consists of at most 100 consecutive amino acids of SEQ ID NO: 4, "a peptide derived from the HBV X consensus protein" consists of at most 100 consecutive amino acids of SEQ ID NO: 45, and "a peptide derived from the HBV large surface protein" consists of at most 100 consecutive amino acids of SEQ ID NO: 1141. Therefore, by definition, a peptide according to the invention is distinct from a full length HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, as these full length proteins are all longer than 100 amino acids. Preferably, the peptide of the present invention is from about 15 to about 100 amino acids in length. More preferably the length of the peptide is from 15 up to 100 length indicated herein as the length of the peptide is 15-100 amino acids, or preferably the length of the peptide is 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15-65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids. Within the context of the present invention "a peptide which comprises at most 100 amino acids derived from an HBV protein" preferably means that the number of consecutive amino acids originating from an HBV protein, preferably being a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81,
80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids or less. Within the context of the present invention "a peptide which comprises at least 15 amino acids derived from an HBV protein" preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids. Within the context of the present invention "a peptide which comprises 15-100 amino acids derived from an HBV protein" preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X consensus protein and HBV large surface protein and present in a peptide as defined herein, is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and no more than 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82,
81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,
35, 34, 33, 32, 31, or 30 amino acids. Within the context of the present invention "a peptide which comprises 15-100 amino acids derived from an HBV protein" preferably means that the number of consecutive amino acids originating from a protein selected from the group consisting of SEQ ID NO: 1, 4, 45 and 1141 and present in a peptide as defined herein, is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acids and no more than about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acids. More preferably, the length of the contiguous amino acid sequence from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein comprised within the peptide is 15-100 amino acids, or preferably 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15- 65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids. Even more preferably, the length of the contiguous amino acid sequence from the sequences selected from the group consisting of SEQ ID NO: 1, 4, 45 and 1141 comprised within the peptide is 15-100 amino acids, or preferably 15-95 amino acids, or 15-90 amino acids, or 15-85 amino acids, or 15-70 amino acids, or 15- 65 amino acids, or 15-60 amino acids, or 15-55 amino acids, or 15-50 amino acids, even more preferably 15-45 amino acids, even more preferably, 15-40 amino acids, even more preferably 17-39, even more preferably 19-43 amino acids, even more preferably 22-40 amino acids, even more preferably 28-40 and even more preferably 30-39 amino acids. A peptide according to the invention may comprise additional amino acids than the ones originating from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein or may entirely be made of or consist of an amino acid sequence originating from a protein selected from the group consisting of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. A peptide according to the invention may comprise several parts of non-contiguous amino acid sequences from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, wherein it is to be understood that said peptide has a length, a TRIA score and/or amount and type of T cell epitopes as defined herein.
According to one embodiment, a peptide according to the invention consists of any of the contiguous amino acid sequence from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein as defined herein and indicated by its representing SEQ ID NO, whereby it is understood that no amino acids are appended to either end of the said peptide.
According to another embodiment, the peptide according to the invention comprises any of the contiguous amino acid sequences from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein as defined herein and indicated by its representing SEQ ID NO and further may comprise a modified amino acid and/or a covalently linked functional group such as a fluorinated group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chains or glycan, a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1, CpG oligodeoxynucleotides (CpG-ODNs), Cyclic dinucleotides (CDNs), 2-aminoisobutyric acid (Abu), Muramyl dipeptide (MDP), a DC pulse cassette, a tetanus toxin derived peptide.
In an embodiment, the peptide of the invention comprises or consists of a non- naturally occurring sequence as a result of the synthesis of non-natural lengths or as a result of comprising additional amino acids not originating from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein or as a result of comprising non-contiguous amino acid sequences from an HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, and/or as a result of comprising a modified amino acid and/or a non-naturally occurring amino acid and/or a covalently linked functional group such as a fluorinated group, a fluorcarbon group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chains or glycan, a pam3cys and/or derivative thereof preferably such as described in WO2013051936A1, CpG oligodeoxynucleotides (CpG-ODNs), Cyclic dinucleotides (CDNs), a DC pulse cassette, a tetanus toxin derived peptide, a human HMGB1 derived peptide; either within the peptide or appended to the peptide, as indicated above. The peptide of the invention may comprise 2-aminoisobutyric acid (Abu, an isostereomer of cysteine). A cysteine of the peptide of the invention may be replaced by Abu. Encompassed within the present invention is a peptide of SEQ ID NO: 77, wherein the N-terminal cysteine has been replaced by Abu.
Preferably, a peptide of the invention is an isolated peptide, wherein "isolated" does not reflect the extent to which the peptide is purified, but indicates that the peptide has been removed from its natural milieu (i.e., that has been subject to human manupilation), and may be a recombinantly produced peptide or a synthetically produced peptide.
Preferably, the invention relates to a peptide that can be effectively used in the prevention, partial clearance and/or treatment or full clearance of a HBV related disease or condition in a subject, preferably as detectable by:
- an activation or an induction of the immune system and/or an increase in HBV specific activated CD4+ and/or CD8+ T-cells in peripheral blood or an increase of the cytokines produced by these T-cells after at least one week of treatment; and/or - an inhibition of proliferation of HBV infection or a detectable decrease of HBV infected cells or a decrease in cell viability of HBV infected cells; and/or
- an induction or increased induction of HBV infected cell death; and/or
- an inhibition or prevention of the increase of HBV infected cells.
In all embodiments of the present invention, a subject is preferably a mammal, more preferably a human. A subject may be an animal model, preferably a non- human mammalian model with humanized HLA class I and class II molecules, or a mammalian, preferably human, organ, such as a liver.
In all embodiments of the present invention, the term "HBV related disease or condition" is preferably defined as acute HBV infection, chronic HBV infection and other conditions where the hepatitis virus is found in the blood or body fluids containing blood of a subject, such as liver cirrhosis and liver cancer, or optionally of an asymptomatic subject that is characterized by the presence of the virus in the body of said subject.
In the context of the invention, a patient may survive and may be considered as being disease free as a consequence of treatment according to the invention. Alternatively, the disease or condition may have been stopped or regressed (i.e. cleared or partially cleared infection). A significant increase of HBV-specific activated CD4+ or CD8+ cells in peripheral blood at least one week after vaccination is preferably at least a 5%, 10%, 20%, 30% increase or more. An inhibition of the proliferation of HBV infected cells is preferably at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% inhibition or more. An induction of HBV infected cell death may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more. HBV infection may be inhibited at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% induction, or more. HBV infected cells may be reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%.
In each embodiment, within this or further aspects disclosed herein, wherein the effect of a peptide according to the invention, a composition according to the invention, a polynucleotide according to the invention, a viral vector comprising a polynucleotide according to the invention and/or a cell according to the invention and/or a cell obtained or obtainable by a method according to the invention, is quantified, the assay may be carried out by comparison to a subject not treated or to the same subject before treatment.
Acute and chronic HBV infection can be treated using the present invention. A peptide according to the invention comprising epitopes which are to be presented to T- cell receptors of CD8+ cytotoxic T cells and/or CD4+ T-helper cells preferably fulfill a number of structural requirements as defined herein. In vitro and ex vivo T cell experiments are preferably used to confirm the capability of peptides according to the invention to induce substantial CD4+ T-helper and CD8+ cytotoxic T cell responses. The peptides of the present invention thereby provide a marked improvement in the selection of relatively short peptides that may be chemically synthesized, comprising the most potent and most widely applicable HLA class I and/or class II presented T cell epitopes derived from HBV.
In an embodiment, a peptide is distinct from a contiguous sequence of amino acids of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
A peptide according to the invention comprising a T-cell epitope from HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, may be modified by deletion or substitution of one or more amino acids, by extension at the N- and/or C-terminus with additional amino acids or functional groups, which may improve bio-availability, targeting to T-cells, or comprise or release immune modulating substances that provide adjuvant or (co) stimulatory functions. The optional additional amino acids at the N- and/or C-terminus are preferably not present in the corresponding positions in the native amino acid sequence of HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein.
A peptide according to the invention comprising a T-cell epitope is obtainable by chemical synthesis and subsequent purification according to methods well-known in the art. (see e.g. Atherton et al., 1989; Barany et al., 1979; Fields et al, 1997). A peptide according to the invention is preferably soluble in physiologically acceptable watery solutions (e.g. PBS) comprising no more than 35, 20, 10, 5 or 0% DMSO. In such a solution the peptide according to the invention is preferably soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml. More preferably, a mixture of more than one different peptide according to the invention is soluble at a concentration of at least 0.5, 1, 2, 4, or 8 mg peptide per ml in such solutions. The peptides according to the invention may be easily synthesized and are large enough to be taken up by professional antigen presenting cells, in particular dendritic cells (DC), processed by the proteasome and/or the endosomal/lysosomal degradation and antigen processing system and preferably have sufficient length to contain at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170 to preferably up to 175 CTL epitopes and/or at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to preferably up to 96 Th-cell epitopes as defined herein. Optionally, a peptide according to the invention may comprise N- or C-terminal extensions, which may be amino acids, modified amino acids or other functional groups that may for instance enhance bio-availability, cellular uptake, processing and/or solubility.
Preferably, a peptide according to the invention is a peptide comprising a peptide with an amino acid sequence selected from the group consisting of:
- a fragment of 15 to 30 amino acids of SEQ ID NO: 51, preferably contiguous amino acids,
- a fragment of 15 to 35 amino acids of SEQ ID NO: 52, preferably contiguous amino acids,
- a fragment of 15 to 33 amino acids of SEQ ID NO: 53, preferably contiguous amino acids,
- a fragment of 15 to 33 amino acids of SEQ ID NO: 54, preferably contiguous amino acids,
- a fragment of 15 to 39 amino acids of SEQ ID NO: 55, preferably contiguous amino acids,
- a fragment of 15 to 35 amino acids of SEQ ID NO: 56, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 57, preferably contiguous amino acids,
- a fragment of 15 to 32 amino acids of SEQ ID NO: 58, preferably contiguous amino acids,
- a fragment of 15 to 33 amino acids of SEQ ID NO: 59, preferably contiguous amino acids,
- a fragment of 15 to 38 amino acids of SEQ ID NO: 60, preferably contiguous amino acids, - a fragment of 15 to 38 amino acids of SEQ ID NO: 61, preferably contiguous amino acids,
- a fragment of 15 to 33 amino acids of SEQ ID NO: 62, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 63, preferably contiguous amino acids,
- a fragment of 15 to 36 amino acids of SEQ ID NO: 64, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 65, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 66, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 67, preferably contiguous amino acids,
- a fragment of 15 to 39 amino acids of SEQ ID NO: 68, preferably contiguous amino acids,
- a fragment of 15 to 35 amino acids of SEQ ID NO: 69, preferably contiguous amino acids,
- a fragment of 15 to 32 amino acids of SEQ ID NO: 70, preferably contiguous amino acids,
- a fragment of 15 to 38 amino acids of SEQ ID NO: 71, preferably contiguous amino acids,
- a fragment of 15 to 36 amino acids of SEQ ID NO: 72, preferably contiguous amino acids,
- a fragment of 15 to 36 amino acids of SEQ ID NO: 73, preferably contiguous amino acids,
- a fragment of 15 to 35 amino acids of SEQ ID NO: 74, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 75, preferably contiguous amino acids,
- a fragment of 15 to 33 amino acids of SEQ ID NO: 76, preferably contiguous amino acids, - a fragment of 15 to 35 amino acids of SEQ ID NO: 77, preferably contiguous amino acids,
- a fragment of 15 to 35 amino acids of SEQ ID NO: 78, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 79, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 1142, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 1 143, preferably contiguous amino acids,
- a fragment of 15 to 34 amino acids of SEQ ID NO: 1 144, preferably contiguous amino acids,
- a fragment of 15 to 36 amino acids of SEQ ID NO: 1145, preferably contiguous amino acids,
- a fragment of 15 to 32 amino acids of SEQ ID NO: 1468, preferably contiguous amino acids,
- a fragment of 15 to 31 amino acids of SEQ ID NO: 1469, preferably contiguous amino acids,
- a fragment of 15 to 30 amino acids of SEQ ID NO: 1470, preferably contiguous amino acids,
- a fragment of 15 to 31 amino acids of SEQ ID NO: 1471, preferably contiguous amino acids, and;
wherein the length of the preferably contiguous amino acid sequence is preferably at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids and/or preferably no more than 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 amino acids, most preferably a length of 30-39 amino acids.
In a second aspect, the invention provides a polynucleotide encoding a peptide according to the invention, preferably a peptide as defined herein above. A polynucleotide may be any polynucleotide comprising e.g. RNA, DNA, and/or cDNA; a polynucleotide may be single stranded or double stranded and may comprise nucleotide analogues and/or nucleotide equivalents such as a peptide nucleic acid (PNA) and a morpholino nucleotide analogue. A polynucleotide may be codon optimized for a host of choice to facilitate expression of the encoded subject matter.
The polynucleotide according to the invention does not encode a wild-type full length HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, but rather encode a peptide according to the invention as such, or flanked by amino acid sequence that are not contiguous with a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein. Such flanking amino acids may be from proteins other than a wild-type HBV and/or they may be from other locations within a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein that are not contiguous with the peptide they flank. Preferably, the polynucleotide encodes two or more peptides according to the invention arranged as beads-on-string, whereby the peptides according to the invention (the beads) are linked directly together and/or are linked through linker sequences that are from proteins other than a wild-type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, and/or from other locations within a wild- type HBV core protein, HBV polymerase, HBV X protein and/or HBV large surface protein, that are not contiguous with the peptide they flank. The amino acid sequences flanking or linking the peptides may comprise proteolytic cleavage sites. A polynucleotide according to the invention may be applied to deliver a peptide according to the invention in various ways. A polynucleotide according to the invention may e.g. be used in the production of recombinant protein or peptide in a suitable host cell (e.g. a bacterial host cell such as E. coli, a suitable yeast host cell such as S. cerevisiae, a suitable filamentous fungal such as an Aspergillus or mammalian host cell) from which the recombinant protein or peptide may be purified. Alternatively the polynucleotide may be operably linked to expression regulatory sequences (promoters and the like) and incorporated in an expression construct for human cells. Such (autologous) cells may be transfected or transduced ex vivo to be (re)-administered to a subject in need thereof. Alternatively such expression construct according to the invention may be incorporated into a suitable gene therapy vector. Viral vectors (based on a defective virus) are more efficient agents for gene transfer as compared to non- viral agents. Suitable viral expression constructs include e.g. vectors that are based on adenovirus, adeno-associated virus (AAV), retroviruses or modified vaccinia Ankara (MVA). The polynucleotide according to the invention may also be operably linked to a sequence encoding and adjuvant such as a Toll-like receptor (TLR) ligand, a NOD ligand, or a RIG-I ligand.
In a third aspect, the present invention provides a cell comprising the polynucleotide according to the second aspect of the invention. Such cell can be used for e.g. production of a peptide according to the invention or for medical purposes such as prevention and/or treatment of an HBV related disease as defined elsewhere herein. Said cell may be any host cell. For the specific applications such as described here above, the selection of the host cell may be made according to such use. The host cell may be a prokaryote or may be a eukaryote. A preferred prokaryote cell is E. coli. When the cell is a eukaryote, the cell preferably is a mammalian, insect, plant, fungal, or algal cell. Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6 cells, and antigen presenting cells such as dendritic cells. Preferred insect cells include e.g. Sf9 and Sf21 cells and derivatives thereof. Preferred fungal cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia and filamentous fungal cells. Most preferably, the eukaryotic cell is a human antigen presenting cell, preferably a dendritic cell.
Methods to introduce a polynucleotide into a cell are known to the person skilled in the art. When expression of the polynucleotide is desired, the person skilled in the art knows how to achieve such; the polynucleotide may e.g. be provided with proper control sequences such as a promoter and terminator sequence and may be inserted into a proper vector such as a plasmid or a method described in the second aspect of the invention may be used.
The present invention also provides for an antigen presenting cell such as a dendritic cell as defined earlier herein that has been contacted and/or loaded with a peptide according to the invention, preferably a peptide according to the first aspect of the present invention. Such preferably autologous dendritic cell may be used for immune therapeutic treatment of a subject in need thereof. Such dendritic cell can be isolated from the subject, loaded with at least one peptide according to the invention and used for treatment. In a fourth aspect, the present invention provides a method for the preparation of an HBV specific T-cell, said method comprising contacting a T-cell with an antigen presenting cell expressing a polynucleotide according to the invention and/or contacting a T-cell with an antigen presenting cell loaded with a peptide according to the invention; and, optionally, culturing said T-cell. The T-cell is preferably a CD8+ cytotoxic T-cell or a CD4+ T-helper cell.
Contacting a cell with a polynucleotide may be performed using any method known to the person skilled in the art, preferably a polynucleotide according to the invention is introduced into the antigen presenting cell (APC), preferably a dendritic cell, using transfection. Before contacting, the polynucleotide according to the invention may be provided with proper control sequences, or be comprised into a proper vector such as described elsewhere herein.
Contacting a T-cell with a peptide according to the invention can be performed by any method known to the person skilled in the art. Preferably, a peptide or an epitope comprised in a peptide is presented to the CD8+ cytotoxic T-cell or CD4+ T- helper cell by an HLA class I or an HLA class II molecule on the surface of an antigen presenting cell, preferably a dendritic cell. The person skilled in the art knows how to load an antigen presenting cell with a peptide.
Culturing said T-cell may be performed using any method known by the person skilled in the art. Maintaining a T-cell under conditions to keep the cell alive is herein also to be construed to be culturing.
Preferably, the T-cell according to this aspect of the invention is contacted with a peptide according to the invention as defined in the first aspect of the invention. In a fifth aspect, the present invention provides a T-cell obtainable by the method depicted in the fourth aspect of the present invention. Preferably, such T-cell is a T-cell that is obtained by the method according to the fourth aspect of the invention. The T-cell is preferably a CD8 cytotoxic T-cell or a CD4+ T-helper cell.
Preferably, the T-cell according to this aspect of the invention has been contacted with a peptide according to the invention as defined in the first aspect of the invention. In a sixth aspect, the present invention provides a composition useful for the prevention and/or treatment of an HBV related disease or related condition, comprising a peptide according to the invention and/or a polynucleotide according to the invention and/or a cell, preferably a T-cell, according to the invention and/or a cell, preferably a T-cell, obtained by the method according to the fourth aspect of the invention and a pharmaceutically acceptable carrier.
When comprising a peptide according to the invention, the composition according to the invention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different peptides according to the invention. Preferably, a composition according to the invention comprises a peptide according to the invention as defined in the first aspect of the invention. In a preferred embodiment, a composition of the invention comprises a combination of peptides wherein said combination of peptides covers at least 70%, 80%, 90%, 92%, 94%, 95%, 96%, 97%,98%, 99% or 100% of the HLA class I molecules that are encoded by HLA alleles predominant in the population of human subjects to be treated as defined herein above.
When comprising a polynucleotide according to the present invention, the composition according to the invention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different polynucleotides according to the invention. Preferably, a composition according to the invention comprises a polynucleotide according to the invention as defined in the second aspect of the invention.
When comprising a cell according to the invention, the composition according to the invention preferably comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different cells, preferably T-cells that have been contacted with a peptide according to the invention. Preferably, said T-cells have been contacted with a peptide according to the invention as defined in the first aspect of the invention. The T-cell is preferably a CD8+ cytotoxic T-cell or a CD4+ T-helper cell.
In a preferred embodiment, the composition of the invention comprises at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different peptides of the peptides consisting of or comprising of a peptide selected from the group consisting of SEQ ID NO: 51-79, 1142-1145 and 1468-1471, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 76, 77, 1142 and 1469, more preferably selected from the group consisting of SEQ ID NO: 51, 55, 60, 63, 64, 68, 71, 74, 75, 77, 1142 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 77 and 1469, even more preferably selected from the group consisting of SEQ ID NO: 60, 63, 71, 74, 75 and 1469, most preferably selected from the group of SEQ ID NO: 75, 1469 and 63. Further preferred is a composition that comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and up to 33 different peptides of the peptides consisting of or comprising of a peptide selected from the group consisting of SEQ ID NO: 51, 60, 63, 64, 68, 71, 74-77, more preferably selected from the group consisting of SEQ ID NO: 63, 71 and 75.
In a preferred embodiment, the composition of the invention comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63 and peptide that comprises or consists of a peptide of SEQ ID NO: 1143.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1143 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 71 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 71 and peptide that comprises or consists of a peptide of SEQ ID NO: 63.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 63. Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 1144 and peptide that comprises or consists of a peptide of SEQ ID NO: 1143.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, a peptide that comprises or consists of a peptide of SEQ ID NO: 1143, and a peptide that comprises or consists of a peptide of SEQ ID NO: 75.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, a peptide that comprises or consists of a peptide of SEQ ID NO: 1143, a peptide that comprises or consists of a peptide of SEQ ID NO: 75, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1144.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 75, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1469. Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 63, and a peptide that comprises or consists of a peptide of SEQ ID NO: 1469.
Also preferred is a composition that comprises at least a peptide that comprises or consists of a peptide of SEQ ID NO: 75, a peptide that comprises or consists of a peptide of SEQ ID NO: 1469, and a peptide that comprises or consists of a peptide of SEQ ID NO: 63. Preferably, said composition further comprising a peptide that comprises or consists of a peptide of SEQ ID NO: 60 and/or that comprises or consists of a peptide of SEQ ID NO: 71, and/or that comprises or consists of a peptide of SEQ ID NO: 74.
A preferred composition of the invention comprises a peptide that consists of or comprises a peptide of SEQ ID NO: 75, a peptide that consists of or comprises a peptide of SEQ ID NO: 63, and a peptide that consists of or comprises a peptide of SEQ ID NO: 1469
A preferred composition of the invention comprises a peptide that consists of or comprises a peptide of SEQ ID NO: 75, a peptide that consists of or comprises a peptide of SEQ ID NO: 63, and a peptide that consists of or comprises a peptide of SEQ ID NO: 71.
A pharmaceutically acceptable carrier can be any such carrier known to the person skilled in the art, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).
Preferably, a composition according to the present invention further comprises at least one adjuvant. Such adjuvant may be any adjuvant known to the person skilled in the art. Preferred adjuvants are defined later herein.
A preferred use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention is the use as a medicament. A specific preferred use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention is for the treatment and/or prevention of an HBV related disease or condition. Accordingly, the invention provides for the use of a peptide, polynucleotide, composition, cell and/or T-cell according to the invention or a T-cell obtainable or obtained by a method according to the invention for the manufacturing of a medicament for the treatment and/or prevention of an HBV related disease.
The invention further provides a method for the prevention and/or treatment of an HBV related disease or condition comprising administering to a subject an effective amount of a peptide, polynucleotide, composition, cell or T-cell according to the invention and/or a T-cell obtainable or obtained by a method according to the invention.
Formulation of medicaments, ways of administration and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia. Pharmaceutical compositions and medicaments according to the invention are preferably formulated to be suitable for intravenous or subcutaneous, or intramuscular administration, although other administration routes can be envisaged, such as mucosal administration or intradermal and/or intracutaneous administration, e.g. by injection. Intradermal administration is preferred herein. Advantages and/or preferred embodiments that are specifically associated with intradermal administration are later on defined in a separate section entitled "intradermal administration".
It is furthermore encompassed by the present invention that the administration of a peptide, a polynucleotide, a composition and/or a cell according to the invention and/or a cell obtainable or obtained by a method according to the invention with an appropriate pharmaceutical excipient such as an adjuvant and/or a carrier may be carried out as a single administration. Alternatively, the administration may be repeated if needed and/or distinct peptides, polynucleotides, compositions and/or cells according to the invention and/or cells obtainable or obtained by a method according to the invention with appropriate pharmaceutical excipients such as adjuvants and/or carriers, may be sequentially administered.
The peptide, polynucleotide, composition and/or cell according to the invention and/or cell obtainable or obtained by a method according to the invention (also referred to as medicaments according to the invention) may preferably comprise at least one immune response stimulating compound or adjuvant. Advantageously the medicaments according to the invention may additionally comprise one or more synthetic adjuvants. Such adjuvant may be admixed to the medicament according to the invention or may be administered separately to the subject, mammal or human, to be treated. Particularly preferred are those adjuvants that are known to act via the Toll-like receptors and/or via a RIG-I (Retinoic acid- Inducible Gene-1) protein and/or via an endothelin receptor. Immune modifying compounds that are capable of activation of the innate immune system can be activated particularly well via Toll like receptors (TLRs), including TLRs 1 - 10. Compounds capable of activating TLR receptors and modifications and derivatives thereof are well documented in the art. TLR1 may be activated by bacterial lipoproteins and acetylated forms thereof, TLR2 may in addition be activated by Gram positive bacterial glycolipids, LPS, LP A, LTA, fimbriae, outer membrane proteins, heat shock proteins from bacteria or from the host, and Mycobacterial lipoarabinomannans. TLR3 may be activated by dsRNA, in particular of viral origin, or by the chemical compound poly(LC). TLR4 may be activated by Gram negative LPS, LTA, Heat shock proteins from the host or from bacterial origin, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides and fibronectins. TLR5 may be activated with bacterial flagellae or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group B Streptococcus heat labile soluble factor (GBS- F) or Staphylococcus modulins. TLR7 may be activated by imidazoquinolines, such as imiquimod, resiquimod and derivatives imiquimod or resiquimod. TLR9 may be activated by unmethylated CpG DNA or chromatin - IgG complexes. In particular TLR3, TLR7 and TLR9 play an important role in mediating an innate immune response against viral infections, and compounds capable of activating these receptors are particularly preferred for use in the methods of treatment and in the compositions or medicaments according to the invention. Particularly preferred adjuvants comprise, but are not limited to, synthetically produced compounds comprising dsRNA, poly(LC), unmethylated CpG DNA which trigger TLR3 and TLR9 receptors, IC31, a TLR 9 agonist, IMSAVAC, a TLR 4 agonist, Montanide ISA-51, Montanide ISA 720 (an adjuvant produced by Seppic 7, France). RIG-I protein is known to be activated by dsRNA just like TLR3 (Kato et al, 2005). A particularly preferred TLR ligand is a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, preferably such as described in WO2013051936A1. Further preferred adjuvants are Cyclic dinucleotides (CDNs), Muramyl dipeptide (MDP) and poly-ICLC. In a preferred embodiment, the adjuvants of the invention are non-naturally occurring adjuvants such as the pam3cys lipopeptide derivative as described in WO2013051936A1, Poly-ICLC, imidazoquinoline such as imiquimod, resiquimod or derivatives thereof, CpG oligodeoxynucleotides (CpG-ODNs) having a non-naturally occurring sequence, and peptide-based adjuvants, such as muramyl dipeptide (MDP) or tetanus toxoid peptide, comprising non-naturally occurring amino acids.
In another preferred embodiment, the synthetic adjuvant compounds are physically linked to the peptides of the invention. Physical linkage of adjuvants and costimulatory compounds or functional groups to the ULA class I and ULA class II epitope comprising peptides provides an enhanced immune response by improved targeting to antigen-presenting cells, in particular dendritic cells, that internalize, metabolize and display antigen and by simultaneously stimulating such cells to up- regulate expression of a variety of co-stimulatory molecules, thereby becoming efficient T cell response inducing and enhancing cells. Another preferred immune modifying compound is an inhibitor of an endothelin receptor such as BQ-788 (Buckanovich RJ et al, 2008; Ishikawa K, 1994). BQ-788 is N-cis-2,6- dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D - 1 - methoxycarbonyltryptophanyl-D-norleucine. However any derivative of BQ-788 or modified BQ-788 compound is also encompassed within the scope of this invention. Another preferred immune response stimulating compound or adjuvant is Interferon alpha (IFNa), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately to the subject as an immuno-modulatory agent. It is to be construed herein that when an immune response stimulating compound is admixed to the medicament according to the invention, it is depicted as an adjuvant; when administered separately, it is depicted as an immuno-modulatory agent, or an immuno-modulator, which terms are used herein interchangeably. Furthermore, the use of antigen presenting cell (co)stimulatory molecules, as set out in WO99/61065 and in WO03/084999, in combination with the peptides and compositions of the invention is preferred. In particular the use of 4- IBB and/or CD40 ligands, agonistic antibodies, OX40 ligands, CD27 ligands or functional fragments and derivatives thereof, as well as synthetic compounds with similar agonistic activity are preferably administered separately or combined with the peptides of the invention to subjects to be treated in order to further stimulate the mounting of an optimal immune response in the subject.
In addition, a preferred embodiment comprises delivery of the medicaments according to the invention, with or without additional immune stimulants such as TLR ligands and/or anti CD40/anti-4-lBB/ anti-OX-40 or anti-CD27 antibodies in a slow release vehicle such as mineral oil (e.g. Montanide ISA 51) or PLGA. Alternatively, the medicament according to the invention may be delivered intradermally, e.g. by injection, with or without immune stimulants (adjuvants and/or immuno-modulators). Preferably, for intradermal delivery the medicaments according to the invention are administered in a composition consisting of the medicaments and one or more immunologically inert pharmaceutically acceptable carriers, e.g. buffered aqueous solutions at physiological ionic strength and/or osmolarity (such as e.g. PBS).
In a preferred embodiment, a medicament according to the invention as defined herein is formulated to be suitable for intradermal administration or application. Intradermal is known to the skilled person. In the context of the invention, intradermal is synonymous with intradermal and is distinct from subcutaneous. A most superficial application of a substance is epicutaneous (on the skin), then would come an intradermal application (in or into the skin), then a subcutaneous application (in the tissues just under the skin), then an intramuscular application (into the body of the muscle). An intradermal application is usually given by injection. An intradermal injection of a substance can be done to test a possible reaction, allergy and/or cellular immunity to it, but can also be performed to elicit a specific antibody or T cell immune response. A subcutaneous application is usually also given by injection: a needle is injected in the tissues under the skin.
The advantage of intradermal administration is that the formulation procedure can be simplified and be made more robust. Furthermore, intradermal vaccine delivery has been repeatedly shown to allow significant dose sparing for eliciting vaccine-induced antibody or T cell responses when compared to conventional administration methods such as intramuscular and subcutaneous administration. This effect is attributed to the relatively dense network of immune cells present in the skin. This was also shown with the HPV16 synthetic long peptides in a human study published by Van der Burg et al. (2007). In this study it was shown that intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of HPV16-specific T cells circulating in the blood.
In an embodiment, a medicament according to the invention does not comprise any adjuvant such as Montanide ISA-51, and specifically Montanide ISA-51. This means that the formulation of the medicament is more simple: an oil-water based emulsion is preferably also not present in a medicament according to the invention. Accordingly, a medicament according to the invention preferably does not comprise an adjuvant such as Montanide ISA-51 and specifically Montanide ISA-51 and/or does not comprise an oil-in-water based emulsion; more preferably a medicament according to the invention comprises neither of these to adjuvant and even more preferably comprises no adjuvant at all. Therefore, in an embodiment, the medicament according to the invention is a, preferably buffered, aqueous solution, preferably at physiological ionic strength and/or osmolality, such as e.g. PBS (Phosphate Buffer Saline) or water for injection (WFI), comprising or consisting of one or more medicaments as defined earlier herein. The skilled person knows how to prepare such a solution.
A medicament according to the invention has another advantage, which is that by intradermally administering low amounts of a medicament, preferably a peptide as earlier herein defined, an immunogenic effect may still be achieved. The amount of each peptide used is preferably ranged from 1 and 1000 μg, more preferably from 5 and 500 μg, even more preferably from 10 and 100 μg.
In an embodiment, the medicament according to the invention comprises a peptide as earlier defined herein and at least one adjuvant, said adjuvant being not formulated in an oil-in water based emulsion and/or not being of an oil-in-water emulsion type as earlier defined herein. This type of medicament according to the invention may be administered as a single administration. Alternatively, the administration of a peptide as earlier herein defined and/or an adjuvant may be repeated if needed and/or distinct peptides and/or distinct adjuvants may be sequentially administered. It is further encompassed by the present invention that a peptide according to the invention is administered intradermally whereas an adjuvant as defined herein is sequentially administered. The adjuvant may be intradermally administered. However any other way of administration may be used for the adjuvant. Intradermal administration of a peptide may be attractive since typically and depending on the disease, the injection of the vaccine is realized at or as close by as possible to the site of the disease resulting in the local activation of the disease draining lymph node, resulting in a stronger local activation of the immune system. A preferred immune response stimulating compound (immuno-modulator) or adjuvant for intradermal administration is Interferon alpha (IFNa), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately, for example by subcutaneous injection to the subject. When administered separately the Interferon alpha is preferably also administered subcutaneously and is preferably administered at a dose of 1 microgram/kilogram of body weight within 10 cm proximity to the site where the medicament according to the invention is administered, such described in Zeestraten et al, 2013.
Another typical advantage of the medicaments according to the invention is that relatively low amounts of peptides may be used, in one single shot, in a simple formulation and without any adjuvant known to give undesired side-effects as Montanide IS A- 51.
The medicament for intradermal administration may be any medicament according to the invention as defined herein. A medicament according to the invention used for subcutaneous administration may be the same as the one used for intradermal administration and may thus be any medicament according to the invention as defined herein. The skilled person knows how to formulate a medicament suited for subcutaneous administration.
Preferably, a medicament according to the invention for subcutaneous administration comprises a peptide as already herein defined in combination with an adjuvant. Preferred adjuvants or immune modulators have already been mentioned herein. Other preferred adjuvants are of the type of an oil-in water emulsions such as incomplete Freund's adjuvant or IF A, Montanide ISA-51 or Montanide ISA 720 (Seppic France). In a further preferred embodiment, a medicament according to the invention suited for subcutaneous administration comprises one or more peptides according to the invention, an adjuvant or immune modulator as earlier defined herein and an inert pharmaceutically acceptable carrier and/or excipients all as earlier defined herein. Formulation of medicaments, and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia. A preferred immune response stimulating compound or adjuvant for subcutaneous administration is Interferon alpha (IFNa), more preferably pegylated Interferon alpha, which may be admixed to the medicament according to the invention, or may be administered separately to the subject. When administered separately, the Interferon alpha is preferably also administered subcutaneously and is preferably administered at a dose of 1 microgram/kilogram of body weight within 10 cm proximity to the site where the medicament according to the invention is administered, such described in Zeestraten et al, 2013.
In an embodiment, the medicament according to the invention suited for intradermal administration may be simultaneously administered with a medicament according to the invention suited for subcutaneous administration. Alternatively, both medicaments may be sequentially intradermally and subsequently subcutaneously administered or vice versa (first subcutaneous administration followed by intradermal administration). In this embodiment as in the earlier described embodiment dedicated to the intradermal administration, the intradermal and/or subcutaneous administration of a medicament according to the invention, preferably a peptide according to the invention, and/or of an adjuvant may be repeated if needed and/or of distinct medicament, preferably peptides and/or of distinct adjuvants may be sequentially intradermally and/or subcutaneously administered. It is further encompassed by the present invention that a medicament according to the invention, preferably a peptide is administered intradermally and/or subcutaneously whereas an adjuvant as defined herein is sequentially administered as immune-modulator. The adjuvant or immune- modulator may be intradermally and/or subcutaneously administered. However any other way of administration may be used for the adjuvant or immune-modulator.
We expect the combination of an intradermal and a subcutaneous administration of a medicament according to the invention is advantageous. DC in the epidermis are clearly different from DC in the dermis and in the subcutis. The intracutaneous (intradermal) immunization will cause antigen processing and activation of epidermal DC (Langerin-positive Langerhans cells) that through their dendritic network are in close contact with the keratinocytes. This will also optimally activate inflammatory pathways in the interactions between Langerhans cell and keratinocytes, followed by trafficking of antigen loaded and activated Langerhans cell to the skin-draining lymph nodes. The subcutaneous administration will activate other DC subsets, that will also become loaded with antigen and travel independently to the skin- draining lymph nodes. Conceivably, the use of a medicament which may be administered both intradermally and subcutaneously may lead to a synergistic stimulation of T-cells in these draining nodes by the different DC subsets.
A medicament according to the present invention and the methods of treatment described herein using a medicament according to the invention may advantageously be combined with other medicaments and methods of treatment. As such, a medicament according to the invention or a method of treatment according to the invention may be combined with e.g. therapy, and/or antibody therapy against an HBV related disease or may be combined with e.g. immunotherapy and/or antibody therapy against another than HBV related disease, or may be combined with immunotherapy against another antigen than HBV to treat an HBV related disease.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.
The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the HBV core, HBV polymerase and HBV large surface protein polypeptides obtainable by expression of the gene present in SEQ ID NO: 5, 2 and 1467 containing the respective nucleic acid sequence coding for the polypeptides should prevail.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.). LEGENDS TO THE FIGURES
Figure 1: Proteasome-mediated cleavage pattern of long vaccine peptides as predicted in silico and observed in experimental in vitro digestions. For each SLP, the observed cleavage sites are indicated by arrows (major and minor cleavage sites are indicated by bold and thin arrows, respectively, as further detailed herein). Further, for each of the ten SLPs tested in the digestion experiments (SLP1, SLP 10 and SLP21 in Figure 1A; SLP 14, SLP 18 and SLP21 in Figure IB; SLP24, SLP25 and SLP26 in Figure 1C; and SLP 27 in Figure ID), the predicted C-score is indicated in the first row, the confirmed predicted cleavage sites are indicated in the second row (indicated as '+'), the amino acid position within the source protein is indicated in the third row, the respective amino acids in the sequence are indicated in the fourth row, and the amino acid number within the SLP is indicated in the fifth row. For SLP27, C* indicates the cysteine replacement by Abu.
Figure 2: Overview of responding naive donors after T cell induction with HBV- derived long peptides. Combined data of IFNy production as measured by ELISA ('ELISA'); hatched box SI (Stimulation Index) > 1.5, and T cell proliferation ('Prolif ) as measured by 3H thymidine incorporation; hatched box SI > 1.5. N.t : not tested
Figure 3: Overview of responding HBV-immune donors after stimulation with HBV- derived long peptides. Data represent IFNy-ELISpot results, with hatched boxes indicating a positive response with SI (Stimulation Index)>3, white boxes SI<3 . Figure 4: The average Stimulation Index (SI) for each peptide, as measured in an IFNy ELISpot assay, plotted against the predicted TRIA score of the corresponding peptide. Abu-SLP27 is indicated (φ).
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
EXAMPLES
Introduction
In the current invention, we developed an optimal T cell inducing vaccine composition consisting of synthetic long peptides to treat chronically HBV-infected patients. A selection of 37 long peptides (Table 3; sequences ranging from 30 to 39 amino acids) was made encompassing the regions of the HBV Polymerase, Core protein, Surface Antigen and X-protein with the highest putative T cell immunity inducing capacity. To this end first putative HLA class I restricted cytotoxic T cell (CTL) epitopes and putative HLA class II restricted T helper epitopes were identified in these proteins using algorithms that predict HLA class I and II peptide binding and C- terminal generation by the proteasome of all HLA class I binding short peptides (with a length of a CTL epitope; 8 - 12 aa) contained in the HBV proteins. Numerical values were assigned to all putative CTL epitopes and T helper epitopes reflecting their immunogenicity quality. The quality of any CTL epitope was assessed using the so- called Class I-BCI score (Class I-Binding-Cleavage-Immunogenicity score), incorporating the combination of the HLA class I binding capacity of the epitope in conjunction with the likelihood of its C-terminal liberation from the source protein by the proteasome. Putative T helper epitopes were assessed using the so-called Class II- B score (Class II-Binding score) reflecting their binding capacity and thus predicted immunogenic quality. The combined cumulative value, i.e. the sum of the cumulative Class I-BCI score and the cumulative Class II-B score, was calculated for all possible long peptides, reflecting the cumulative quantity and quality of all CTL epitopes and T helper epitopes, and is expressed in the T cell Regional Immunogenicity Assessment (TRIA) score. Accordingly, the TRIA score enabled the assessment of the total T cell immunogenicity of any possible long peptide (length 30 - 39 aa) in the HBV proteins. The 37 HBV-derived long peptides with the highest TRIA scores were selected.
Next, we assessed whether naive donors and donors that have cleared an HBV infection in the past are able to respond to one or more of a subset of 13 long peptides with varying TRIA scores selected from the broad set of 37 long peptides.
PBMC were obtained from buffy coats of twelve healthy donors, six of which were HBV-nai've and six others had cleared an HBV-infection in the past. Using long- term T cell induction assays, we found responses against 12 out of 13 peptides in naive donors (eleven of which induced responses in multiple donors), confirming the ability of these SLPs to induce T cell responses in a great majority of individuals that had not encountered the antigens before. The strength of T cell responses found in PBMC of HBV-immune donors against these 13 long vaccine peptides correlated with their predicted strength of overall T cell immunogenicity, as expressed in their TRIA scores, validating the predictive value of the TRIA score for selecting immunogenic peptides. Thus the TRIA score is a reliable criterion for the selection of optimal immunogenic long peptides, invigorating us to select the long peptides with the highest TRIA scores into our HBV SLP -based vaccine. Material and methods
Peptide synthesis
Peptides were synthesized using solid phase Fmoc/tBu chemistry on a PTI Prelude peptide synthesizer and purified on a Gilson preparative HPLC system to > 95% purity. The identity and purity of the peptides were confirmed with UPLC-MS on a Waters Acquity UPLC/TQD system.
Prediction of CTL and T helper cell immunity inducing capacity in selected HBV- derived SLPs by net-based algorithm
The putative CTL immunity inducing capacity per SLP was predicted by calculating the cumulative Class I-BCI score per SLP. As detailed below, the cumulative BCI score is based on the Class I-B score, which is a measure for HLA class I peptide binding, and the C-score, which is a measure for proteosomal epitope liberation.
The putative T helper cell immunity inducing capacity per SLP was predicted by calculating the cumulative Class II-B score per SLP. As detailed below, the cumulative Class II-B score is based on the Class II-B score, which is a measure for HLA class II peptide binding.
The total putative immunity inducing capacity per SLP was predicted by summation of the cumulative Class I-BCI score and the cumulative Class II-B score, which value is denominated herein as the TRIA score.
Class I-B score
Peptide binding to 50 HLA class I molecules (see text) of HBV polymerase- derived peptides, HBV Core protein-derived peptides, HBV Surface Antigen-derived peptides and HBV X protein-derived peptides was assessed in silico using a proprietary algorithm. The upper 1.5th percentile of the predicted binding peptides to each HLA class I molecule was selected. The 'Class I-Binding score (Class I-B score)' is derived from the ranking of the predicted binding affinity of the peptides. Briefly, the ranking was first reversed and subsequently normalized to 100, so that the peptide predicted to bind best has a score of 100. Example: five peptides were selected (5 within the 1.5th percentile). Peptides were first assigned the 'reverse ranking score' 5 to 1 (5 for the best binding peptide). Subsequently, each reverse ranking score is normalized to the number of peptides within the upper 1.5th percentile, so that the best binder scores 100. To that end, the ranking score for each peptide is multiplied with 100/5 (=20). The best binder then obtains a Class I-B score: 5 x 20 = 100, the second best binder has a Class I-B score of 4 x 20 = 80, etc. In general, the ranking score is multiplied with 100/n (n= number of peptide within the 1,5th percentile). As a consequence, the best predicted binder (to a certain HLA class I molecule) always scores 100, irrespective of the precise number of peptides within the 1,5th percentile that are selected. C-score
C-terminal generation by the proteasome of the upper 1.5th percentile of predicted high affinity binding peptides of HBV polymerase, HBV Core protein, HB V Surface Antigen and HBV X-protein (for each HLA class I molecule) was assessed using two proprietary algorithms, which predict the likelihood of a proteasomal cleavage after a certain amino acid position in HBV polymerase, HBV Core protein, HBV Surface Antigen and HBV X-protein, respectively, and can score between 0 and 1, where a higher value represents a higher likelihood of cleavage after the amino acid. The value 0.5 may be used as an arbitrary threshold value: > 0.5 the cleavage is likely to occur, and < 0.5 the cleavage is likely to not occur. Accordingly, a value close to 1 indicates a high likelihood of cleavage after the specific residue. Because great differences between the predictions by both different algorithms occur, we developed the Cleavage score (C-score) that takes into account the prediction results of both proprietary algorithms. The C-score is the summation of the separate scores by both methods. Therefore, the C-score for each position in HBV polymerase is maximally (close to) 2, and minimally (close to) 0, where close to 2 reflects a very high likelihood by BOTH methods that the cleavage after the residue will be produced by the proteasome, and a C-score close to 1 is considered as an indifferent tendency for cleavage by the proteasome (as predicted on average by both network methods). Class I-BCI score
To incorporate in one quantitative measure both the Class I-B score and the C- score, which are the measures indicative for the likelihood that a peptide will bind with high affinity to HLA class I molecules and will be C-terminally produced, the Class I- Binding-Cleavage-Immunogenicity (Class I-BCI) score was developed. The Class I- BCI score is the Class I-B score multiplied by the C-score. As such the Class I-BCI can attain a maximal value of 200 (100 x 2) (arbitrary units).
Cumulative Class I-BCI score The cumulative Class I-BCI score for each long peptide according to the invention was used as (one of two) selection criterion to identify the peptides of the invention. The cumulative Class I-BCI score is a quantitative reflection of both the total number of CD8+ cytotoxic T cell epitopes that are contained in a long peptide according to the invention and their predicted quality, in terms of binding capacity and likelihood of intracellular generation by the proteasome, and is as such indicative for the CD8+ cytotoxic T cell-inducing power of each peptide according to the invention (its CD8+ T cell immunogenicity). A relatively high cumulative Class I-BCI score of a peptide according to the invention indicates a high CD8+ T cell immunogenicity.
Class II-B score
Peptide binding to 13 prevalent HLA class II molecules of HBV polymerase- derived peptides, HBV Core protein-derived peptides, HBV Surface Antigen-derived peptides and HBV X protein-derived peptides was assessed in silico using a proprietary algorithm. The 'Class II-Binding score' (Class II-B score) is derived from the ranking of the predicted binding affinity of the peptides. Briefly, the ranking was first reversed and subsequently normalized to 100, so that the peptide predicted to bind best has a score of 100. To reduce the number of peptides in the list, all length variants of peptides predicted to bind to a particular HLA class II molecule with a lower predicted binding (lower Class II-B score) are discarded in the list.
Cumulative Class II-B score
The cumulative Class II-B score for each long peptide according to the invention was used as the second selection criterion to identify the peptides of the invention. The cumulative Class II-B score is a quantitative reflection of both the total number of CD4+ T-helper cell epitopes that are contained in a long peptide according to the invention and their predicted quality, in terms of binding capacity, and is as such indicative for the CD4+ T cell-inducing power of each peptide according to the invention (its CD4+ T cell immunogenicity). A relatively high cumulative Class II-B score of a peptide according to the invention indicates a high CD4+ T cell immunogenicity
T cell Regional Immunogenicity Assessment (TRIA) score The TRIA score for a particular peptide of the invention (SLP) is the sum of cumulative Class I-BCI score and the cumulative Class II-B score of that particular long peptide of the invention (SLP). Proteasomal digestion
Dithiotreitol (DTT; Sigma- Aldrich) was freshly dissolved in UPLC-grade water and added to a 2X concentrated proteasome digestion buffer (60 mM Trizma-base; pH 7.5; Sigma-Aldrich, 20 mM KC1; Sigma-Aldrich, 10 mM MgCl2; Sigma-Aldrich, 10 mM NaCl; Sigma-Aldrich) to an end concentration DTT of 2 mM. Then, 130 μΐ UPLC- grade water was added to 3 reaction vials, along with 150 μΐ of the 2X concentrated proteasome digestion buffer containing 2 mM DTT, and 10 μΐ of the peptide to be tested (stock concentration 300 nmol/ml). After vortexing the vials, 10 μΐ of water was added to vial 1 (mock control digest), 1 μg (10 μΐ) of constitutive 20S-proteasome (stock 0.1 mg/ml; Enzo Life Sciences) to vial 2, and 1 μg (10 μΐ) of immune 20S- proteasome (stock 0.1 mg/ml; Enzo Life Sciences) to vial 3. A 50 μΐ sample for T=0 was taken directly after vortexing and 4 μΐ of Formic acid (Sigma-Aldrich) was added to stop the reaction. The reaction vials were vortexed and incubated at 37°C. Samples of 50 μΐ were collected after lh, 3h, 6h and 24h incubation. The reactions were stopped with 4 μΐ Formic acid, and all samples were stored at 20°C. Mass spectrometry analysis of digested fragments
A Q-TOF1 mass spectrometer (Waters) equipped with an online nanoelectrospray interface with an approximate flow rate of 250 nl/min was used for electrospray ionization-mass spectrometry. Peptide-digestion samples were trapped on a precolumn
(MCA-300-05-C18; Dionex) and were eluted with a steep gradient of 70-90% buffer B over 10 min (buffer A, water, acetonitrile and formic acid, 95:3 : 1 (vol/vol/vol); buffer B, water, acetonitrile and formic acid, 10:90: 1 (vol/vol/vol)). Mass spectra were recorded from a mass of 50-2000 daltons. In tandem mass spectrometry mode, ions were selected with a window of 3 daltons. The collision gas was argon (4 χ 10~5 mbar), and the collision voltage was -30 V. For peptide digestion by purified constitutive proteasome and immunoproteasome, peaks in the mass spectra were searched in source substrate peptides with BioLynx software (Waters) and the abundance of a specific digestion fragment was assessed quantitatively as its percentage of the total summed intensities, including undigested substrate. Cells
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by centrifugation over a Ficoll gradient. To generate dendritic cells (DCs), approximately 50* 106 PBMC were brought to a concentration of 3* 106 cells/ ml complete medium (EVIDM, Lonza, supplemented with 8% HS, Seralab; penicillin/streptomycin, Lonza; L-glutamin, Lonza) and seeded 3 ml /well in a 6-wells plate (Corning). After incubation for 1.5 hours at 37°C, non-adherent cells were washed away in three washing steps using complete medium (day 0). The adherent cells were cultured for three days at 37°C in 2 ml/well of complete medium containing 800 U/ml GM-CSF and 500 U/ml IL-4 (Peprotech). On day 3, 1 ml of complete medium containing 2400 U/ml GM-CSF and 1500 U/ml IL-4 was added to each well and cultured for another three days at 37°C. Induction of T cells
On day 6, long peptides distributed over 3 pools were added to monocyte- derived DCs of naive donors at a 3 nmol/ml concentration and incubated overnight at 37°C. Pool 1 comprises SLP 26 (SEQ ID NO: 76), SLP 24 (SEQ ID NO: 74), SLP 1 (SEQ ID NO: 51) and SLP 30 (SEQ ID NO: 1142); Pool 2 comprises Abu-SLP 27 (SEQ ID NO: 77, wherein the cysteine on amino acid position 1 is replaced by Abu), SLP 25 (SEQ ID NO: 75), SLP 10 (SEQ ID NO: 60) and SLP 34 (SEQ ID NO: 1469); and Pool 3 comprises SLP 5 (SEQ ID NO: 55), SLP 13 (SEQ ID NO: 63), SLP 14 (SEQ ID NO: 64) and SLP 21 (SEQ ID NO: 21). On day 7, the cells were washed twice with complete medium to remove peptides. DC and autologous PBMC were co- cultured in a 1 : 10 ratio for 10 days at 37°C in the presence of 10 ng/ml IL-7 and 100 pg/ml IL-12p70. The T cell lines generated by this process were checked every 2-3 days and split when necessary.
Restimulation of T cell lines
Three days after T cell induction (day 10), a second batch of autologous DC was differentiated and loaded with peptide pools as described above. On day 10 after initiation of the T cell line (day 17), the peptide-loaded DC were washed twice with complete medium and added to the T cells in a 1 : 10 (DC:T cell) ratio in the presence of 10 ng/ml IL-7 and 100 pg/ml IL-12p70. The cells were co-cultured for 7 days. On day 17, also a new batch of DC was differentiated and loaded with peptide pools as described above. For this second restimulation, starting on day 24, DC and T cells were co-cultured in a 1 : 10 ratio. Two days after restimulation, 30 IU/ml IL-2 (Peprotech) and 5 ng/ml IL-15 (Peprotech) were added to the culture medium. A third restimulation, starting on day 31, was performed identically to the second restimulation.
T cell proliferation and IFNy production
To measure T cell activation and proliferation, autologous DC were loaded for 6 hours with each of the 13 HBV-derived peptides separately on day 23, 30 and 37 of the T cell induction protocol described above. The DCs were washed and 5,000 peptide- loaded DC were co-cultured with 50,000 T cells for 48 hours. Then, supernatant was collected for ELISA (IFNy ELISA, Diaclone) and culture medium containing 3H thymidine was added to all wells. Radioactive 3H thymidine is incorporated in the DNA of newly formed (proliferated) cells, which is measured after 16 hours of incubation on a MicroBeta liquid scintillation counter (Wallac/Perkin Elmer). IFNy-ELISpot
To detect antigen-specific IFNy-producing human T cells, the PBMC were first pre-stimulated with 3 nmol/ml of the indicated peptide for 72 hours at 37°C. During this stimulation, ELISpot PVDF plates (Mabtech) were coated with 5 ug/ml anti-human IFNy mAb 1-DlK coating antibody (Mabtech) in PBS and incubated overnight at 4°C. After stimulation of the PBMC, the coating antibody was aspirated from the plate, and washed 4 times with PBS. To block aspecific binding, 100 μΐ of IMDM containing 8% FCS was added to all wells and incubated at 37°C for 1 hour. In the meantime, stimulated PBMC were harvested, centrifuged, resuspended in X-vivo 15 medium (Lonza) and counted. All PBMC samples were brought to a concentration of 1.5* 106 cells/ml in X-vivo 15 medium. The medium in the wells of the PVDF plate was aspirated and 100 μΐ of each PBMC sample was added to the plate in quadruplicates. The plates were incubated at 37°C overnight. The next day, the supernatant was discarded and plates are washed 6 times with PBS/Tween20 0.05%. The biotinylated anti-human IFNy mAb 7-B6-1 (Mabtech) was added to all wells (100 μΐ/well) at a 0.3 μg/ml concentration in PBS with 1% FBS, and incubated for 2 hours at RT. Next, plates were washed 6 times using PBS/Tween20 0.05% and 1 μg/ml Extravidin- Alkaline phosphatase (ALP) (Sigma-Aldrich) was added to all wells (100 μΐ/well) in PBS with 1% FBS. The plates were incubated for 1 hour at RT. ALP substrate solution BCIP/NBT-plus (Mabtech) was prepared and 100 μΐ/well was added to all wells after the plates were washed 4 times with PBS/Tween20 0.05%. To terminate the colorimetric reaction (after 1-20 minutes), tap water was used to wash the plates extensively. After drying, the formed spots were measured on a Biosys Bioreader 5000.
Results
Selection of long peptides based on HLA class I peptide binding prediction and predicted C-terminal generation of all possible CD8+ T cell epitopes contained in the long peptides
A high quality CD8+ T cell epitope is defined as a peptide that possesses both a predicted high affinity for the HLA class I molecule to which it binds and is also predicted to be generated at its C-terminus by a proteolytic cleavage of the proteasome. Peptides according to the present invention were selected in HBV protein regions that contain optimally high numbers of high quality CD8+ and CD4+ T cell epitopes. To this end, first the HLA class I binding and C-terminal generation of all possible CD8+ T cell epitopes was assessed using a proprietary HLA class I peptide binding algorithm and two proprietary algorithms predicting the cleavages by the proteasome. Subsequently, we devised a single quantitative measure, the so-called binding-cleavage- immunogenicity (BCI) Class I-score, that for each short peptide (8 - 13 amino acids) incorporates both its predicted binding affinity for the HLA class I molecule to which it binds and the likelihood that the peptide is generated by the proteasome in the cells. The Class I-BCI score is calculated from (1) the binding Class I-score (Class I-B score), which is derived from the results of the in silico prediction of HLA class I peptide binding using the aforementioned algorithm, and (2) the cleavage score (C- score), which is derived from the results of the in silico prediction of the proteasome- mediated C-terminal generation of the peptide by the proteasome using the aforementioned algorithms. Tables 4a, 5a, 6a, and 7a present the Class I-BCI score for all possible CD8+ T cell epitopes of these Polymerase, Core protein, Surface Antigen or X-protein derived SLPs, respectively, together with the cumulative BCI Class I score. Tables 4b, 5b, 6b and 7b present the Class II-B score for all possible CD4+ T cell epitopes of these Polymerase, Core protein, Surface Antigen or X-protein derived SLPs, respectively, together with the cumulative Class II-B score. Together the cumulative Class I-BCI score (for CTL epitopes) and the cumulative B Class II-score (for T helper epitopes) resulted in one quantitative value, the so-called Total Regional Immunogenicity Assessment (TRIA) score, reflecting the overall T cell immunogenicity of a long vaccine peptide (Table 3). Based on the highest TRIA scores, 37 SLPs derived from HBV Polymerase, Core protein, Surface Antigen or X-protein were selected for further evaluation (Table 3). From these, to validate the predictive power of the TRIA score, we chose a representative set of 13 SLPs, which included SLP with relatively low and high TRIA scores, for in vitro immunogenicity assessment. These 13 peptides were divided over three peptide pools for T cell induction assays, as described (see below).
Peptide fragment analysis after proteasomal digestion of SLPs reveals high accuracy of in silico predictions
An important component of the identification of putative CTL epitopes is the prediction of their C-terminal generation by a proteasome-mediated cleavage. To validate the reliability of this prediction, proteasomal digestion patterns were experimentally assessed for all but 3 of the 13 functionally tested long vaccine peptides (three long peptides were not being cleaved due to technical reasons).
Digestion experiments were performed separately with 20S constitutive proteasomes and 20S immuno proteasome preparations. The combined analysis of cleavages produced by both types of proteasomes allows the assessment of the C- terminal generation of CTL epitopes that are expressed both on the surface of antigen presenting cells (mainly dendritic cells), containing immunoproteasomes, and on the surface of cancer cells, especially from solid tumors, which mainly express constitutive proteasomes. For vaccination purposes these epitopes are preferred because vaccination with such epitopes will allow the induction of CTL by vaccination and the subsequent eradication of cancer cells by these CTL after recognition of the epitopes on the surface of cancer cells.
As indicated in Figure 1, 10 long peptides of the invention (length 30 - 39 aa) were co-incubated in an appropriate buffer with the proteasome preparations at 37°C for 0, 1, 3, 6 and 24 h. After incubation for the indicated interval reactions were stopped and digestion mixtures, containing the digestion fragments, were measured by mass spectrometry as described herein. The mass spectra were (semi-quantitatively) analyzed to assess the position and abundance of the cleavage sites. The results of 24 h digestion are shown in Figure 1.
The observed cleavage sites observed after 24 h incubation are indicated with arrows. Only cleavage sites that were observed in both the digestion with constitutive proteasomes and the digestion with immunoproteasomes are shown. Major cleavage sites and minor cleavages sites at 24 h digestion are depicted with bold and thin arrows, respectively, according to the following classification:
Major cleavage site: fragments containing as COOH terminus the residue MTi-terminal from the cleavage site together with the (possible) complementary fragment(s) are present for >7% at 24 h incubation, as calculated from the intensities of the fragment peaks in the mass spectra.
Minor cleavage site: fragments containing as COOH terminus the residue MTi-terminal from the cleavage site together with the (possible) complementary fragment(s) are present for <7% at 24 h incubation. Cleavage sites with a cumulative fragment abundance of <1% are not shown.
Figure 1 also indicates the C-score of proteasomal cleavage prediction. This score indicates the likelihood of cleavage C-terminal of the residue directly under the C- score. If the C-score > 1 the cleavage site is considered predicted to be cleaved by both the constitutive proteasome and the immunoproteasome. As described herein, the C- score is a summation of the predictions by two in silico algorithms separately predicting the proteasome-mediated cleavages by constitutive proteasomes and the cleavages by immunoproteasomes. Each separate prediction can attain a maximal value of 1. Accordingly, the maximal value of the C-score is 2. C-scores > 1 are together counted as the total number of predicted cleavage sites (the C-terminus of the long substrate peptide is not taken into account, because cleavage after this residue cannot be tested).
Figure 1 further indicates the confirmed cleavages (indicated by '+'), which are those predicted cleavage sites (C-score > 1) that are confirmed to be cleaved after 24 h in the proteasome-mediated digestion assay.
As indicated by Figure 1, for SLP1 36% (4/11), for SLP10 71% (10/14), for
SLP13 62% (10/ 16), for SLP14 100% (13/13), for SLP18 65% (13/20), for SLP21 87% (14/16), for SLP24 62% (10/16), for SLP25 57% (8/14), for SLP26 72% (8/11), and for SLP27 75% (9/12) of the predicted cleavage sites have been confirmed here. Induction of T cell responses against 12 out of 13 selected peptides in a naive population
To assess whether the 13 selected peptides are able to induce a T cell response in naive donors, PBMC were isolated from buffy coats derived from six healthy donors that had not experienced an HBV-infection. These PBMC were restimulated with either of the 3 peptide pools to obtain T cell lines, that were subsequently stimulated with the selected 13 peptides. The production of IFNy and T cell proliferation (3H thymidine incorporation) were measured as a read-out for T cell activation. Results are shown in Figure 2, indicating the percentage of donors showing a positive T cell response with a stimulation index (SI) of 1.5 or higher . The SI is calculated by dividing the measured sample value by the value of non-stimulated control cells. Induced responses in naive donors were detected against 12 of the 13 pre-selected peptides, while 11 pre-selected peptides induced a response in multiple naive donors.
Strength of pre-existing T cell responses in HBV-immune donors correlates with TRIA score
Subjects that have gone through an HBV infection and successfully cleared it, possess circulating memory T cells specific for HBV. To assess the relevance of the selected vaccine peptides for the clearance of a naturally occurring HBV infection, we tested the presence of T cell responses against the 13 selected HBV peptides in PBMC derived from six HBV-immune donors. After isolation of the PBMC, cells were stimulated with each of the 13 peptides and an IFNy-ELISpot was performed to detect T cell responses. PBMC samples from 3 out of 6 tested donors showed a positive IFNy response against one or more of the 13 peptides (SI > 3). Responses were observed against 11 (SLP 5, 10, 13, 14, 18, 21, 24, 25, 26, 27 and 34, represented by SEQ ID NO: 55, 60, 63, 64, 68, 71, 74, 75, 76, 77 and 1469, respectively) out of the 13 peptides, 6 of which induced responses in multiple donors (SLP 10, 13, 21, 24, 25 and 34, represented by SEQ ID NO: 60, 63, 71, 74, 75 and 1469, respectively). The results are summarized in Figure 3, indicating the donors showing a positive IFNy+ T cell response against a peptide. For each SLP tested, the average SI of the IFNy-responses was calculated; 1.21 for SLPl, 2.1 1 for SLP5, 2.40 for SLP10, 2.44 for SLP13, 1.97 for SLP 14, 1.86 for SLP 18, 2.87 for SLP21, 2.54 for SLP24, 2.66 for SLP25, 1.56 for SLP26, 1,66 for Abu-SLP27, 1.49 for SLP30 and 3.02 for SLP34. Importantly, when the average SI of the IFNy-responses to each peptide is plotted against the corresponding TRIA score, we see a significant correlation (Figure 4), both while including (R2=0.37, p=0.028; see Figure 4) the results of Abu-SLP27 and while excluding (R2=0.34, p=0.048; not shown) the results of Abu-SLP27. This validates the use of the TRIA score for the selection of immunogenic peptides.
Discussion
The experimental results presented herein validate, and therefore support, the selection and underscore the immunological relevance of the HBV-derived long vaccine peptides of the present invention. These long vaccine peptides encompass the HBV protein regions with the highest number of high quality HLA class I and HLA class II binding epitopes in an outbred population. A preferred combination of the vaccine peptides will be incorporated in a novel HBV SLP vaccine composition to treat chronically HBV-infected patients. Using algorithms to predict the peptide binding affinity of short peptides (8 - 12 aa) for all prevalent HLA class I molecules and the likelihood of the C-terminal generation of these short peptides by cleavage by the proteasome, in combination with the identification of the putative T helper epitopes, we identified highly immunogenic regions from which the optimal 37 long vaccine peptides were selected.
To enable proper selection of long vaccine peptides in the present invention, a quantitative measure was developed, the TRIA score. Without such a quantitative measure attributed to all possible long HBV peptides, a proper selection of the optimal long peptides is not possible. The TRIA score is a quantitative representation of the quality and quantity of all putative HLA class I restricted CD8+ cytotoxic T cell epitopes and HLA class II restricted CD4+ T helper epitopes contained in a long peptide. The TRIA score was calculated for all possible HBV peptides with a length of 30 - 39 aa, which is the optimal peptide length for vaccination purposes, enabling the rational selection of a set of highly immunogenic long vaccine peptides.
For further testing and validation of the immunological relevance, we selected a set of 13 SLP with varying TRIA scores. First, we experimentally digested these SLP using either constitutive proteasome or immunoproteasome. The generated fragments showed a clear overlap with the predicted C-terminal cleavage sites, expressed in the BCI score. Thereafter, T cell assays were performed using PBMC from both naive and HBV-immune donors. Nearly all 13 selected SLPs were able to induce T cell responses in PBMC derived from naive healthy donors, which proves that the selected SLP set has the potential to induce responses by the T cell repertoire that had not been stimulated before. Within the same set of vaccine peptides, a strong correlation was observed between the TRIA score of a certain vaccine peptide and the strength of the IFNy-response in HBV-immune donors, indicating that the TRIA score is a predictive value for in vivo immunogenicity and thus functionality of the vaccine peptides.
Table 1. Protein and DNA sequences of HBV polymerase, HBV core and HBV large surface proteins.
SEQ SEQ type Gene / Gene Sequence
ID product
NO:
1 Protein HBV MPLSYQHFRKLLLLDDGTEAGPLEEELPRLADADLHRRVAE
Polymerase DLNLGNLNVSIPWTHKVGNFTGLYSSTVPIFNPEWQTPSFP
KIHLQEDIINRCQQFVGPLT EKRRLKLIMPARFYPTHTK YLPLDKGIKPYYPDQV HYFQTRHYLHTLWKAGILYKRET TRSASFCGSPYSWEQELQHGRLVIKTSQRHGDESFCSQSSG ILSRSSVGPCIRSQLKQSRLGLQPRQGRLASSQPSRSGSIR AKAHPSTRRYFGVEPSGSGHIDHS NSSSCLHQSAVRKAA YSHLSTSKRQSSSGHAVEFHCLPPNSAGSQSQGSVSSCWWL QFRNSKPCSEYCLSHL LREDWGPCDEHGEHHIRIPRTPA RVTGGVFLVDKNPHNTAESRLWDFSQFSRGISRVSWPKFA VPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPAAMPHLLI GSSGLSRYVARLSSNSRINNNQYGTMQNLHDSCSRQLYVSL MLLYKTYGWKLHLYSHPIVLGFRKIPMGVGLSPFLLAQFTS AICSWRRAFPHCLAFSYMDDWLGAKSVQHRESLYTAVTN FLLSLGIHLNPNKTKRWGYSLNFMGYIIGSWGTLPQDHIVQ KIKHCFRKLP RPIDWKVCQRIVGLLGFAAPFTQCGYPAL MPLYACIQAKQAFTFSPTYKAFLSKQYMNLYPVARQRPGLC QVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAELLAACFA RSRSGAKLIGTDNSWLSRKYTSFPWLLGCTANWILRGTSF VYVPSALNPADDPSRGRLGLSRPLLRLPFQPTTGRTSLYAV SPSVPSHLPVRVHFASPLHVAWRPP
2 CDS HBV See sequence listing
Polymerase
3 Genomic HBV genome See sequence listing
4 Protein HBV Core MQLFHLCLIISCTCPTVQASKLCLGWLWGMDIDPYKEFGAT
VELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHT ALRQAILCWGELMTLATWVGNNLEDPASRDLWNYVNTNVG LKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPP NAPILSTLPETTWRRRDRGRSPRRRTPSPRRRRSPSPRRR RSQSRESQC
5 CDS HBV Core See sequence listing
1141 Protein HBV MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNP large surface DWDFNPVKDDWPAANQVGVGAFGPRLTPPHGGILGWSPQAQ protein GILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWN
STAFHQTLQDPRVRGLYLPAGGSSSGTVNPAPNIASHISSI SARTGDPVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQS LDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGY RWMCLRRFI I FLFILLLCLI FLLVLLDYQGMLPVCPLI PGS TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPS SWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAI WMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI Table 2. Different HBV X protein variants and the deduced consensus protein sequence.
Figure imgf000063_0001
Figure imgf000064_0001
Table 3. Synthetic long peptide (SLP) sequences.
Figure imgf000065_0001
Figure imgf000066_0001
A Cumulative Class I-BCI score: See Material and Methods (Examples section).
B Cumulative Class II-B score: See Material and Methods (Examples section).
CTRIA score is the sum of the Cumulative Class I-BCI score and the Cumulative Class II-B score.
Table 4a. Predicted HLA class I-restricted CD8+ cytotoxic T cell epitopes contained in SLP sequences from HBV polymerase protein.
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000067_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000068_0001
Figure imgf000069_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000072_0002
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000073_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000074_0001
Figure imgf000075_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000076_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000077_0001
Figure imgf000078_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000079_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000080_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000081_0001
Figure imgf000082_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000083_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000084_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000085_0001
Figure imgf000086_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000087_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000088_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000089_0001
Figure imgf000090_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000096_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000097_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000098_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000099_0001
Figure imgf000100_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000101_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000102_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000106_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000107_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000108_0001
Figure imgf000109_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000110_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000111_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000112_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000113_0001
Figure imgf000114_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000115_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000116_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000117_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000118_0001
Figure imgf000119_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000120_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000121_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000122_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000123_0001
Figure imgf000124_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000125_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000126_0001
Figure imgf000127_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000128_0001
Figure imgf000129_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000130_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000131_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000132_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000133_0001
Figure imgf000134_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000135_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000136_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000137_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000138_0001
Figure imgf000139_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000140_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000141_0001
Figure imgf000142_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000143_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000144_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000145_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000146_0001
Figure imgf000147_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000148_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000149_0001
Figure imgf000150_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000151_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000152_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000153_0001
HLA class I binding peptides in SLP sequences derived from HBV Polymerase protein Peptide of invention (SLP)
Figure imgf000154_0001
"Start" and "End" are relative to the amino acid sequence of HBV polymerase as depicted in SEQ ID NO: 1
A Peptide amino acid sequence. Each HLA class I binding peptide of HBV polymerase is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
B Class I-B score. See Material and Methods (Examples section).
c C-score. See Material and Methods (Examples section).
D Class I-BCI score. See Material and Methods (Examples section).
E Cumulative Class I-BCI score. See Material and Methods (Examples section).
Table 4b. Predicted HLA class Il-restricted CD4+ T cell epitopes contained sequences from HB V polymerase protein.
HLA class II binding peptides in SLP sequences derived from Peptide of invention (SLP)
HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
1 15 MPLSYQHFRKLLLLD 686 *0701 79 1 1 30
1 15 MPLSYQHFRKLLLLD * 1101 86 1
1 15 MPLSYQHFRKLLLLD * 1501 9 1
2 16 PLSYQHFRKLLLLDD 687 *0701 40 1
2 16 PLSYQHFRKLLLLDD * 1101 84 1
2 16 PLSYQHFRKLLLLDD * 1501 4 1
3 17 LSYQHFRKLLLLDDG 688 *0701 9 1
3 17 LSYQHFRKLLLLDDG * 1101 83 1
3 17 LSYQHFRKLLLLDDG * 1501 3 1
4 18 SYQHFRKLLLLDDGT 689 * 1101 81 1
5 19 YQHFRKLLLLDDGTE 690 * 1101 80 1
6 20 QHFRKLLLLDDGTEA 691 *0301 31 1
6 20 QHFRKLLLLDDGTEA * 1101 56 1
7 21 HFRKLLLLDDGTEAG 692 *0301 30 1
8 22 FRKLLLLDDGTEAGP 693 *0301 29 1
9 23 RKLLLLDDGTEAGPL 694 *0301 27 1
10 24 KLLLLDDGTEAGPLE 695 *0301 26 1
11 25 LLLLDDGTEAGPLEE 696 *0301 24 1
Cumulative Class II-B scorec: 780
55 69 THKVGNFTGLYSSTV 697 *0101 14 2 52 86
55 69 THKVGNFTGLYSSTV * 1501 54 2
56 70 HKVGNFTGLYSSTVP 698 *0101 10 2
56 70 HKVGNFTGLYSSTVP * 1501 51 2
57 71 KVGNFTGLYSSTVPI 699 *0101 64 2
57 71 KVGNFTGLYSSTVPI *0105 78 2
57 71 KVGNFTGLYSSTVPI *0107 78 2
57 71 KVGNFTGLYSSTVPI *0401 11 2
57 71 KVGNFTGLYSSTVPI *0701 26 2
57 71 KVGNFTGLYSSTVPI * 1501 47 2
58 72 VGNFTGLYSSTVPIF 700 *0101 93 2
58 72 VGNFTGLYSSTVPIF *0105 88 2
58 72 VGNFTGLYSSTVPIF *0107 88 2
58 72 VGNFTGLYSSTVPIF *0301 14 2
58 72 VGNFTGLYSSTVPIF *0401 23 2
58 72 VGNFTGLYSSTVPIF *0701 34 2
58 72 VGNFTGLYSSTVPIF * 1501 59 2
59 73 GNFTGLYSSTVPIFN 701 *0101 80 2
59 73 GNFTGLYSSTVPIFN *0105 55 2
59 73 GNFTGLYSSTVPIFN *0107 55 2
59 73 GNFTGLYSSTVPIFN *0301 16 2
59 73 GNFTGLYSSTVPIFN *0401 24 2
59 73 GNFTGLYSSTVPIFN *0701 54 2
60 74 NFTGLYSSTVPIFNP 702 *0101 57 2 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
60 74 NFTGLYSSTVPIFNP *0301 11 2
60 74 NFTGLYSSTVPIFNP *0401 16 2
60 74 NFTGLYSSTVPIFNP *0701 30 2
61 75 FTGLYSSTVPIFNPE 703 *0101 43 2
61 75 FTGLYSSTVPIFNPE *0301 13 2
61 75 FTGLYSSTVPIFNPE *0401 19 2
61 75 FTGLYSSTVPIFNPE *0701 11 2
62 76 TGLYSSTVPIFNPEW 704 *0301 7 2
Cumulative Class II-B score: 1323
103 117 VNEKRRLKLIMPARF 705 *0101 47 3 103 135
103 117 VNEKRRLKLIMPARF *0102 33 3
103 117 VNEKRRLKLIMPARF *0104 8 3
103 117 VNEKRRLKLIMPARF *0106 50 3
103 117 VNEKRRLKLIMPARF *0301 36 3
103 117 VNEKRRLKLIMPARF *0701 17 3
103 117 VNEKRRLKLIMPARF * 1301 7 3
103 117 VNEKRRLKLIMPARF * 1501 64 3
104 118 NEKRRLKLIMPARFY 706 *0101 53 3
104 118 NEKRRLKLIMPARFY *0102 83 3
104 118 NEKRRLKLIMPARFY *0104 78 3
104 118 NEKRRLKLIMPARFY *0106 83 3
104 118 NEKRRLKLIMPARFY *0701 27 3
104 118 NEKRRLKLIMPARFY * 1301 64 3
104 118 NEKRRLKLIMPARFY * 1501 76 3
105 119 EKRRLKLIMPARFYP 707 *0101 46 3
105 119 EKRRLKLIMPARFYP *0102 93 3
105 119 EKRRLKLIMPARFYP *0104 85 3
105 119 EKRRLKLIMPARFYP *0106 85 3
105 119 EKRRLKLIMPARFYP *0701 3 3
105 119 EKRRLKLIMPARFYP * 1101 8 3
105 119 EKRRLKLIMPARFYP * 1301 61 3
105 119 EKRRLKLIMPARFYP * 1501 71 3
106 120 KRRLKLIMPARFYPT 708 *0101 49 3
106 120 KRRLKLIMPARFYPT *0102 100 3
106 120 KRRLKLIMPARFYPT *0103 4 3
106 120 KRRLKLIMPARFYPT *0104 100 3
106 120 KRRLKLIMPARFYPT *0105 45 3
106 120 KRRLKLIMPARFYPT *0106 93 3
106 120 KRRLKLIMPARFYPT *0107 45 3
106 120 KRRLKLIMPARFYPT * 1101 25 3
106 120 KRRLKLIMPARFYPT * 1301 71 3
106 120 KRRLKLIMPARFYPT * 1501 73 3
107 121 RRLKLIMPARFYPTH 709 *0101 44 3
107 121 RRLKLIMPARFYPTH *0102 80 3
107 121 RRLKLIMPARFYPTH *0104 88 3
107 121 RRLKLIMPARFYPTH *0106 78 3 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
107 121 RRLKLIMPARFYPTH * 1301 32 3
107 121 RRLKLIMPARFYPTH * 1501 69 3
108 122 RLKLIMPARFYPTHT 710 *0102 53 3
108 122 RLKLIMPARFYPTHT *0104 53 3
108 122 RLKLIMPARFYPTHT *0106 60 3
108 122 RLKLIMPARFYPTHT * 1501 41 3
109 123 LKLIMPARFYPTHTK 711 * 1501 40 3
111 125 LIMPARFYPTHTKYL 712 *0701 93 3
112 126 IMPARFYPTHTKYLP 713 *0701 91 3
113 127 MPARFYPTHTKYLPL 714 *0701 96 3
114 128 PARFYPTHTKYLPLD 715 *0701 94 3
115 129 ARFYPTHTKYLPLDK 716 *0701 90 3
116 130 RFYPTHTKYLPLDKG 717 *0701 86 3
117 131 FYPTHTKYLPLDKGI 718 *0701 81 3
121 135 HTKYLPLDKGIKPYY 719 * 1101 23 3
Cumulative Class II-B score: 3070
121 135 HTKYLPLDKGIKPYY 719 * 1101 23 4 118 150
122 136 TKYLPLDKGIKPYYP 720 * 1101 21 4
130 144 GIKPYYPDQV HYF 721 *0401 26 4
131 145 IKPYYPDQV HYFQ 722 *0401 51 4
132 146 KPYYPDQV HYFQT 723 *0401 21 4
Cumulative Class II-B score: 143
139 153 V HYFQTRHYLHTL 726 * 1101 87 5 139 111
139 153 V HYFQTRHYLHTL * 1501 70 5
140 154 HYFQTRHYLHTLW 727 *0701 70 5
140 154 HYFQTRHYLHTLW * 1101 76 5
140 154 WHYFQTRHYLHTLW * 1501 58 5
141 155 NHYFQTRHYLHTLWK 728 *0701 40 5
141 155 NHYFQTRHYLHTLWK * 1101 79 5
141 155 NHYFQTRHYLHTLWK * 1501 28 5
142 156 HYFQTRHYLHTLWKA 729 * 1101 54 5
142 156 HYFQTRHYLHTLWKA * 1501 30 5
143 157 YFQTRHYLHTLWKAG 730 * 1101 24 5
144 158 FQTRHYLHTLWKAGI 731 * 1101 55 5
144 158 FQTRHYLHTLWKAGI * 1501 5 5
145 159 QTRHYLHTLWKAGIL 732 * 1101 78 5
145 159 QTRHYLHTLWKAGIL * 1501 38 5
146 160 TRHYLHTLWKAGILY 733 * 1101 88 5
146 160 TRHYLHTLWKAGILY * 1501 45 5
147 161 RHYLHTLWKAGILYK 734 * 1101 85 5
147 161 RHYLHTLWKAGILYK * 1501 40 5
148 162 HYLHTLWKAGILYKR 735 * 1101 50 5
154 168 WKAGILYKRETTRSA 736 *0401 81 5
155 169 KAGILYKRETTRSAS 737 *0401 83 5
156 170 AGILYKRETTRSASF 738 *0401 84 5 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
157 171 GILYKRETTRSASFC 739 *0401 80 5
158 172 ILYKRETTRSASFCG 740 *0401 79 5
159 173 LYKRETTRSASFCGS 741 *0401 14 5
Cumulative Class II-B score: 1519
143 157 YFQTRHYLHTLWKAG 730 *1101 24 6 143 111
144 158 FQTRHYLHTLWKAGI 731 *1101 55 6
144 158 FQTRHYLHTLWKAGI *1501 5 6
145 159 QTRHYLHTLWKAGIL 732 *1101 78 6
145 159 QTRHYLHTLWKAGIL *1501 38 6
146 160 TRHYLHTLWKAGILY 733 *1101 88 6
146 160 TRHYLHTLWKAGILY *1501 45 6
147 161 RHYLHTLWKAGILYK 734 *1101 85 6
147 161 RHYLHTLWKAGILYK *1501 40 6
148 162 HYLHTLWKAGILYKR 735 *1101 50 6
154 168 WKAGILYKRETTRSA 736 *0401 81 6
155 169 KAGILYKRETTRSAS 737 *0401 83 6
156 170 AGILYKRETTRSASF 738 *0401 84 6
157 171 GILYKRETTRSASFC 739 *0401 80 6
158 172 ILYKRETTRSASFCG 740 *0401 79 6
159 173 LYKRETTRSASFCGS 741 *0401 14 6
Cumulative Class II-B score: 928
137 151 DQV HYFQTRHYLH 724 *0701 53 7 137 170
137 151 DQV HYFQTRHYLH *1101 75 7
137 151 DQVWHYFQTRHYLH *1501 63 7
138 152 QWNHYFQTRHYLHT 725 *0701 63 7
138 152 QWNHYFQTRHYLHT *1101 59 7
138 152 QWNHYFQTRHYLHT *1501 73 7
139 153 WNHYFQTRHYLHTL 726 *0701 88 7
139 153 WNHYFQTRHYLHTL *1101 87 7
139 153 WNHYFQTRHYLHTL *1501 70 7
140 154 VNHYFQTRHYLHTLW 727 *0701 70 7
140 154 WHYFQTRHYLHTLW *1101 76 7
140 154 VNHYFQTRHYLHTLW *1501 58 7
141 155 NHYFQTRHYLHTLWK 728 *0701 40 7
141 155 NHYFQTRHYLHTLWK *1101 79 7
141 155 NHYFQTRHYLHTLWK *1501 28 7
142 156 HYFQTRHYLHTLWKA 729 *1101 54 7
142 156 HYFQTRHYLHTLWKA *1501 30 7
143 157 YFQTRHYLHTLWKAG 730 *1101 24 7
144 158 FQTRHYLHTLWKAGI 731 *1101 55 7
144 158 FQTRHYLHTLWKAGI *1501 5 7
145 159 QTRHYLHTLWKAGIL 732 *1101 78 7
145 159 QTRHYLHTLWKAGIL *1501 38 7
146 160 TRHYLHTLWKAGILY 733 *1101 88 7
146 160 TRHYLHTLWKAGILY *1501 45 7 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
147 161 RHYLHTLWKAGILYK 734 * 1101 85 7
147 161 RHYLHTLWKAGILYK * 1501 40 7
148 162 HYLHTLWKAGILYKR 735 * 1101 50 7
154 168 WKAGILYKRETTRSA 736 *0401 81 7
155 169 KAGILYKRETTRSAS 737 *0401 83 7
156 170 AGILYKRETTRSASF 738 *0401 84 7
Cumulative Class II-B score: 1817
324 338 SCWWLQFRNSKPCSE 742 *0401 3 8 316 347
385 399 TAESRLWDFSQFSR 743 *0301 97 9 385 417
386 400 AESRLWDFSQFSRG 744 *0301 87 9
387 401 ESRLWDFSQFSRGI 745 *0301 96 9
388 402 SRLWDFSQFSRGIS 746 *0301 94 9
389 403 RLWDFSQFSRGISR 747 *0301 77 9
390 404 LWDFSQFSRGISRV 748 *0301 76 9
Cumulative Class II-B score: 527
421 435 LSSNLSWLSLDVSAA 749 *0401 27 10 419 456
422 436 SSNLSWLSLDVSAAF 750 *0301 57 10
422 436 SSNLSWLSLDVSAAF *0401 71 10
423 437 SNLSWLSLDVSAAFY 751 *0301 56 10
423 437 SNLSWLSLDVSAAFY *0401 70 10
424 438 NLSWLSLDVSAAFYH 752 *0301 54 10
424 438 NLSWLSLDVSAAFYH *0401 69 10
425 439 LSWLSLDVSAAFYHI 753 *0301 53 10
425 439 LSWLSLDVSAAFYHI *0401 67 10
425 439 LSWLSLDVSAAFYHI * 1301 18 10
426 440 SWLSLDVSAAFYHIP 754 *0301 51 10
426 440 SWLSLDVSAAFYHIP *0401 66 10
427 441 WLSLDVSAAFYHIPL 755 *0301 50 10
427 441 WLSLDVSAAFYHIPL *0401 17 10
428 442 LSLDVSAAFYHIPLH 756 *0301 49 10
432 446 VSAAFYHI PLHPAAM 757 *0101 8 10
432 446 VSAAFYHI PLHPAAM *0105 8 10
432 446 VSAAFYHI PLHPAAM *0107 8 10
433 447 SAAFYHI PLHPAAMP 758 *0101 35 10
433 447 SAAFYHI PLHPAAMP *0105 35 10
433 447 SAAFYHI PLHPAAMP *0107 35 10
433 447 SAAFYHI PLHPAAMP * 1101 3 10
434 448 AAFYHI PLHPAAMPH 759 *0101 13 10
434 448 AAFYHI PLHPAAMPH *0105 13 10
434 448 AAFYHI PLHPAAMPH *0107 13 10
Cumulative Class II-B score: 943
422 436 SSNLSWLSLDVSAAF 750 *0301 57 11 422 459
422 436 SSNLSWLSLDVSAAF *0401 71 11 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
423 437 SNLSWLSLDVSAAFY 751 *0301 56 11
423 437 SNLSWLSLDVSAAFY *0401 70 11
424 438 NLSWLSLDVSAAFYH 752 *0301 54 11
424 438 NLSWLSLDVSAAFYH *0401 69 11
425 439 LSWLSLDVSAAFYHI 753 *0301 53 11
425 439 LSWLSLDVSAAFYHI *0401 67 11
425 439 LSWLSLDVSAAFYHI * 1301 18 11
426 440 SWLSLDVSAAFYHIP 754 *0301 51 11
426 440 SWLSLDVSAAFYHIP *0401 66 11
427 441 WLSLDVSAAFYHIPL 755 *0301 50 11
427 441 WLSLDVSAAFYHIPL *0401 17 11
428 442 LSLDVSAAFYHIPLH 756 *0301 49 11
432 446 VSAAFYHI PLHPAAM 757 *0101 8 11
432 446 VSAAFYHI PLHPAAM *0105 8 11
432 446 VSAAFYHI PLHPAAM *0107 8 11
433 447 SAAFYHI PLHPAAMP 758 *0101 35 11
433 447 SAAFYHI PLHPAAMP *0105 35 11
433 447 SAAFYHI PLHPAAMP *0107 35 11
433 447 SAAFYHI PLHPAAMP * 1101 3 11
434 448 AAFYHI PLHPAAMPH 759 *0101 13 11
434 448 AAFYHI PLHPAAMPH *0105 13 11
434 448 AAFYHI PLHPAAMPH *0107 13 11
444 458 AAMPHLLIGSSGLSR 760 *0101 9 11
444 458 AAMPHLLIGSSGLSR *0301 64 11
445 459 AMPHLLIGSSGLSRY 761 *0101 7 11
445 459 AMPHLLIGSSGLSRY *0301 63 11
Cumulative Class II-B score: 1058
427 441 WLSLDVSAAFYHIPL 755 *0301 50 12 427 459
427 441 WLSLDVSAAFYHIPL *0401 17 12
428 442 LSLDVSAAFYHIPLH 756 *0301 49 12
432 446 VSAAFYHI PLHPAAM 757 *0101 8 12
432 446 VSAAFYHI PLHPAAM *0105 8 12
432 446 VSAAFYHI PLHPAAM *0107 8 12
433 447 SAAFYHI PLHPAAMP 758 *0101 35 12
433 447 SAAFYHI PLHPAAMP *0105 35 12
433 447 SAAFYHI PLHPAAMP *0107 35 12
433 447 SAAFYHI PLHPAAMP * 1101 3 12
434 448 AAFYHI PLHPAAMPH 759 *0101 13 12
434 448 AAFYHI PLHPAAMPH *0105 13 12
434 448 AAFYHI PLHPAAMPH *0107 13 12
444 458 AAMPHLLIGSSGLSR 760 *0101 9 12
444 458 AAMPHLLIGSSGLSR *0301 64 12
445 459 AMPHLLIGSSGLSRY 761 *0101 7 12
445 459 AMPHLLIGSSGLSRY *0301 63 12
Cumulative Class II-B score: 426 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
483 497 SCSRQLYVSLMLLYK 762 * 1101 94 13 481 514
484 498 CSRQLYVSLMLLYKT 763 * 1101 93 13
484 498 CSRQLYVSLMLLYKT * 1501 20 13
485 499 SRQLYVSLMLLYKTY 764 * 1101 91 13
485 499 SRQLYVSLMLLYKTY * 1501 10 13
486 500 RQLYVSLMLLYKTYG 765 *0301 47 13
486 500 RQLYVSLMLLYKTYG * 1101 100 13
486 500 RQLYVSLMLLYKTYG * 1301 21 13
486 500 RQLYVSLMLLYKTYG * 1501 23 13
487 501 QLYVSLMLLYKTYGW 766 *0301 10 13
487 501 QLYVSLMLLYKTYGW * 1101 99 13
487 501 QLYVSLMLLYKTYGW * 1301 20 13
487 501 QLYVSLMLLYKTYGW * 1501 61 13
488 502 LYVSLMLLYKTYGWK 767 *0301 9 13
488 502 LYVSLMLLYKTYGWK * 1101 97 13
488 502 LYVSLMLLYKTYGWK * 1301 19 13
488 502 LYVSLMLLYKTYGWK * 1501 60 13
489 503 YVSLMLLYKTYGWKL 768 *0701 100 13
489 503 YVSLMLLYKTYGWKL * 1101 96 13
489 503 YVSLMLLYKTYGWKL * 1301 17 13
489 503 YVSLMLLYKTYGWKL * 1501 99 13
490 504 VSLMLLYKTYGWKLH 769 *0701 99 13
490 504 VSLMLLYKTYGWKLH * 1301 16 13
490 504 VSLMLLYKTYGWKLH * 1501 100 13
491 505 SLMLLYKTYGWKLHL 770 *0701 97 13
491 505 SLMLLYKTYGWKLHL * 1301 14 13
491 505 SLMLLYKTYGWKLHL * 1501 97 13
492 506 LMLLYKTYGWKLHLY 771 *0106 3 13
492 506 LMLLYKTYGWKLHLY *0701 89 13
492 506 LMLLYKTYGWKLHLY * 1101 5 13
492 506 LMLLYKTYGWKLHLY * 1301 13 13
492 506 LMLLYKTYGWKLHLY * 1501 77 13
493 507 MLLYKTYGWKLHLYS 772 *0701 87 13
493 507 MLLYKTYGWKLHLYS * 1101 9 13
493 507 MLLYKTYGWKLHLYS * 1501 66 13
494 508 LLYKTYGWKLHLYSH 773 *0701 84 13
494 508 LLYKTYGWKLHLYSH * 1101 7 13
494 508 LLYKTYGWKLHLYSH * 1501 85 13
495 509 LYKTYGWKLHLYSHP 774 *0701 74 13
495 509 LYKTYGWKLHLYSHP * 1101 6 13
495 509 LYKTYGWKLHLYSHP * 1501 13 13
496 510 YKTYGWKLHLYSHPI 775 * 1101 4 13
497 511 KTYGWKLHLYSHPIV 776 *0101 30 13
497 511 KTYGWKLHLYSHPIV *0701 13 13
497 511 KTYGWKLHLYSHPIV * 1101 3 13
497 511 KTYGWKLHLYSHPIV * 1501 90 13
498 512 TYGWKLHLYSHPIVL 777 *0101 70 13 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
498 512 TYGWKLHLYSHPIVL *0701 71 13
498 512 TYGWKLHLYSHPIVL * 1101 1 13
498 512 TYGWKLHLYSHPIVL * 1501 89 13
499 513 YGWKLHLYSHPIVLG 778 *0101 81 13
499 513 YGWKLHLYSHPIVLG *0701 24 13
499 513 YGWKLHLYSHPIVLG * 1301 50 13
499 513 YGWKLHLYSHPIVLG * 1501 87 13
500 514 GWKLHLYSHPIVLGF 779 *0101 66 13
500 514 GWKLHLYSHPIVLGF *0701 7 13
500 514 GWKLHLYSHPIVLGF * 1301 49 13
500 514 GWKLHLYSHPIVLGF * 1501 86 13
Cumulative Class II-B score: 3046
524 538 SPFLLAQFTSAICSV 780 *0101 61 14 524 559
524 538 SPFLLAQFTSAICSV *0401 43 14
524 538 SPFLLAQFTSAICSV * 1501 16 14
525 539 PFLLAQFTSAICSW 781 *0101 24 14
525 539 PFLLAQFTSAICSW *0401 41 14
525 539 PFLLAQFTSAICSW *0701 59 14
525 539 PFLLAQFTSAICSW * 1501 14 14
526 540 FLLAQFTSAICSWR 782 *0401 13 14
526 540 FLLAQFTSAICSWR *0701 53 14
527 541 LLAQFTSAICSWRR 783 *0701 49 14
528 542 LAQFTSAICSWRRA 784 *0701 43 14
529 543 AQFTSAICSWRRAF 785 *0701 51 14
533 547 SAICSWRRAFPHCL 786 *0701 47 14
533 547 SAICSWRRAFPHCL * 1301 60 14
534 548 AICSWRRAFPHCLA 787 *0701 16 14
534 548 AICSWRRAFPHCLA * 1301 59 14
535 549 ICSWRRAFPHCLAF 788 *0106 10 14
535 549 ICSWRRAFPHCLAF *0701 21 14
535 549 ICSWRRAFPHCLAF * 1301 57 14
536 550 CSWRRAFPHCLAFS 789 *0103 30 14
536 550 CSWRRAFPHCLAFS *0106 30 14
536 550 CSWRRAFPHCLAFS * 1301 56 14
537 551 SWRRAFPHCLAFSY 790 * 1301 54 14
538 552 WRRAFPHCLAFSYM 791 * 1301 53 14
539 553 VRRAFPHCLAFSYMD 792 * 1301 51 14
545 559 HCLAFSYMDDWLGA 793 *0401 30 14
Cumulative Class II-B score: 1042
526 540 FLLAQFTSAICSWR 782 *0401 13 15 526 559
526 540 FLLAQFTSAICSWR *0701 53 15
527 541 LLAQFTSAICSWRR 783 *0701 49 15
528 542 LAQFTSAICSWRRA 784 *0701 43 15
529 543 AQFTSAICSWRRAF 785 *0701 51 15
533 547 SAICSWRRAFPHCL 786 *0701 47 15 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
533 547 SAICSWRRAFPHCL * 1301 60 15
534 548 AICSWRRAFPHCLA 787 *0701 16 15
534 548 AICSWRRAFPHCLA * 1301 59 15
535 549 ICSWRRAFPHCLAF 788 *0106 10 15
535 549 ICSWRRAFPHCLAF *0701 21 15
535 549 ICSWRRAFPHCLAF * 1301 57 15
536 550 CSWRRAFPHCLAFS 789 *0103 30 15
536 550 CSWRRAFPHCLAFS *0106 30 15
536 550 CSWRRAFPHCLAFS * 1301 56 15
537 551 SWRRAFPHCLAFSY 790 * 1301 54 15
538 552 WRRAFPHCLAFSYM 791 * 1301 53 15
539 553 VRRAFPHCLAFSYMD 792 * 1301 51 15
545 559 HCLAFSYMDDWLGA 793 *0401 30 15
Cumulative Class II-B score: 783
565 579 RESLYTAVTNFLLSL 794 *0103 9 16 565 598
565 579 RESLYTAVTNFLLSL *0701 67 16
566 580 ESLYTAVTNFLLSLG 795 *0701 66 16
567 581 SLYTAVTNFLLSLGI 796 *0701 64 16
568 582 LYTAVTNFLLSLGIH 797 *0701 63 16
569 583 YTAVTNFLLSLGIHL 798 *0101 90 16
569 583 YTAVTNFLLSLGIHL *0701 61 16
569 583 YTAVTNFLLSLGIHL * 1501 37 16
570 584 TAVTNFLLSLGIHLN 799 *0101 91 16
570 584 TAVTNFLLSLGIHLN *0102 28 16
570 584 TAVTNFLLSLGIHLN *0104 23 16
570 584 TAVTNFLLSLGIHLN * 1501 49 16
571 585 AVTNFLLSLGIHLNP 800 *0101 89 16
571 585 AVTNFLLSLGIHLNP *0102 35 16
571 585 AVTNFLLSLGIHLNP *0104 25 16
571 585 AVTNFLLSLGIHLNP * 1501 46 16
572 586 VTNFLLSLGIHLNPN 801 *0101 84 16
572 586 VTNFLLSLGIHLNPN *0102 38 16
572 586 VTNFLLSLGIHLNPN *0104 38 16
572 586 VTNFLLSLGIHLNPN *0106 5 16
572 586 VTNFLLSLGIHLNPN * 1501 63 16
573 587 TNFLLSLGIHLNPNK 802 *0101 83 16
573 587 TNFLLSLGIHLNPNK *0102 10 16
573 587 TNFLLSLGIHLNPNK *0104 5 16
573 587 TNFLLSLGIHLNPNK *0401 4 16
573 587 TNFLLSLGIHLNPNK * 1501 43 16
574 588 NFLLSLGIHLNPNKT 803 *0101 33 16
574 588 NFLLSLGIHLNPNKT * 1501 30 16
575 589 FLLSLGIHLNPNKTK 804 *0101 23 16
575 589 FLLSLGIHLNPNKTK * 1501 29 16
576 590 LLSLGIHLNPNKTKR 805 * 1301 25 16
577 591 LSLGIHLNPNKTKRW 806 * 1301 50 16 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
578 592 SLGIHLNPNKTKRWG 807 * 1301 43 16
579 593 LGIHLNPNKTKRWGY 808 * 1301 36 16
Cumulative Class II-B score: 1482
589 603 KRWGYSLNFMGYIIG 809 * 1501 57 17 584 617
590 604 RWGYSLNFMGYIIGS 810 * 1501 56 17
591 605 WGYSLNFMGYIIGSW 811 * 1501 34 17
592 606 GYSLNFMGYIIGSWG 812 * 1501 21 17
593 607 YSLNFMGYIIGSWGT 813 * 1501 24 17
594 608 SLNFMGYIIGSWGTL 814 * 1501 11 17
595 609 LNFMGYIIGSWGTLP 815 * 1501 19 17
Cumulative Class II-B score: 223
653 667 YPALMPLYACIQAKQ 816 * 1101 11 18 653 691
656 670 LMPLYACIQAKQAFT 817 *0101 58 18
656 670 LMPLYACIQAKQAFT *0105 58 18
656 670 LMPLYACIQAKQAFT *0107 58 18
657 671 MPLYACIQAKQAFTF 818 *0101 75 18
657 671 MPLYACIQAKQAFTF *0105 75 18
657 671 MPLYACIQAKQAFTF *0107 75 18
657 671 MPLYACIQAKQAFTF * 1301 41 18
658 672 PLYACIQAKQAFTFS 819 *0101 48 18
658 672 PLYACIQAKQAFTFS *0105 48 18
658 672 PLYACIQAKQAFTFS *0107 48 18
658 672 PLYACIQAKQAFTFS * 1301 40 18
659 673 LYACIQAKQAFTFSP 820 * 1301 39 18
660 674 YACIQAKQAFTFSPT 821 * 1301 37 18
661 675 ACIQAKQAFTFSPTY 822 * 1301 36 18
662 676 CIQAKQAFTFSPTYK 823 * 1301 34 18
663 677 IQAKQAFTFSPTYKA 824 *0701 6 18
663 677 IQAKQAFTFSPTYKA * 1301 33 18
664 678 QAKQAFTFSPTYKAF 825 *0401 44 18
664 678 QAKQAFTFSPTYKAF *0701 37 18
665 679 AKQAFTFSPTYKAFL 826 *0401 79 18
665 679 AKQAFTFSPTYKAFL *0701 77 18
666 680 KQAFTFSPTYKAFLS 827 *0401 33 18
666 680 KQAFTFSPTYKAFLS *0701 60 18
666 680 KQAFTFSPTYKAFLS * 1101 13 18
666 680 KQAFTFSPTYKAFLS * 1501 8 18
667 681 QAFTFSPTYKAFLSK 828 *0401 54 18
667 681 QAFTFSPTYKAFLSK *0701 46 18
668 682 AFTFSPTYKAFLSKQ 829 *0701 14 18
669 683 FTFSPTYKAFLSKQY 830 *0701 19 18
672 686 SPTYKAFLSKQYMNL 831 * 1501 3 18
673 687 PTYKAFLSKQYMNLY 832 * 1501 23 18
677 691 AFLSKQYMNLYPVAR 833 *0101 40 18
Cumulative Class II-B score: 1365 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
657 671 MPLYACIQAKQAFTF 818 *0101 75 19 657 691
657 671 MPLYACIQAKQAFTF *0105 75 19
657 671 MPLYACIQAKQAFTF *0107 75 19
657 671 MPLYACIQAKQAFTF * 1301 41 19
658 672 PLYACIQAKQAFTFS 819 *0101 48 19
658 672 PLYACIQAKQAFTFS *0105 48 19
658 672 PLYACIQAKQAFTFS *0107 48 19
658 672 PLYACIQAKQAFTFS * 1301 40 19
659 673 LYACIQAKQAFTFSP 820 * 1301 39 19
660 674 YACIQAKQAFTFSPT 821 * 1301 37 19
661 675 ACIQAKQAFTFSPTY 822 * 1301 36 19
662 676 CIQAKQAFTFSPTYK 823 * 1301 34 19
663 677 IQAKQAFTFSPTYKA 824 *0701 6 19
663 677 IQAKQAFTFSPTYKA * 1301 33 19
664 678 QAKQAFTFSPTYKAF 825 *0401 44 19
664 678 QAKQAFTFSPTYKAF *0701 37 19
665 679 AKQAFTFSPTYKAFL 826 *0401 79 19
665 679 AKQAFTFSPTYKAFL *0701 77 19
666 680 KQAFTFSPTYKAFLS 827 *0401 33 19
666 680 KQAFTFSPTYKAFLS *0701 60 19
666 680 KQAFTFSPTYKAFLS * 1101 13 19
666 680 KQAFTFSPTYKAFLS * 1501 8 19
667 681 QAFTFSPTYKAFLSK 828 *0401 54 19
667 681 QAFTFSPTYKAFLSK *0701 46 19
668 682 AFTFSPTYKAFLSKQ 829 *0701 14 19
669 683 FTFSPTYKAFLSKQY 830 *0701 19 19
672 686 SPTYKAFLSKQYMNL 831 * 1501 3 19
673 687 PTYKAFLSKQYMNLY 832 * 1501 23 19
677 691 AFLSKQYMNLYPVAR 833 *0101 40 19
Cumulative Class II-B score: 1181
717 731 MRGTFVAPLPIHTAE 834 *0101 10 20 715 746
717 731 MRGTFVAPLPIHTAE *0105 10 20
717 731 MRGTFVAPLPIHTAE *0107 10 20
718 732 RGTFVAPLPIHTAEL 835 *0101 4 20
718 732 RGTFVAPLPIHTAEL *0102 20 20
718 732 RGTFVAPLPIHTAEL *0104 30 20
718 732 RGTFVAPLPIHTAEL *0105 60 20
718 732 RGTFVAPLPIHTAEL *0106 33 20
718 732 RGTFVAPLPIHTAEL *0107 60 20
719 733 GTFVAPLPIHTAELL 836 *0101 6 20
732 746 LLAACFARSRSGAKL 837 *0701 1 20
Cumulative Class II-B score: 244
767 781 GCTANWILRGTSFVY 838 * 1501 1 21 754 791
768 782 CTANWILRGTSFVYV 839 * 1301 21 21 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRB1 Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
768 782 CTANWI LRGTS FVYV * 1501 10 21
773 787 I L RGT S FVYVP SALN 840 *0101 100 21
773 787 I L RGT S FVYVP SALN *0401 23 21
773 787 I L RGT S FVYVP SALN *0701 31 21
774 788 L RGT S FVYVP SALNP 841 *0101 96 21
774 788 L RGT S FVYVP SALNP *0105 40 21
774 788 L RGT S FVYVP SALNP *0107 40 21
774 788 L RGT S FVYVP SALNP *0401 56 21
774 788 L RGT S FVYVP SALNP *0701 23 21
775 789 RGT S FVYVP SALN PA 842 *0101 99 21
775 789 RGT S FVYVP SALN PA *0102 30 21
775 789 RGT S FVYVP SALN PA *0104 43 21
775 789 RGT S FVYVP SALN PA *0105 93 21
775 789 RGT S FVYVP SALN PA *0107 93 21
775 789 RGT S FVYVP SALN PA *0401 76 21
775 789 RGT S FVYVP SALN PA *0701 29 21
776 790 GTS FVYVP SALN PAD 843 *0101 97 21
776 790 GTS FVYVP SALN PAD *0102 13 21
776 790 GTS FVYVP SALN PAD *0104 28 21
776 790 GTS FVYVP SALN PAD *0105 85 21
776 790 GTS FVYVP SALN PAD *0107 85 21
776 790 GTS FVYVP SALN PAD *0401 74 21
776 790 GTS FVYVP SALN PAD *0701 4 21
777 791 T S FVYVP SALN PAD D 844 *0101 94 21
777 791 T S FVYVP SALN PAD D *0105 68 21
777 791 T S FVYVP SALN PAD D *0107 68 21
777 791 T S FVYVP SALN PAD D *0401 73 21
Cumulative Class II-B score: 1591
767 781 GCTANWI LRGTS FVY 838 * 1501 1 22 757 792
768 782 CTANWI LRGTS FVYV 839 * 1301 21 22
768 782 CTANWI LRGTS FVYV * 1501 10 22
773 787 I L RGT S FVYVP SALN 840 *0101 100 22
773 787 I L RGT S FVYVP SALN *0401 23 22
773 787 I L RGT S FVYVP SALN *0701 31 22
774 788 L RGT S FVYVP SALNP 841 *0101 96 22
774 788 L RGT S FVYVP SALNP *0105 40 22
774 788 L RGT S FVYVP SALNP *0107 40 22
774 788 L RGT S FVYVP SALNP *0401 56 22
774 788 L RGT S FVYVP SALNP *0701 23 22
775 789 RGT S FVYVP SALN PA 842 *0101 99 22
775 789 RGT S FVYVP SALN PA *0102 30 22
775 789 RGT S FVYVP SALN PA *0104 43 22
775 789 RGT S FVYVP SALN PA *0105 93 22
775 789 RGT S FVYVP SALN PA *0107 93 22
775 789 RGT S FVYVP SALN PA *0401 76 22
775 789 RGT S FVYVP SALN PA *0701 29 22 HLA < :lass II binding peptides in SLP sequences derived from Peptide of invention (SLP) HBV ] Olymerase
Start End Sequence A SEQ HLA-DRBl Class SLP# SLP Start SLP End
ID molecule II-B
NO: scoreB
776 790 GTS FVYVP SALN PAD 843 *0101 97 22
776 790 GTS FVYVP SALN PAD *0102 13 22
776 790 GTS FVYVP SALN PAD *0104 28 22
776 790 GTS FVYVP SALN PAD *0105 85 22
776 790 GTS FVYVP SALN PAD *0107 85 22
776 790 GTS FVYVP SALN PAD *0401 74 22
776 790 GTS FVYVP SALN PAD *0701 4 22
777 791 T S FVYVP SALN PAD D 844 *0101 94 22
777 791 T S FVYVP SALN PAD D *0105 68 22
777 791 T S FVYVP SALN PAD D *0107 68 22
777 791 T S FVYVP SALN PAD D *0401 73 22
778 792 S FVYVP SALN PAD D P 845 *0101 54 22
778 792 S FVYVP SALN PAD D P *0401 64 22
Cumulative Class II-B score: 1709
767 781 GCTANWI LRGTS FVY 838 * 1501 1 23 754 789
768 782 CTANWI LRGTS FVYV 839 * 1301 21 23
768 782 CTANWI LRGTS FVYV * 1501 10 23
773 787 I L RGT S FVYVP SALN 840 *0101 100 23
773 787 I L RGT S FVYVP SALN *0401 23 23
773 787 I L RGT S FVYVP SALN *0701 31 23
774 788 L RGT S FVYVP SALNP 841 *0101 96 23
774 788 L RGT S FVYVP SALNP *0105 40 23
774 788 L RGT S FVYVP SALNP *0107 40 23
774 788 L RGT S FVYVP SALNP *0401 56 23
774 788 L RGT S FVYVP SALNP *0701 23 23
775 789 RGT S FVYVP SALN PA 842 *0101 99 23
775 789 RGT S FVYVP SALN PA *0102 30 23
775 789 RGT S FVYVP SALN PA *0104 43 23
775 789 RGT S FVYVP SALN PA *0105 93 23
775 789 RGT S FVYVP SALN PA *0107 93 23
775 789 RGT S FVYVP SALN PA *0401 76 23
775 789 RGT S FVYVP SALN PA *0701 29 23
Cumulative Class II-B score: 903
"Start" and "End" are relative to the amino acid sequence of human HBV polymerase as depicted in SEQ ID NO: 1
A Peptide amino acid sequence. Each HLA-DRB l binding peptide of HBV polymerase is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
Class II-B score. See Material and Methods (Examples section). Cumulative Class II-B score. See Material and Methods (Examples section). Table 5a. Predicted HLA class I-restricted CD8+ cytotoxic T cell epitopes contained in SLP sequences from HBV Core protein.
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
"Start" and "End" are relative to the amino acid sequence of HBV core protein as depicted in SEQ ID NO: 4
A Peptide amino acid sequence. Each HLA class I binding peptide of HBV core protein is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
B Class I-B score. See Material and Methods of Examples section.
c C-score. See Material and Methods of Examples section.
D Class I-BCI score. See Material and Methods (Examples section).
E Cumulative Class I-BCI score. See Material and Methods (Examples section).
Table 5b. Predicted HLA class Il-restricted CD4+ T cell epitopes contained sequences from HBV Core protein.
HLA class II binding peptides in SLP sequences derived from Peptide of invention HBV Core protein (SLP)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
107 121 DPAS RDLWN YVNTN 924 *0301 46 24 107 141
108 122 PAS RDLW YV TNV 925 *0401 69 24
108 122 PAS RDLW YV TNV *1301 10 24
109 123 AS RDLWN Y TNVG 926 *0401 85 24
109 123 AS RDLWN Y TNVG *1301 20 24
110 124 SRDLWNYV TNVGL 927 *0401 92 24
110 124 SRDLWNYV TNVGL *1301 40 24
111 125 RDLWN Y TNVGLK 928 *0103 17 24
111 125 RDLWN Y TNVGLK *0401 100 24
111 125 RDLWN Y TNVGLK *1301 50 24
112 126 DLWNYVNTNVGLKI 929 *0103 50 24
112 126 DLWNYVNTNVGLKI *0106 25 24
112 126 DLWNYVNTNVGLKI *0301 62 24
112 126 DLWNYVNTNVGLKI *0401 77 24
112 126 DLWNYVNTNVGLKI *0701 15 24
112 126 DLWNYVNTNVGLKI *1301 80 24
113 127 LWNYVNTNVGLKI R 930 *0103 100 24
113 127 LWNYVNTNVGLKI R *0104 35 24
113 127 LWNYVNTNVGLKI R *0106 75 24
113 127 LWNYVNTNVGLKI R *0301 69 24
113 127 LWNYVNTNVGLKI R *0701 50 24
113 127 LWNYVNTNVGLKI R *1301 100 24
114 128 WNYVNTNVGLKI RQ 931 *0103 83 24
114 128 WNYVNTNVGLKI RQ *0104 45 24
114 128 WNYVNTNVGLKI RQ *0106 80 24
114 128 WNYVNTNVGLKI RQ *1301 90 24
115 129 VNYVNTNVGLKI RQL 932 *0101 21 24
115 129 VNYVNTNVGLKI RQL *0102 10 24
115 129 VNYVNTNVGLKI RQL *0103 67 24
115 129 VNYVNTNVGLKI RQL *0104 65 24
115 129 VNYVNTNVGLKI RQL *0105 19 24
115 129 VNYVNTNVGLKI RQL *0106 85 24
115 129 VNYVNTNVGLKI RQL *0107 21 24
115 129 VNYVNTNVGLKI RQL *0701 33 24
115 129 VNYVNTNVGLKI RQL *1301 70 24
116 130 NYVNTNVGLKI RQLL 933 *0103 33 24
116 130 NYVNTNVGLKI RQLL *0104 25 24
116 130 NYVNTNVGLKI RQLL *0106 65 24
116 130 NYVNTNVGLKI RQLL *1301 60 24
117 131 YVNTNVGLKI RQLLW 934 *1101 54 24
117 131 YVNTNVGLKI RQLLW *1301 30 24
118 132 VNTNVGLKI RQLLWF 935 *0301 38 24
118 132 VNTNVGLKI RQLLWF *1301 46 24
119 133 NTNVGLKI RQLLWFH 936 *0301 31 24 HLA class II binding peptides in SLP sequences derived from Peptide of invention HBV Core protein (SLP)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
119 133 NTNVGLKIRQLLWFH *1301 38 24
120 134 TNVGLKIRQLLWFHI 937 *0102 70 24
120 134 TNVGLKIRQLLWFHI *0104 55 24
120 134 TNVGLKIRQLLWFHI *0106 40 24
120 134 TNVGLKIRQLLWFHI *0301 23 24
120 134 TNVGLKIRQLLWFHI *0701 38 24
120 134 TNVGLKIRQLLWFHI *1301 31 24
120 134 TNVGLKIRQLLWFHI *1501 38 24
121 135 NVGLKIRQLLWFHIS 938 *0101 11 24
121 135 NVGLKIRQLLWFHIS *0102 85 24
121 135 NVGLKIRQLLWFHIS *0104 75 24
121 135 NVGLKIRQLLWFHIS *0105 10 24
121 135 NVGLKIRQLLWFHIS *0106 50 24
121 135 NVGLKIRQLLWFHIS *0107 11 24
121 135 NVGLKIRQLLWFHIS *0301 15 24
121 135 NVGLKIRQLLWFHIS *0701 23 24
121 135 NVGLKIRQLLWFHIS *1301 23 24
121 135 NVGLKIRQLLWFHIS *1501 85 24
122 136 VGLKIRQLLWFHISC 939 *0102 65 24
122 136 VGLKIRQLLWFHISC *0104 50 24
122 136 VGLKIRQLLWFHISC *0106 20 24
122 136 VGLKIRQLLWFHISC *0301 8 24
122 136 VGLKIRQLLWFHISC *1301 15 24
122 136 VGLKIRQLLWFHISC *1501 46 24
123 137 GLKIRQLLWFHISCL 940 *0101 31 24
123 137 GLKIRQLLWFHISCL *0102 75 24
123 137 GLKIRQLLWFHISCL *0104 80 24
123 137 GLKIRQLLWFHISCL *0106 55 24
123 137 GLKIRQLLWFHISCL *0701 100 24
123 137 GLKIRQLLWFHISCL *1301 8 24
123 137 GLKIRQLLWFHISCL *1501 100 24
124 138 LKIRQLLWFHISCLT 941 *0101 26 24
124 138 LKIRQLLWFHISCLT *0102 80 24
124 138 LKIRQLLWFHISCLT *0104 70 24
124 138 LKIRQLLWFHISCLT *0105 24 24
124 138 LKIRQLLWFHISCLT *0106 60 24
124 138 LKIRQLLWFHISCLT *0107 26 24
124 138 LKIRQLLWFHISCLT *0701 92 24
124 138 LKIRQLLWFHISCLT *1501 92 24
125 139 KIRQLLWFHISCLTF 942 *0101 5 24
125 139 KIRQLLWFHISCLTF *0102 50 24
125 139 KIRQLLWFHISCLTF *0104 40 24
125 139 KIRQLLWFHISCLTF *0105 5 24
125 139 KIRQLLWFHISCLTF *0106 35 24
125 139 KIRQLLWFHISCLTF *0107 5 24
125 139 KIRQLLWFHISCLTF *0701 85 24
125 139 KIRQLLWFHISCLTF *1501 31 24 HLA class II binding peptides in SLP sequences derived from Peptide of invention
HBV Core protein (SLP)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
126 140 IRQLLWFHISCLTFG 943 *0102 40 24
126 140 IRQLLWFHISCLTFG *0701 46 24
126 140 IRQLLWFHISCLTFG *1101 69 24
127 141 RQLLWFHISCLTFGR 944 *0701 8 24
127 141 RQLLWFHISCLTFGR *1101 46 24
Cumulative Class II-B scorec: 4649
139 153 FGRETVLEYLVSFGV 945 *0101 42 25 136 169
139 153 FGRETVLEYLVSFGV *0102 35 25
139 153 FGRETVLEYLVSFGV *0105 38 25
139 153 FGRETVLEYLVSFGV *0107 42 25
139 153 FGRETVLEYLVSFGV *0701 69 25
139 153 FGRETVLEYLVSFGV *1501 23 25
140 154 GRETVLEYLVSFGVW 946 *0101 46 25
140 154 GRETVLEYLVSFGVW *0102 55 25
140 154 GRETVLEYLVSFGVW *0104 60 25
140 154 GRETVLEYLVSFGVW *0105 67 25
140 154 GRETVLEYLVSFGVW *0106 45 25
140 154 GRETVLEYLVSFGVW *0107 74 25
140 154 GRETVLEYLVSFGVW *0701 62 25
140 154 GRETVLEYLVSFGVW *1501 15 25
141 155 RETVLEYLVSFGVWI 947 *0101 92 25
141 155 RETVLEYLVSFGVWI *0102 95 25
141 155 RETVLEYLVSFGVWI *0104 90 25
141 155 RETVLEYLVSFGVWI *0105 81 25
141 155 RETVLEYLVSFGVWI *0106 90 25
141 155 RETVLEYLVSFGVWI *0107 89 25
141 155 RETVLEYLVSFGVWI *0701 77 25
141 155 RETVLEYLVSFGVWI *1501 8 25
142 156 ETVLEYLVSFGVWIR 948 *0101 100 25
142 156 ETVLEYLVSFGVWIR *0102 100 25
142 156 ETVLEYLVSFGVWIR *0104 100 25
142 156 ETVLEYLVSFGVWIR *0105 90 25
142 156 ETVLEYLVSFGVWIR *0106 100 25
142 156 ETVLEYLVSFGVWIR *0107 100 25
142 156 ETVLEYLVSFGVWIR *0701 54 25
142 156 ETVLEYLVSFGVWIR *1301 100 25
142 156 ETVLEYLVSFGVWIR *1501 77 25
143 157 TVLEYLVSFGVWIRT 949 *0101 85 25
143 157 TVLEYLVSFGVWIRT *0102 90 25
143 157 TVLEYLVSFGVWIRT *0104 95 25
143 157 TVLEYLVSFGVWIRT *0105 86 25
143 157 TVLEYLVSFGVWIRT *0106 95 25
143 157 TVLEYLVSFGVWIRT *0107 95 25
143 157 TVLEYLVSFGVWIRT *0701 31 25
143 157 TVLEYLVSFGVWIRT *1301 92 25
143 157 TVLEYLVSFGVWIRT *1501 69 25 HLA class II binding peptides in SLP sequences derived from Peptide of invention HBV Core protein (SLP)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
144 158 VLEYLVSFGVWIRTP 950 *0101 69 25
144 158 VLEYLVSFGVWIRTP *0102 60 25
144 158 VLEYLVSFGVWIRTP *0104 85 25
144 158 VLEYLVSFGVWIRTP *0105 76 25
144 158 VLEYLVSFGVWIRTP *0106 70 25
144 158 VLEYLVSFGVWIRTP *0107 84 25
144 158 VLEYLVSFGVWIRTP *1301 85 25
144 158 VLEYLVSFGVWIRTP *1501 62 25
145 159 LEYLVSFGVWIRTPP 951 *0101 62 25
145 159 LEYLVSFGVWIRTPP *0102 15 25
145 159 LEYLVSFGVWIRTPP *0104 20 25
145 159 LEYLVSFGVWIRTPP *0105 48 25
145 159 LEYLVSFGVWIRTPP *0107 53 25
145 159 LEYLVSFGVWIRTPP *1301 77 25
145 159 LEYLVSFGVWIRTPP *1501 54 25
146 160 EYLVSFGVWIRTPPA 952 *1301 69 25
147 161 YLVSFGVWIRTPPAY 953 *1301 62 25
148 162 LVSFGVWIRTPPAYR 954 *0101 32 25
148 162 LVSFGVWIRTPPAYR *0105 29 25
148 162 LVSFGVWIRTPPAYR *0107 32 25
148 162 LVSFGVWIRTPPAYR *0401 40 25
148 162 LVSFGVWIRTPPAYR *1301 54 25
149 163 VSFGVWIRTPPAYRP 955 *0101 63 25
149 163 VSFGVWIRTPPAYRP *0102 25 25
149 163 VSFGVWIRTPPAYRP *0105 57 25
149 163 VSFGVWIRTPPAYRP *0107 63 25
149 163 VSFGVWIRTPPAYRP *0401 60 25
149 163 VSFGVWIRTPPAYRP *1101 25 25
150 164 SFGVWIRTPPAYRPP 956 *0101 68 25
150 164 SFGVWIRTPPAYRPP *0102 30 25
150 164 SFGVWIRTPPAYRPP *0105 62 25
150 164 SFGVWIRTPPAYRPP *0107 68 25
150 164 SFGVWIRTPPAYRPP *0401 80 25
150 164 SFGVWIRTPPAYRPP *1101 75 25
151 165 FGVWIRTPPAYRPPN 957 *0101 79 25
151 165 FGVWIRTPPAYRPPN *0102 45 25
151 165 FGVWIRTPPAYRPPN *0104 30 25
151 165 FGVWIRTPPAYRPPN *0105 71 25
151 165 FGVWIRTPPAYRPPN *0106 5 25
151 165 FGVWIRTPPAYRPPN *0107 79 25
151 165 FGVWIRTPPAYRPPN *0401 100 25
151 165 FGVWIRTPPAYRPPN *1101 100 25
152 166 GVWIRTPPAYRPPNA 958 *0101 58 25
152 166 GVWIRTPPAYRPPNA *0105 52 25
152 166 GVWIRTPPAYRPPNA *0107 58 25
152 166 GVWIRTPPAYRPPNA *1101 50 25
Cumulative Class II-B score: 5493 "Start" and "End" are relative to the amino acid sequence of human HBV core protein as depicted in SEQ ID NO: 4
A Peptide amino acid sequence. Each HLA-DRB l binding peptide of HBV core is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
B Class II-B score. See Material and Methods (Examples section).
Cumulative Class II-B score. See Material and Methods (Examples section).
Table 6a. Predicted HLA class I-restricted CD8+ cytotoxic T cell epitopes contained in SLP sequences from HBV X protein (consensus sequence).
HLA c ass I binding peptides in SLP sequences derived from HBV X protein (consensus sequence) Peptide of invention (SLP)
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
HLA c ass I bindin e tides in SLP se uences derived from HBV X rotein consensus se uence Pe tide of invention SLP
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
"Start" and "End" are relative to the amino acid sequence of HBV X protein, consensus sequence, as depicted in SEQ ID NO: 45
A Peptide amino acid sequence. Each HLA class I binding peptide of HBV X protein is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
B Class I-B score. See Material and Methods (Examples section).
c C-score. See Material and Methods (Examples section).2' Class I-BCI score. See Material and Methods (Examples section).
E Cumulative Class I-BCI score. See Material and Methods (Examples section).
Table 6b. Predicted HLA class II-restricted CD4+ T cell epitopes contained sequences from HBV X protein (consensus sequence).
HLA class II binding peptides in SLP derived from HBV X protein Peptide of invention (SLP) (consens. seq.)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
46 60 PADHGAHLSLRGLPV 1091 *0701 8 26 36 68
47 61 ADHGAHLSLRGLPVC 1092 *0101 13 26
47 61 ADHGAHLSLRGLPVC *0102 20 26
47 61 ADHGAHLSLRGLPVC *0105 13 26
47 61 ADHGAHLSLRGLPVC *0107 13 26
48 62 DHGAHLSLRGLPVCA 1093 *0101 87 26
48 62 DHGAHLSLRGLPVCA *0102 67 26
48 62 DHGAHLSLRGLPVCA *0104 43 26
48 62 DHGAHLSLRGLPVCA *0105 87 26
48 62 DHGAHLSLRGLPVCA *0106 53 26
48 62 DHGAHLSLRGLPVCA *0107 87 26
49 63 HGAHLSLRGLPVCAF 1094 *0101 100 26
49 63 HGAHLSLRGLPVCAF *0102 93 26
49 63 HGAHLSLRGLPVCAF *0103 40 26
49 63 HGAHLSLRGLPVCAF *0104 90 26
49 63 HGAHLSLRGLPVCAF *0105 90 26
49 63 HGAHLSLRGLPVCAF *0106 90 26
49 63 HGAHLSLRGLPVCAF *0107 90 26
49 63 HGAHLSLRGLPVCAF *1101 85 26
49 63 HGAHLSLRGLPVCAF *1301 100 26
49 63 HGAHLSLRGLPVCAF *1501 46 26
50 64 GAHLSLRGLPVCAFS 1095 *0101 77 26
50 64 GAHLSLRGLPVCAFS *0102 97 26
50 64 GAHLSLRGLPVCAFS *0103 70 26
50 64 GAHLSLRGLPVCAFS *0104 93 26
50 64 GAHLSLRGLPVCAFS *0105 93 26
50 64 GAHLSLRGLPVCAFS *0106 97 26
50 64 GAHLSLRGLPVCAFS *0107 93 26
50 64 GAHLSLRGLPVCAFS *1101 77 26
50 64 GAHLSLRGLPVCAFS *1301 92 26
50 64 GAHLSLRGLPVCAFS *1501 54 26
51 65 AHLSLRGLPVCAFS S 1096 *0101 100 26
51 65 AHLSLRGLPVCAFS S *0102 100 26
51 65 AHLSLRGLPVCAFS S *0103 100 26
51 65 AHLSLRGLPVCAFS S *0104 100 26
51 65 AHLSLRGLPVCAFS S *0105 100 26
51 65 AHLSLRGLPVCAFS S *0106 100 26
51 65 AHLSLRGLPVCAFS S *0107 100 26
51 65 AHLSLRGLPVCAFS S *1301 85 26
52 66 HLSLRGLPVCAFS SA 1097 *0101 97 26
52 66 HLSLRGLPVCAFS SA *0102 90 26
52 66 HLSLRGLPVCAFS SA *0103 90 26
52 66 HLSLRGLPVCAFS SA *0104 97 26
52 66 HLSLRGLPVCAFS SA *0105 97 26 HLA class II binding peptides in SLP derived from HBV X protein Peptide of invention (SLP)
(consens. seq.)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
52 66 HLSLRGLPVCAFS SA *0106 93 26
52 66 HLSLRGLPVCAFS SA *0107 97 26
52 66 HLSLRGLPVCAFS SA *1301 77 26
53 67 LSLRGLPVCAFS SAG 1098 *0101 83 26
53 67 LSLRGLPVCAFS SAG *0102 50 26
53 67 LSLRGLPVCAFS SAG *0104 77 26
53 67 LSLRGLPVCAFS SAG *0105 83 26
53 67 LSLRGLPVCAFS SAG *0106 73 26
53 67 LSLRGLPVCAFS SAG *0107 83 26
53 67 LSLRGLPVCAFS SAG *1301 69 26
54 68 SLRGLPVCAFS SAGP 1099 *0101 37 26
54 68 SLRGLPVCAFS SAGP *0102 7 26
54 68 SLRGLPVCAFS SAGP *0104 30 26
54 68 SLRGLPVCAFS SAGP *0105 37 26
54 68 SLRGLPVCAFS SAGP *0106 7 26
54 68 SLRGLPVCAFS SAGP *0107 37 26
54 68 SLRGLPVCAFS SAGP *1301 62 26
Cumulative Class II-B scorec: 4414
61 75 CAFS SAGPCALRFTS 1100 *0101 20 27 61 95
61 75 CAFS SAGPCALRFTS *0105 20 27
61 75 CAFS SAGPCALRFTS *0107 20 27
61 75 CAFS SAGPCALRFTS *0701 15 27
64 78 S SAGPCALRFTSARR 1101 *1501 69 27
65 79 SAGPCALRFTSARRM 1102 *0101 23 27
65 79 SAGPCALRFTSARRM *0102 23 27
65 79 SAGPCALRFTSARRM *0104 27 27
65 79 SAGPCALRFTSARRM *0106 23 27
65 79 SAGPCALRFTSARRM *0401 31 27
65 79 SAGPCALRFTSARRM *0701 85 27
65 79 SAGPCALRFTSARRM *1301 46 27
65 79 SAGPCALRFTSARRM *1501 92 27
66 80 AGPCALRFTSARRME 1103 *0101 69 27
66 80 AGPCALRFTSARRME *0102 37 27
66 80 AGPCALRFTSARRME *0104 40 27
66 80 AGPCALRFTSARRME *0105 3 27
66 80 AGPCALRFTSARRME *0106 37 27
66 80 AGPCALRFTSARRME *0107 3 27
66 80 AGPCALRFTSARRME *0401 46 27
66 80 AGPCALRFTSARRME *0701 69 27
66 80 AGPCALRFTSARRME *1301 38 27
66 80 AGPCALRFTSARRME *1501 85 27
67 81 GPCALRFTSARRMET 1104 *0101 62 27
67 81 GPCALRFTSARRMET *0102 57 27
67 81 GPCALRFTSARRMET *0104 70 27
67 81 GPCALRFTSARRMET *0105 43 27
67 81 GPCALRFTSARRMET *0106 80 27 HLA class II binding peptides in SLP derived from HBV X protein Peptide of invention (SLP) (consens. seq.)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
67 81 GPCALRFTSARRMET *0107 43 27
67 81 GPCALRFTSARRMET *0301 46 27
67 81 GPCALRFTSARRMET *0401 92 27
67 81 GPCALRFTSARRMET *0701 77 27
67 81 GPCALRFTSARRMET *1101 8 27
67 81 GPCALRFTSARRMET *1301 31 27
67 81 GPCALRFTSARRMET *1501 100 27
68 82 PCALRFTSARRMETT 1105 *0101 54 27
68 82 PCALRFTSARRMETT *0102 60 27
68 82 PCALRFTSARRMETT *0104 83 27
68 82 PCALRFTSARRMETT *0105 57 27
68 82 PCALRFTSARRMETT *0106 83 27
68 82 PCALRFTSARRMETT *0107 57 27
68 82 PCALRFTSARRMETT *0301 38 27
68 82 PCALRFTSARRMETT *0401 69 27
68 82 PCALRFTSARRMETT *0701 100 27
68 82 PCALRFTSARRMETT *1101 31 27
68 82 PCALRFTSARRMETT *1301 23 27
68 82 PCALRFTSARRMETT *1501 77 27
69 83 CALRFTSARRMETTV 1106 *0101 46 27
69 83 CALRFTSARRMETTV *0102 43 27
69 83 CALRFTSARRMETTV *0104 67 27
69 83 CALRFTSARRMETTV *0105 47 27
69 83 CALRFTSARRMETTV *0106 57 27
69 83 CALRFTSARRMETTV *0107 47 27
69 83 CALRFTSARRMETTV *0301 31 27
69 83 CALRFTSARRMETTV *0401 23 27
69 83 CALRFTSARRMETTV *0701 92 27
69 83 CALRFTSARRMETTV *1101 38 27
69 83 CALRFTSARRMETTV *1301 15 27
69 83 CALRFTSARRMETTV *1501 62 27
70 84 ALRFT SARRMETT 1107 *0101 38 27
70 84 ALRFT SARRMETTW *0102 17 27
70 84 ALRFT SARRMETTW *0104 37 27
70 84 ALRFT SARRMETTW *0106 3 27
70 84 ALRFT SARRMETTW *0301 62 27
70 84 ALRFT SARRMETTW *0401 8 27
70 84 ALRFT SARRMETTW *0701 62 27
70 84 ALRFT SARRMETTW *1101 46 27
70 84 ALRFT SARRMETTW *1301 8 27
70 84 ALRFT SARRMETTW *1501 23 27
71 85 LRFTSARRMETTWA 1108 *0301 23 27
71 85 LRFTSARRMETTWA *0401 15 27
71 85 LRFTSARRMETTWA *0701 54 27
71 85 LRFTSARRMETTWA *1101 23 27
73 87 FTSARRMETTWAHQ 1109 *0401 85 27
74 88 TSARRMETTWAHQI 1110 *0401 100 27 HLA class II binding peptides in SLP derived from HBV X protein Peptide of invention (SLP)
(consens. seq.)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
75 89 SARRMETT AHQI L 1111 *0401 38 27
75 89 SARRMETT AHQI L *1301 40 27
76 90 ARRMETT AHQI LP 1112 *0401 80 27
76 90 ARRMETT AHQI LP *1301 10 27
77 91 RRMETT AHQI LPK 1113 *1301 50 27
78 92 RMETT AHQI LPKV 1114 *1301 20 27
81 95 TT AHQI LPKVLHK 1115 *1101 15 27
Cumulative Class II-B score: 3788
86 100 HQI LPKVLHKRTLGL 1116 *1101 54 28 86 120
87 101 QI LPKVLHKRTLGLS 1117 *0102 10 28
87 101 QI LPKVLHKRTLGLS *1101 22 28
87 101 QI LPKVLHKRTLGLS *1301 70 28
88 102 I LPKVLHKRTLGLSA 1118 *0102 53 28
88 102 I LPKVLHKRTLGLSA *0104 17 28
88 102 I LPKVLHKRTLGLSA *0106 33 28
88 102 I LPKVLHKRTLGLSA *1101 44 28
88 102 I LPKVLHKRTLGLSA *1301 80 28
89 103 LPKVLHKRTLGLSAM 1119 *0101 10 28
89 103 LPKVLHKRTLGLSAM *0102 77 28
89 103 LPKVLHKRTLGLSAM *0104 60 28
89 103 LPKVLHKRTLGLSAM *0105 10 28
89 103 LPKVLHKRTLGLSAM *0106 77 28
89 103 LPKVLHKRTLGLSAM *0107 10 28
89 103 LPKVLHKRTLGLSAM *0701 10 28
89 103 LPKVLHKRTLGLSAM *1101 78 28
89 103 LPKVLHKRTLGLSAM *1301 100 28
89 103 LPKVLHKRTLGLSAM *1501 40 28
90 104 PKVLHKRTLGLSAMS 1120 *0101 33 28
90 104 PKVLHKRTLGLSAMS *0102 73 28
90 104 PKVLHKRTLGLSAMS *0104 63 28
90 104 PKVLHKRTLGLSAMS *0105 33 28
90 104 PKVLHKRTLGLSAMS *0106 67 28
90 104 PKVLHKRTLGLSAMS *0107 33 28
90 104 PKVLHKRTLGLSAMS *1101 72 28
90 104 PKVLHKRTLGLSAMS *1301 90 28
91 105 KVLHKRTLGLSAMST 1121 *0101 63 28
91 105 KVLHKRTLGLSAMST *0102 70 28
91 105 KVLHKRTLGLSAMST *0104 50 28
91 105 KVLHKRTLGLSAMST *0105 63 28
91 105 KVLHKRTLGLSAMST *0106 60 28
91 105 KVLHKRTLGLSAMST *0107 63 28
91 105 KVLHKRTLGLSAMST *1101 11 28
91 105 KVLHKRTLGLSAMST *1301 60 28
92 106 VLHKRTLGLSAMSTT 1122 *0101 15 28
92 106 VLHKRTLGLSAMSTT *0102 47 28
92 106 VLHKRTLGLSAMSTT *0104 23 28 HLA class II binding peptides in SLP derived from HBV X protein Peptide of invention (SLP) (consens. seq.)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRB1 II-B Start End molecule score3
92 106 VLHKRTLGLSAMSTT *0105 73 28
92 106 VLHKRTLGLSAMSTT *0106 43 28
92 106 VLHKRTLGLSAMSTT *0107 73 28
93 107 LHKRTLGLSAMSTTD 1123 *0101 60 28
93 107 LHKRTLGLSAMSTTD *0102 27 28
93 107 LHKRTLGLSAMSTTD *0105 60 28
93 107 LHKRTLGLSAMSTTD *0107 60 28
94 108 HKRTLGLSAMSTTDL 1124 *0101 92 28
94 108 HKRTLGLSAMSTTDL *0102 33 28
94 108 HKRTLGLSAMSTTDL *0104 3 28
94 108 HKRTLGLSAMSTTDL *0105 67 28
94 108 HKRTLGLSAMSTTDL *0106 40 28
94 108 HKRTLGLSAMSTTDL *0107 67 28
94 108 HKRTLGLSAMSTTDL *0401 77 28
95 109 KRTLGLSAMSTTDLE 1125 *0101 31 28
95 109 KRTLGLSAMSTTDLE *0105 23 28
95 109 KRTLGLSAMSTTDLE *0106 17 28
95 109 KRTLGLSAMSTTDLE *0107 23 28
95 109 KRTLGLSAMSTTDLE *0401 54 28
96 110 RTLGLSAMSTTDLEA 1126 *0101 8 28
96 110 RTLGLSAMSTTDLEA *0105 30 28
96 110 RTLGLSAMSTTDLEA *0106 27 28
96 110 RTLGLSAMSTTDLEA *0107 30 28
96 110 RTLGLSAMSTTDLEA *0401 62 28
97 111 TLGLSAMSTTDLEAY 1127 *0101 17 28
97 111 TLGLSAMSTTDLEAY *0105 17 28
97 111 TLGLSAMSTTDLEAY *0106 20 28
97 111 TLGLSAMSTTDLEAY *0107 17 28
97 111 TLGLSAMSTTDLEAY *0401 50 28
105 119 TTDLEAYFKDCVFKD 1128 *0301 69 28
106 120 TDLEAYFKDCVFKDW 1129 *0301 54 28
Cumulative Class II-B score: 3170
108 122 LEAYFKDCVFKDWEE 1130 *0301 15 29 108 141
110 124 AYFKDCVFKDWEELG 1131 *0301 100 29
111 125 YFKDCVFKDWEELGE 1132 *0301 92 29
119 133 DWEELGEEI RLKVFV 1133 *0301 77 29
120 134 WEELGEEI RLKVFVL 1134 *0301 85 29
120 134 WEELGEEI RLKVFVL *1501 8 29
121 135 EELGEEI RLKVFVLG 1135 *1501 31 29
123 137 LGEEI RLKVFVLGGC 1136 *1501 15 29
124 138 GEEI RLKVFVLGGCR 1137 *1501 38 29
125 139 EEI RLKVFVLGGCRH 1138 *0102 3 29
126 140 EI RLKVFVLGGCRHK 1139 *0102 40 29
126 140 EI RLKVFVLGGCRHK *0104 20 29
126 140 EI RLKVFVLGGCRHK *1501 10 29
127 141 I RLKVFVLGGCRHKL 1140 *0101 40 29 HLA class II binding peptides in SLP derived from HBV X protein Peptide of invention (SLP)
(consens. seq.)
Start End Sequence A SEQ ID HLA- Class SLP# SLP SLP
NO: DRBl II-B Start End
molecule score3
127 141 IRLKVFVLGGCRHKL *0102 80 29
127 141 IRLKVFVLGGCRHKL *0104 73 29
127 141 IRLKVFVLGGCRHKL *0105 40 29
127 141 IRLKVFVLGGCRHKL *0106 70 29
127 141 IRLKVFVLGGCRHKL *0107 40 29
127 141 IRLKVFVLGGCRHKL *1101 56 29
127 141 IRLKVFVLGGCRHKL *1501 90 29
Cumulative Class II-B score: 1024
"Start" and "End" are relative to the amino acid sequence of human HBV X protein as depicted in SEQ ID NO: 45
A Peptide amino acid sequence. Each HLA-DRB l binding peptide of HBV X protein is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
B Class II-B score. See Material and Methods (Examples section).
Cumulative Class II-B score. See Material and Methods (Examples section).
Table 7a. Predicted HLA class I-restricted CD8+ cytotoxic T cell epitopes contained in SLP sequences from HBV large surface protein.
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000200_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000201_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000202_0001
Figure imgf000203_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000204_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000208_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000209_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000210_0001
Figure imgf000211_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000212_0001
Figure imgf000213_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000214_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000215_0001
Figure imgf000216_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
HLA class I binding peptides contained in SLP sequences derived from HBV Surface antigen Peptide of invention (SLP)
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
"Start" and "End" are relative to the amino acid sequence of HBV large surface protein as depicted in SEQ ID NO: 1141
A Peptide amino acid sequence. Each HLA class I binding peptide of HBV large surface protein is listed separately for each HLA class I molecule to which it is predicted to bind, and can be listed multiple times for that reason.
B Class I-B score. See Material and Methods (Examples section).
c C-score. See Material and Methods (Examples section).
D Class I-BCI score. See Material and Methods (Examples section).
E Cumulative Class I-BCI score. See Material and Methods (Examples section).
Table 7b. Predicted HLA class II-restricted CD4+ T cell epitopes contained in
SLP sequences from HBV large surface protein.
Figure imgf000236_0001
EU EU-nummer HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
190 204 QAGFFLLTRILTIPQ *1101 75
190 204 QAGFFLLTRILTIPQ *0101 68
190 204 QAGFFLLTRILTIPQ *0102 63
190 204 QAGFFLLTRILTIPQ *0104 58
190 204 QAGFFLLTRILTIPQ *1501 20
190 204 QAGFFLLTRILTIPQ *0105 8
190 204 QAGFFLLTRILTIPQ *0107 8
191 205 AGFFLLTRILTI PQS 1406 *0401 98
191 205 AGFFLLTRILTI PQS *0701 73
191 205 AGFFLLTRILTI PQS *1101 73
191 205 AGFFLLTRILTI PQS *0102 70
191 205 AGFFLLTRILTI PQS *0104 65
191 205 AGFFLLTRILTI PQS *0101 55
191 205 AGFFLLTRILTI PQS *0106 23
191 205 AGFFLLTRILTI PQS *0105 10
191 205 AGFFLLTRILTI PQS *0107 10
192 206 GFFLLTRILTIPQSL 1407 *0401 95
192 206 GFFLLTRILTIPQSL *1301 85
192 206 GFFLLTRILTIPQSL *0102 83
192 206 GFFLLTRILTIPQSL *0104 80
192 206 GFFLLTRILTIPQSL *0701 70
192 206 GFFLLTRILTIPQSL *1101 70
192 206 GFFLLTRILTIPQSL *0106 50
192 206 GFFLLTRILTIPQSL *0105 28
192 206 GFFLLTRILTIPQSL *0107 28
192 206 GFFLLTRILTIPQSL *0101 18
192 206 GFFLLTRILTIPQSL *1501 10
193 207 FFLLTRILTIPQSLD 1408 *0401 88
193 207 FFLLTRILTIPQSLD *0102 75
193 207 FFLLTRILTIPQSLD *1301 75
193 207 FFLLTRILTIPQSLD *0701 68
193 207 FFLLTRILTIPQSLD *1101 68
193 207 FFLLTRILTIPQSLD *0104 63
193 207 FFLLTRILTIPQSLD *0106 48
193 207 FFLLTRILTIPQSLD *0101 18
193 207 FFLLTRILTIPQSLD *0105 18
193 207 FFLLTRILTIPQSLD *0107 18
194 208 FLLTRILTIPQSLDS 1409 *0401 85
194 208 FLLTRILTIPQSLDS *1301 65
194 208 FLLTRILTIPQSLDS *0102 58
194 208 FLLTRILTIPQSLDS *1101 53
194 208 FLLTRILTIPQSLDS *0106 43
194 208 FLLTRILTIPQSLDS *0104 40
194 208 FLLTRILTIPQSLDS *0701 3
195 209 LLTRILTIPQSLDSW *1301 70
195 209 LLTRILTIPQSLDSW *0102 45
195 209 LLTRILTIPQSLDSW *0106 35
195 209 LLTRILTIPQSLDSW 1410 *0401 15
196 210 LTRILTI PQSLDSWW 1411 *1301 45
4798 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
Cumulative Class II-B score:
240 254 PPICPGYRWMCLRRF 1412 *1301 83 31 239 274
240 254 PPICPGYRWMCLRRF *1101 70
241 255 PICPGYRWMCLRRFI 1413 *1301 80
241 255 PICPGYRWMCLRRFI *1101 80
241 255 PICPGYRWMCLRRFI *1501 60
241 255 PICPGYRWMCLRRFI *0701 35
242 256 ICPGYRWMCLRRFII 1414 *1301 78
242 256 ICPGYRWMCLRRFII *1501 95
242 256 ICPGYRWMCLRRFII *1101 90
242 256 ICPGYRWMCLRRFII *0701 85
242 256 ICPGYRWMCLRRFII *0104 70
242 256 ICPGYRWMCLRRFII *0102 68
242 256 ICPGYRWMCLRRFII *0101 50
242 256 ICPGYRWMCLRRFII *0105 50
242 256 ICPGYRWMCLRRFII *0107 50
242 256 ICPGYRWMCLRRFII *0301 35
243 257 CPGYRWMCLRRFIIF 1415 *1301 75
243 257 CPGYRWMCLRRFIIF *1101 5
243 257 CPGYRWMCLRRFIIF *1501 100
243 257 CPGYRWMCLRRFIIF *0701 95
243 257 CPGYRWMCLRRFIIF *0104 85
243 257 CPGYRWMCLRRFIIF *0102 80
243 257 CPGYRWMCLRRFIIF *0101 58
243 257 CPGYRWMCLRRFIIF *0105 58
243 257 CPGYRWMCLRRFIIF *0107 58
243 257 CPGYRWMCLRRFIIF *0301 40
244 258 PGYRWMCLRRFIIFL 1416 *1301 73
244 258 PGYRWMCLRRFIIFL *1101 95
244 258 PGYRWMCLRRFIIFL *1501 90
244 258 PGYRWMCLRRFIIFL *0701 80
244 258 PGYRWMCLRRFIIFL *0104 75
244 258 PGYRWMCLRRFIIFL *0102 73
244 258 PGYRWMCLRRFIIFL *0101 45
244 258 PGYRWMCLRRFIIFL *0105 45
244 258 PGYRWMCLRRFIIFL *0107 45
244 258 PGYRWMCLRRFIIFL *0301 15
245 259 GYRWMCLRRFIIFLF 1417 *1301 100
245 259 GYRWMCLRRFIIFLF *0701 48
245 259 GYRWMCLRRFIIFLF *1501 20
245 259 GYRWMCLRRFIIFLF *1101 85
245 259 GYRWMCLRRFIIFLF *0102 60
245 259 GYRWMCLRRFIIFLF *0104 60
245 259 GYRWMCLRRFIIFLF *0101 30
245 259 GYRWMCLRRFIIFLF *0105 30
245 259 GYRWMCLRRFIIFLF *0107 30
246 260 YRWMCLRRFI IFLFI 1418 *1301 98
246 260 YRWMCLRRFI IFLFI *0701 50 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
246 260 YRWMCLRRFI I FLFI *1501 23
246 260 YRWMCLRRFI I FLFI *1101 75
247 261 RWMCLRRFIIFLFIL 1419 *1301 95
247 261 RWMCLRRFIIFLFIL *0701 30
247 261 RWMCLRRFIIFLFIL *1501 10
247 261 RWMCLRRFIIFLFIL *1101 45
248 262 WMCLRRFIIFLFILL 1420 *1301 93
248 262 WMCLRRFIIFLFILL *1501 73
248 262 WMCLRRFIIFLFILL *0701 60
248 262 WMCLRRFIIFLFILL *0301 28
249 263 MCLRRFIIFLFILLL 1421 *1501 95
249 263 MCLRRFIIFLFILLL *1301 90
249 263 MCLRRFIIFLFILLL *0301 80
249 263 MCLRRFIIFLFILLL *0701 58
249 263 MCLRRFIIFLFILLL *0101 53
249 263 MCLRRFIIFLFILLL *1101 50
249 263 MCLRRFIIFLFILLL *0401 3
250 264 CLRRFIIFLFILLLC 1422 *1301 88
250 264 CLRRFIIFLFILLLC *0301 78
250 264 CLRRFIIFLFILLLC *1501 68
250 264 CLRRFIIFLFILLLC *0701 55
250 264 CLRRFIIFLFILLLC *0101 48
250 264 CLRRFIIFLFILLLC *1101 48
251 265 LRRFIIFLFILLLCL 1423 *1501 100
251 265 LRRFIIFLFILLLCL *1301 85
251 265 LRRFIIFLFILLLCL *0101 83
251 265 LRRFIIFLFILLLCL *0301 83
251 265 LRRFIIFLFILLLCL *1101 58
251 265 LRRFIIFLFILLLCL *0701 53
252 266 RRFIIFLFILLLCLI 1424 *1501 98
252 266 RRFIIFLFILLLCLI *0101 80
252 266 RRFIIFLFILLLCLI *1101 55
252 266 RRFIIFLFILLLCLI *0301 40
252 266 RRFIIFLFILLLCLI *0701 38
252 266 RRFIIFLFILLLCLI *1301 35
253 267 RFIIFLFILLLCLIF 1425 *1101 100
253 267 RFIIFLFILLLCLIF *0101 78
253 267 RFIIFLFILLLCLIF *1501 60
253 267 RFIIFLFILLLCLIF *0301 38
253 267 RFIIFLFILLLCLIF *0701 35
253 267 RFIIFLFILLLCLIF *1301 33
253 267 RFIIFLFILLLCLIF *0401 8
254 268 FIIFLFILLLCLIFL 1426 *1101 98
254 268 FIIFLFILLLCLIFL *1501 88
254 268 FIIFLFILLLCLIFL *0101 65
254 268 FIIFLFILLLCLIFL *0301 35
254 268 FIIFLFILLLCLIFL *1301 30
254 268 FIIFLFILLLCLIFL *0401 13
255 269 IIFLFILLLCLIFLL 1427 *1101 95
255 269 IIFLFILLLCLIFLL *1501 93 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
255 269 IIFLFILLLCLIFLL *0301 70
255 269 IIFLFILLLCLIFLL *0101 35
255 269 IIFLFILLLCLIFLL *0401 35
255 269 IIFLFILLLCLIFLL *1301 28
256 270 IFLFILLLCLIFLLV 1428 *1101 93
256 270 IFLFILLLCLIFLLV *0301 68
256 270 IFLFILLLCLIFLLV *1501 53
256 270 IFLFILLLCLIFLLV *0101 30
256 270 IFLFILLLCLIFLLV *1301 25
256 270 IFLFILLLCLIFLLV *0401 10
257 271 FLFILLLCLI FLLVL 1429 *1101 90
257 271 FLFILLLCLI FLLVL *0301 65
257 271 FLFILLLCLI FLLVL *1501 58
257 271 FLFILLLCLI FLLVL *0101 28
257 271 FLFILLLCLI FLLVL *1301 23
257 271 FLFILLLCLI FLLVL *0401 18
258 272 LFILLLCLIFLLVLL 1430 *1101 88
258 272 LFILLLCLIFLLVLL *0301 63
258 272 LFILLLCLIFLLVLL *1501 50
258 272 LFILLLCLIFLLVLL *0101 25
258 272 LFILLLCLIFLLVLL *1301 20
259 273 FILLLCLI FLLVLLD 1431 *1101 85
259 273 FILLLCLI FLLVLLD *0301 55
259 273 FILLLCLI FLLVLLD *1501 30
259 273 FILLLCLI FLLVLLD *0101 23
259 273 FILLLCLI FLLVLLD *1301 18
260 274 ILLLCLI FLLVLLDY 1432 *1101 45
260 274 ILLLCLI FLLVLLDY *1301 15
260 274 ILLLCLI FLLVLLDY *1501 13
Cumulative Class II-B score: 7238
330 344 WAFAKYLWEWASVRF 1433 *1501 5 32 323 358
331 345 AFAKYLWEWASVRFS 1434 *0401 65
331 345 AFAKYLWEWASVRFS *0301 30
331 345 AFAKYLWEWASVRFS *1501 15
331 345 AFAKYLWEWASVRFS *1101 5
332 346 FAKYLWEWASVRFSW 1435 *0401 80
332 346 FAKYLWEWASVRFSW *0301 55
332 346 FAKYLWEWASVRFSW *1501 50
332 346 FAKYLWEWASVRFSW *1101 40
332 346 FAKYLWEWASVRFSW *0104 25
332 346 FAKYLWEWASVRFSW *0101 23
332 346 FAKYLWEWASVRFSW *0105 23
332 346 FAKYLWEWASVRFSW *0107 23
332 346 FAKYLWEWASVRFSW *0701 15
333 347 AKYLWEWASVRFSWL 1436 *0701 28
333 347 AKYLWEWASVRFSWL *0401 60 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
333 347 AKYLWEWASVRFSWL *0301 25
333 347 AKYLWEWASVRFSWL *1101 25
333 347 AKYLWEWASVRFSWL *0101 3
333 347 AKYLWEWASVRFSWL *0105 3
333 347 AKYLWEWASVRFSWL *0107 3
334 348 KYLWEWASVRFSWLS 1437 *0701 25
334 348 KYLWEWASVRFSWLS *0401 5
336 350 LWEWASVRFSWLSLL 1438 *0301 33
337 351 WEWASVRFSWLS LLV 1439 *0301 73
337 351 WEWASVRFSWLS LLV *1501 45
337 351 WEWASVRFSWLS LLV *1301 25
338 352 EWASVRFSWLSLLVP 1440 *0301 30
338 352 EWASVRFSWLSLLVP *1501 40
338 352 EWASVRFSWLSLLVP *1301 35
339 353 WASVRFSWLS LLVP F 1441 *0301 58
339 353 WASVRFSWLS LLVP F *1501 55
339 353 WASVRFSWLS LLVP F *1301 50
339 353 WASVRFSWLS LLVP F *0104 38
339 353 WASVRFSWLS LLVP F *0102 5
340 354 ASVRFSWLSLLVPFV 1442 *0301 75
340 354 ASVRFSWLSLLVPFV *0401 33
340 354 ASVRFSWLSLLVPFV *1101 18
340 354 ASVRFSWLSLLVPFV *0104 53
340 354 ASVRFSWLSLLVPFV *1501 30
340 354 ASVRFSWLSLLVPFV *0102 28
340 354 ASVRFSWLSLLVPFV *1301 20
340 354 ASVRFSWLSLLVPFV *0106 10
341 355 SVRFSWLS LLVP FVQ 1443 *0401 30
341 355 SVRFSWLS LLVP FVQ *1101 15
341 355 SVRFSWLS LLVP FVQ *0104 50
341 355 SVRFSWLS LLVP FVQ *0102 23
341 355 SVRFSWLS LLVP FVQ *0106 5
342 356 VRFSWLS LLVPFVQW 1444 *0401 28
342 356 VRFSWLS LLVPFVQW *0101 15
342 356 VRFSWLS LLVPFVQW *1101 13
342 356 VRFSWLS LLVPFVQW *0104 15
343 357 RFSWLSLLVP FVQWF 1445 *0401 25
343 357 RFSWLSLLVP FVQWF *0101 20
343 357 RFSWLSLLVP FVQWF *1101 10
344 358 FSWLS LLVPFVQWFV 1446 *1501 90
344 358 FSWLS LLVPFVQWFV *1301 70
344 358 FSWLS LLVPFVQWFV *0401 23
344 358 FSWLS LLVPFVQWFV *1101 8
Cumulative Class II-B score: 1815
330 344 WAFAKYLWEWASVRF 1433 *1501 5 33 327 358
331 345 AFAKYLWEWASVRFS 1434 *0401 65
331 345 AFAKYLWEWASVRFS *0301 30
331 345 AFAKYLWEWASVRFS *1501 15 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
331 345 AFAKYLWEWASVRFS *1101 5
332 346 FAKYLWEWASVRFSW 1435 *0401 80
332 346 FAKYLWEWASVRFSW *0301 55
332 346 FAKYLWEWASVRFSW *1501 50
332 346 FAKYLWEWASVRFSW *1101 40
332 346 FAKYLWEWASVRFSW *0104 25
332 346 FAKYLWEWASVRFSW *0101 23
332 346 FAKYLWEWASVRFSW *0105 23
332 346 FAKYLWEWASVRFSW *0107 23
332 346 FAKYLWEWASVRFSW *0701 15
333 347 AKYLWEWASVRFSWL 1436 *0701 28
333 347 AKYLWEWASVRFSWL *0401 60
333 347 AKYLWEWASVRFSWL *0301 25
333 347 AKYLWEWASVRFSWL *1101 25
333 347 AKYLWEWASVRFSWL *0101 3
333 347 AKYLWEWASVRFSWL *0105 3
333 347 AKYLWEWASVRFSWL *0107 3
334 348 KYLWEWASVRFSWLS 1437 *0701 25
334 348 KYLWEWASVRFSWLS *0401 5
336 350 LWEWASVRFSWLSLL 1438 *0301 33
337 351 WEWASVRFSWLS LLV 1439 *0301 73
337 351 WEWASVRFSWLS LLV *1501 45
337 351 WEWASVRFSWLS LLV *1301 25
338 352 EWASVRFSWLSLLVP 1440 *0301 30
338 352 EWASVRFSWLSLLVP *1501 40
338 352 EWASVRFSWLSLLVP *1301 35
339 353 WASVRFSWLS LLVP F 1441 *0301 58
339 353 WASVRFSWLS LLVP F *1501 55
339 353 WASVRFSWLS LLVP F *1301 50
339 353 WASVRFSWLS LLVP F *0104 38
339 353 WASVRFSWLS LLVP F *0102 5
340 354 ASVRFSWLSLLVPFV 1442 *0301 75
340 354 ASVRFSWLSLLVPFV *0401 33
340 354 ASVRFSWLSLLVPFV *1101 18
340 354 ASVRFSWLSLLVPFV *0104 53
340 354 ASVRFSWLSLLVPFV *1501 30
340 354 ASVRFSWLSLLVPFV *0102 28
340 354 ASVRFSWLSLLVPFV *1301 20
340 354 ASVRFSWLSLLVPFV *0106 10
341 355 SVRFSWLS LLVP FVQ 1443 *0401 30
341 355 SVRFSWLS LLVP FVQ *1101 15
341 355 SVRFSWLS LLVP FVQ *0104 50
341 355 SVRFSWLS LLVP FVQ *0102 23
341 355 SVRFSWLS LLVP FVQ *0106 5
342 356 VRFSWLS LLVPFVQW 1444 *0401 28
342 356 VRFSWLS LLVPFVQW *0101 15
342 356 VRFSWLS LLVPFVQW *1101 13
342 356 VRFSWLS LLVPFVQW *0104 15
343 357 RFSWLSLLVP FVQWF 1445 *0401 25
343 357 RFSWLSLLVP FVQWF *0101 20 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
343 357 RFSWLSLLVP FVQWF *1101 10
344 358 FSWLS LLVPFVQWFV 1446 *1501 90
344 358 FSWLS LLVPFVQWFV *1301 70
344 358 FSWLS LLVPFVQWFV *0401 23
344 358 FSWLS LLVPFVQWFV *1101 8
Cumulative Class II-B score: 1815
330 344 WAFAKYLWEWASVRF 1433 *1501 5 34 328 358
331 345 AFAKYLWEWASVRFS 1434 *0401 65
331 345 AFAKYLWEWASVRFS *0301 30
331 345 AFAKYLWEWASVRFS *1501 15
331 345 AFAKYLWEWASVRFS *1101 5
332 346 FAKYLWEWASVRFSW 1435 *0401 80
332 346 FAKYLWEWASVRFSW *0301 55
332 346 FAKYLWEWASVRFSW *1501 50
332 346 FAKYLWEWASVRFSW *1101 40
332 346 FAKYLWEWASVRFSW *0104 25
332 346 FAKYLWEWASVRFSW *0101 23
332 346 FAKYLWEWASVRFSW *0105 23
332 346 FAKYLWEWASVRFSW *0107 23
332 346 FAKYLWEWASVRFSW *0701 15
333 347 AKYLWEWASVRFSWL 1436 *0701 28
333 347 AKYLWEWASVRFSWL *0401 60
333 347 AKYLWEWASVRFSWL *0301 25
333 347 AKYLWEWASVRFSWL *1101 25
333 347 AKYLWEWASVRFSWL *0101 3
333 347 AKYLWEWASVRFSWL *0105 3
333 347 AKYLWEWASVRFSWL *0107 3
334 348 KYLWEWASVRFSWLS 1437 *0701 25
334 348 KYLWEWASVRFSWLS *0401 5
336 350 LWEWASVRFSWLSLL 1438 *0301 33
337 351 WEWASVRFSWLS LLV 1439 *0301 73
337 351 WEWASVRFSWLS LLV *1501 45
337 351 WEWASVRFSWLS LLV *1301 25
338 352 EWASVRFSWLSLLVP 1440 *0301 30
338 352 EWASVRFSWLSLLVP *1501 40
338 352 EWASVRFSWLSLLVP *1301 35
339 353 WASVRFSWLS LLVP F 1441 *0301 58
339 353 WASVRFSWLS LLVP F *1501 55
339 353 WASVRFSWLS LLVP F *1301 50
339 353 WASVRFSWLS LLVP F *0104 38
339 353 WASVRFSWLS LLVP F *0102 5
340 354 ASVRFSWLSLLVPFV 1442 *0301 75
340 354 ASVRFSWLSLLVPFV *0401 33
340 354 ASVRFSWLSLLVPFV *1101 18
340 354 ASVRFSWLSLLVPFV *0104 53
340 354 ASVRFSWLSLLVPFV *1501 30
340 354 ASVRFSWLSLLVPFV *0102 28
340 354 ASVRFSWLSLLVPFV *1301 20 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
340 354 ASVRFSWLSLLVPFV *0106 10
341 355 SVRFSWLSLLVPFVQ 1443 *0401 30
341 355 SVRFSWLSLLVPFVQ *1101 15
341 355 SVRFSWLSLLVPFVQ *0104 50
341 355 SVRFSWLSLLVPFVQ *0102 23
341 355 SVRFSWLSLLVPFVQ *0106 5
342 356 VRFSWLSLLVPFVQW 1444 *0401 28
342 356 VRFSWLSLLVPFVQW *0101 15
342 356 VRFSWLSLLVPFVQW *1101 13
342 356 VRFSWLSLLVPFVQW *0104 15
343 357 RFSWLSLLVPFVQWF 1445 *0401 25
343 357 RFSWLSLLVPFVQWF *0101 20
343 357 RFSWLSLLVPFVQWF *1101 10
344 358 FSWLSLLVPFVQWFV 1446 *1501 90
344 358 FSWLSLLVPFVQWFV *1301 70
344 358 FSWLSLLVPFVQWFV *0401 23
344 358 FSWLSLLVPFVQWFV *1101 8
Cumulative Class II-B score: 1815
365 379 WLSAIWMMWYWGPSL 1447 *1501 35 35 365 400
366 380 LSAIW MWYWGPSLY 1448 *1501 48
367 381 SAIWMMWYWGPSLYS 1449 *1501 40
368 382 AIW MWYWGPSLYSI 1450 *1501 45
369 383 IWMMWYWGPSLYSIV 1451 *1501 38
369 383 IWMMWYWGPSLYSIV *0101 20
369 383 IWMMWYWGPSLYSIV *0105 20
369 383 IWMMWYWGPSLYSIV *0107 20
370 384 WMMWYWGPSLYSIVS 1452 *1501 28
370 384 WMMWYWGPSLYSIVS *0101 33
370 384 WMMWYWGPSLYSIVS *0105 33
370 384 WMMWYWGPSLYSIVS *0107 33
371 385 MMWYWGPSLYSIVSP 1453 *1501 8
373 387 WYWGPSLYSIVSPFI 1454 *0701 90
373 387 WYWGPSLYSIVSPFI *0401 40
373 387 WYWGPSLYSIVSPFI *0106 45
373 387 WYWGPSLYSIVSPFI *0101 43
373 387 WYWGPSLYSIVSPFI *0105 43
373 387 WYWGPSLYSIVSPFI *0107 43
373 387 WYWGPSLYSIVSPFI *0102 18
374 388 YWGPSLYSIVSPFIP 1455 *0701 65
374 388 YWGPSLYSIVSPFIP *0101 60
374 388 YWGPSLYSIVSPFIP *0105 60
374 388 YWGPSLYSIVSPFIP *0107 60
374 388 YWGPSLYSIVSPFIP *0401 55
374 388 YWGPSLYSIVSPFIP *0106 53
374 388 YWGPSLYSIVSPFIP *0102 48
374 388 YWGPSLYSIVSPFIP *0104 43
374 388 YWGPSLYSIVSPFIP *1501 25
375 389 WGPSLYSIVSPFIPL 1456 *0701 85 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
375 389 WGPSLYSIVSPFIPL *0401 20
375 389 WGPSLYSIVSPFIPL *1501 18
375 389 WGPSLYSIVSPFIPL *0104 88
375 389 WGPSLYSIVSPFIPL *0102 85
375 389 WGPSLYSIVSPFIPL *0101 83
375 389 WGPSLYSIVSPFIPL *0105 83
375 389 WGPSLYSIVSPFIPL *0106 83
375 389 WGPSLYSIVSPFIPL *0107 83
375 389 WGPSLYSIVSPFIPL *0103 15
375 389 WGPSLYSIVSPFIPL *1101 15
376 390 GPSLYSIVSPFIPLL 1457 *0701 83
376 390 GPSLYSIVSPFIPLL *0401 43
376 390 GPSLYSIVSPFIPLL *1501 43
376 390 GPSLYSIVSPFIPLL *0101 33
376 390 GPSLYSIVSPFIPLL *0104 100
376 390 GPSLYSIVSPFIPLL *0102 98
376 390 GPSLYSIVSPFIPLL *0105 93
376 390 GPSLYSIVSPFIPLL *0107 93
376 390 GPSLYSIVSPFIPLL *0106 90
376 390 GPSLYSIVSPFIPLL *0103 50
376 390 GPSLYSIVSPFIPLL *1101 35
376 390 GPSLYSIVSPFIPLL *1301 15
377 391 PSLYSIVSPFIPLLP 1458 *0701 63
377 391 PSLYSIVSPFIPLLP *1501 33
377 391 PSLYSIVSPFIPLLP *0101 10
377 391 PSLYSIVSPFIPLLP *0401 5
377 391 PSLYSIVSPFIPLLP *0104 93
377 391 PSLYSIVSPFIPLLP *0102 90
377 391 PSLYSIVSPFIPLLP *0106 85
377 391 PSLYSIVSPFIPLLP *0105 80
377 391 PSLYSIVSPFIPLLP *0107 80
377 391 PSLYSIVSPFIPLLP *0103 20
377 391 PSLYSIVSPFIPLLP *1101 20
377 391 PSLYSIVSPFIPLLP *1301 10
378 392 SLYSIVSPFIPLLPI 1459 *0701 43
378 392 SLYSIVSPFIPLLPI *0104 83
378 392 SLYSIVSPFIPLLPI *0102 78
378 392 SLYSIVSPFIPLLPI *0106 73
378 392 SLYSIVSPFIPLLPI *0101 63
378 392 SLYSIVSPFIPLLPI *0105 63
378 392 SLYSIVSPFIPLLPI *0107 63
378 392 SLYSIVSPFIPLLPI *1501 35
378 392 SLYSIVSPFIPLLPI *0401 20
378 392 SLYSIVSPFIPLLPI *0103 5
379 393 LYSIVSPFIPLLPIF 1460 *0701 33
379 393 LYSIVSPFIPLLPIF *0106 63
379 393 LYSIVSPFIPLLPIF *0102 55
379 393 LYSIVSPFIPLLPIF *0104 55
379 393 LYSIVSPFIPLLPIF *0101 53
379 393 LYSIVSPFIPLLPIF *0105 53 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
379 393 LYSIVSPFIPLLPIF *0107 53
380 394 YSIVSPFIPLLPIFF 1461 *1101 35
380 394 YSIVSPFIPLLPIFF *0101 8
380 394 YSIVSPFIPLLPIFF *0104 23
380 394 YSIVSPFIPLLPIFF *0106 18
380 394 YSIVSPFIPLLPIFF *0105 5
380 394 YSIVSPFIPLLPIFF *0107 5
381 395 SIVSPFIPLLPIFFC 1462 *1101 33
381 395 SIVSPFIPLLPIFFC *0101 5
382 396 IVSPFIPLLPIFFCL 1463 *1101 30
382 396 IVSPFIPLLPIFFCL *0101 3
382 396 IVSPFIPLLPIFFCL *0104 18
382 396 IVSPFIPLLPIFFCL *0105 15
382 396 IVSPFIPLLPIFFCL *0107 15
382 396 IVSPFIPLLPIFFCL *0102 3
383 397 VSPFIPLLPIFFCLW 1464 *1101 28
383 397 VSPFIPLLPIFFCLW *0101 13
383 397 VSPFIPLLPIFFCLW *0105 13
383 397 VSPFIPLLPIFFCLW *0107 13
383 397 VSPFIPLLPIFFCLW *0104 10
384 398 SPFIPLLPIFFCLWV 1465 *1101 25
384 398 SPFIPLLPIFFCLWV *1501 5
386 400 FIPLLPIFFCLWVYI 1466 *1101 23
Cumulative Class II-B score: 4425
371 385 MMWYWGPSLYSIVSP 1453 *1501 8 36 371 400
373 387 WYWGPSLYSIVSPFI 1454 *0701 90
373 387 WYWGPSLYSIVSPFI *0401 40
373 387 WYWGPSLYSIVSPFI *0106 45
373 387 WYWGPSLYSIVSPFI *0101 43
373 387 WYWGPSLYSIVSPFI *0105 43
373 387 WYWGPSLYSIVSPFI *0107 43
373 387 WYWGPSLYSIVSPFI *0102 18
374 388 YWGPSLYSIVSPFIP 1455 *0701 65
374 388 YWGPSLYSIVSPFIP *0101 60
374 388 YWGPSLYSIVSPFIP *0105 60
374 388 YWGPSLYSIVSPFIP *0107 60
374 388 YWGPSLYSIVSPFIP *0401 55
374 388 YWGPSLYSIVSPFIP *0106 53
374 388 YWGPSLYSIVSPFIP *0102 48
374 388 YWGPSLYSIVSPFIP *0104 43
374 388 YWGPSLYSIVSPFIP *1501 25
375 389 WGPSLYSIVSPFIPL 1456 *0701 85
375 389 WGPSLYSIVSPFIPL *0401 20
375 389 WGPSLYSIVSPFIPL *1501 18
375 389 WGPSLYSIVSPFIPL *0104 88
375 389 WGPSLYSIVSPFIPL *0102 85
375 389 WGPSLYSIVSPFIPL *0101 83
375 389 WGPSLYSIVSPFIPL *0105 83 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
375 389 WGPSLYSIVSPFIPL *0106 83
375 389 WGPSLYSIVSPFIPL *0107 83
375 389 WGPSLYSIVSPFIPL *0103 15
375 389 WGPSLYSIVSPFIPL *1101 15
376 390 GPSLYSIVSPFIPLL 1457 *0701 83
376 390 GPSLYSIVSPFIPLL *0401 43
376 390 GPSLYSIVSPFIPLL *1501 43
376 390 GPSLYSIVSPFIPLL *0101 33
376 390 GPSLYSIVSPFIPLL *0104 100
376 390 GPSLYSIVSPFIPLL *0102 98
376 390 GPSLYSIVSPFIPLL *0105 93
376 390 GPSLYSIVSPFIPLL *0107 93
376 390 GPSLYSIVSPFIPLL *0106 90
376 390 GPSLYSIVSPFIPLL *0103 50
376 390 GPSLYSIVSPFIPLL *1101 35
376 390 GPSLYSIVSPFIPLL *1301 15
377 391 PSLYSIVSPFIPLLP 1458 *0701 63
377 391 PSLYSIVSPFIPLLP *1501 33
377 391 PSLYSIVSPFIPLLP *0101 10
377 391 PSLYSIVSPFIPLLP *0401 5
377 391 PSLYSIVSPFIPLLP *0104 93
377 391 PSLYSIVSPFIPLLP *0102 90
377 391 PSLYSIVSPFIPLLP *0106 85
377 391 PSLYSIVSPFIPLLP *0105 80
377 391 PSLYSIVSPFIPLLP *0107 80
377 391 PSLYSIVSPFIPLLP *0103 20
377 391 PSLYSIVSPFIPLLP *1101 20
377 391 PSLYSIVSPFIPLLP *1301 10
378 392 SLYSIVSPFIPLLPI 1459 *0701 43
378 392 SLYSIVSPFIPLLPI *0104 83
378 392 SLYSIVSPFIPLLPI *0102 78
378 392 SLYSIVSPFIPLLPI *0106 73
378 392 SLYSIVSPFIPLLPI *0101 63
378 392 SLYSIVSPFIPLLPI *0105 63
378 392 SLYSIVSPFIPLLPI *0107 63
378 392 SLYSIVSPFIPLLPI *1501 35
378 392 SLYSIVSPFIPLLPI *0401 20
378 392 SLYSIVSPFIPLLPI *0103 5
379 393 LYSIVSPFIPLLPIF 1460 *0701 33
379 393 LYSIVSPFIPLLPIF *0106 63
379 393 LYSIVSPFIPLLPIF *0102 55
379 393 LYSIVSPFIPLLPIF *0104 55
379 393 LYSIVSPFIPLLPIF *0101 53
379 393 LYSIVSPFIPLLPIF *0105 53
379 393 LYSIVSPFIPLLPIF *0107 53
380 394 YSIVSPFIPLLPIFF 1461 *1101 35
380 394 YSIVSPFIPLLPIFF *0101 8
380 394 YSIVSPFIPLLPIFF *0104 23
380 394 YSIVSPFIPLLPIFF *0106 18
380 394 YSIVSPFIPLLPIFF *0105 5 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
380 394 YSIVSPFIPLLPIFF *0107 5
381 395 SIVSPFIPLLPIFFC 1462 *1101 33
381 395 SIVSPFIPLLPIFFC *0101 5
382 396 IVSPFIPLLPIFFCL 1463 *1101 30
382 396 IVSPFIPLLPIFFCL *0101 3
382 396 IVSPFIPLLPIFFCL *0104 18
382 396 IVSPFIPLLPIFFCL *0105 15
382 396 IVSPFIPLLPIFFCL *0107 15
382 396 IVSPFIPLLPIFFCL *0102 3
383 397 VSPFIPLLPIFFCLW 1464 *1101 28
383 397 VSPFIPLLPIFFCLW *0101 13
383 397 VSPFIPLLPIFFCLW *0105 13
383 397 VSPFIPLLPIFFCLW *0107 13
383 397 VSPFIPLLPIFFCLW *0104 10
384 398 SPFIPLLPIFFCLWV 1465 *1101 25
384 398 SPFIPLLPIFFCLWV *1501 5
386 400 FIPLLPIFFCLWVYI 1466 *1101 23
Cumulative Class II-B score: 4035
370 384 W MWYWGPSLYSIVS 1452 *1501 28 37 370 400
370 384 W MWYWGPSLYSIVS *0101 33
370 384 WMMWYWGPSLYSIVS *0105 33
370 384 WMMWYWGPSLYSIVS *0107 33
371 385 MMWYWGPSLYSIVSP 1453 *1501 8
373 387 WYWGPSLYSIVSPFI 1454 *0701 90
373 387 WYWGPSLYSIVSPFI *0401 40
373 387 WYWGPSLYSIVSPFI *0106 45
373 387 WYWGPSLYSIVSPFI *0101 43
373 387 WYWGPSLYSIVSPFI *0105 43
373 387 WYWGPSLYSIVSPFI *0107 43
373 387 WYWGPSLYSIVSPFI *0102 18
374 388 YWGPSLYSIVSPFIP 1455 *0701 65
374 388 YWGPSLYSIVSPFIP *0101 60
374 388 YWGPSLYSIVSPFIP *0105 60
374 388 YWGPSLYSIVSPFIP *0107 60
374 388 YWGPSLYSIVSPFIP *0401 55
374 388 YWGPSLYSIVSPFIP *0106 53
374 388 YWGPSLYSIVSPFIP *0102 48
374 388 YWGPSLYSIVSPFIP *0104 43
374 388 YWGPSLYSIVSPFIP *1501 25
375 389 WGPSLYSIVSPFIPL 1456 *0701 85
375 389 WGPSLYSIVSPFIPL *0401 20
375 389 WGPSLYSIVSPFIPL *1501 18
375 389 WGPSLYSIVSPFIPL *0104 88
375 389 WGPSLYSIVSPFIPL *0102 85
375 389 WGPSLYSIVSPFIPL *0101 83
375 389 WGPSLYSIVSPFIPL *0105 83
375 389 WGPSLYSIVSPFIPL *0106 83
375 389 WGPSLYSIVSPFIPL *0107 83 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRB1 SLP# SLP End
NO: B score3 Start
molecule
375 389 WGPSLYSIVSPFIPL *0103 15
375 389 WGPSLYSIVSPFIPL *1101 15
376 390 GPSLYSIVSPFIPLL 1457 *0701 83
376 390 GPSLYSIVSPFIPLL *0401 43
376 390 GPSLYSIVSPFIPLL *1501 43
376 390 GPSLYSIVSPFIPLL *0101 33
376 390 GPSLYSIVSPFIPLL *0104 100
376 390 GPSLYSIVSPFIPLL *0102 98
376 390 GPSLYSIVSPFIPLL *0105 93
376 390 GPSLYSIVSPFIPLL *0107 93
376 390 GPSLYSIVSPFIPLL *0106 90
376 390 GPSLYSIVSPFIPLL *0103 50
376 390 GPSLYSIVSPFIPLL *1101 35
376 390 GPSLYSIVSPFIPLL *1301 15
377 391 PSLYSIVSPFIPLLP 1458 *0701 63
377 391 PSLYSIVSPFIPLLP *1501 33
377 391 PSLYSIVSPFIPLLP *0101 10
377 391 PSLYSIVSPFIPLLP *0401 5
377 391 PSLYSIVSPFIPLLP *0104 93
377 391 PSLYSIVSPFIPLLP *0102 90
377 391 PSLYSIVSPFIPLLP *0106 85
377 391 PSLYSIVSPFIPLLP *0105 80
377 391 PSLYSIVSPFIPLLP *0107 80
377 391 PSLYSIVSPFIPLLP *0103 20
377 391 PSLYSIVSPFIPLLP *1101 20
377 391 PSLYSIVSPFIPLLP *1301 10
378 392 SLYSIVSPFIPLLPI 1459 *0701 43
378 392 SLYSIVSPFIPLLPI *0104 83
378 392 SLYSIVSPFIPLLPI *0102 78
378 392 SLYSIVSPFIPLLPI *0106 73
378 392 SLYSIVSPFIPLLPI *0101 63
378 392 SLYSIVSPFIPLLPI *0105 63
378 392 SLYSIVSPFIPLLPI *0107 63
378 392 SLYSIVSPFIPLLPI *1501 35
378 392 SLYSIVSPFIPLLPI *0401 20
378 392 SLYSIVSPFIPLLPI *0103 5
379 393 LYSIVSPFIPLLPIF 1460 *0701 33
379 393 LYSIVSPFIPLLPIF *0106 63
379 393 LYSIVSPFIPLLPIF *0102 55
379 393 LYSIVSPFIPLLPIF *0104 55
379 393 LYSIVSPFIPLLPIF *0101 53
379 393 LYSIVSPFIPLLPIF *0105 53
379 393 LYSIVSPFIPLLPIF *0107 53
380 394 YSIVSPFIPLLPIFF 1461 *1101 35
380 394 YSIVSPFIPLLPIFF *0101 8
380 394 YSIVSPFIPLLPIFF *0104 23
380 394 YSIVSPFIPLLPIFF *0106 18
380 394 YSIVSPFIPLLPIFF *0105 5
380 394 YSIVSPFIPLLPIFF *0107 5
381 395 SIVSPFIPLLPIFFC 1462 *1101 33 HLA class II binding peptides in SLP derived from HBV Surface
Peptide of invention (SLP) antigen
HLA-
SEQ ID Class II- SLP
Start End Sequence A DRBl SLP# SLP End
NO: B score3 Start
molecule
381 395 SIVSPFIPLLPIFFC *0101 5
382 396 IVSPFIPLLPIFFCL 1463 *1101 30
382 396 IVSPFIPLLPIFFCL *0101 3
382 396 IVSPFIPLLPIFFCL *0104 18
382 396 IVSPFIPLLPIFFCL *0105 15
382 396 IVSPFIPLLPIFFCL *0107 15
382 396 IVSPFIPLLPIFFCL *0102 3
383 397 VSPFIPLLPIFFCLW 1464 *1101 28
383 397 VSPFIPLLPIFFCLW *0101 13
383 397 VSPFIPLLPIFFCLW *0105 13
383 397 VSPFIPLLPIFFCLW *0107 13
383 397 VSPFIPLLPIFFCLW *0104 10
384 398 SPFIPLLPIFFCLWV 1465 *1101 25
384 398 SPFIPLLPIFFCLWV *1501 5
386 400 FIPLLPIFFCLWVYI 1466 *1101 23
Cumulative Class II-B score: 4160
"Start" and "End" are relative to the amino acid sequence of human HBV large surface protein as depicted in SEQ ID NO: 1141
A Peptide amino acid sequence. Each HLA-DRB l binding peptide of HBV large surface protein is listed separately for each HLA class II molecule to which it is predicted to bind, and each peptide can be listed multiple times for that reason.
B B-score. See Material and Methods (Examples section).
Cumulative B-score. See Material and Methods (Examples section).
REFERENCE LIST
Atherton, E. and Sheppard, R., 1989, Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford.
Barany, G. and Merrifield, R., 1979. In The Peptides, Vol. 2 (E. Gross and J. Meienhofer, eds.) pp. 1-284. Academic Press, New York.
Buckanovich RJ et al., 2008, Nature Medicine 14: 28.
Bui et al. 2006, BMC Bioinformatics 7: 153.
Chapiro J et al, 2006; J Immunol 176: 1053-1061.
Craiu A, et al, 1997, Proc Natl Acad Sci U S A 94: 10850-10855.
Fields, G.B., 1997,. Methods Enzymol. Vol. 289.
Huang et al., 201 lCurr Opin Immunol 23 :237-243.
Ganem et al, 2004, N Engl J Med 350: 1118-1129.
Grimm et al, 2013 Clin Sci (Lond) 124:77-85.
Ishikawa K, 1994, PNAS 91 : 4892.
Kato et al., 2005, Immunity, 1 : 19-28.
Kessler JH et al., 2003, Hum Immunol 64: 245-255.
Lanzavecchia, 1998, Nature 393 : 413.
Lok AS: 2002, N Engl J Med 346: 1682-1683.
Lundegaard C, et al. - 2010; Immunology 130: 309.
Michel ML et al, 2001, J. Hepatol 34: 917-921.
Michel ML, et al, 2011. J Hepatol 54: 1286-1296.
Morel S et al, 2000 Immunity 12: 107-117, 2000.
Mo XY et al., 1999 J Immunol 163 :5851-5859.
Nielsen M, et al, 2005, Immunogenetics 57: 33
Nielsen M, et al, 2010, Immunome Res 6: 9
Rehermann et al, 2005, 5:215-229. Remington; The Science and Practice of Pharmacy, 21st Edition 2005, University of Sciences in Philadelphia.
Ridge et al. 1998, Nature 393 : 474.
Rock et al, 2004, Nat. Immunol. 5:670.
Rosalia et al, 2013, Eur J Immunol 43 :2554-2565.
Schirle M et al., 2000, Eur J Immunol 30:2216-2225.
Schirle M et al., 2001 J Immunol Methods 257: 1-16.
Schoenberger et al.1998, Nature 393 : 480.
Stoltze L et al, 1998 Eur J Immunol 28:4029-4036.
Sun et al. 2004, Nat. Immunol. 5: 927
Thimme et al:. J Virol 77:68-76, 2003.
Toes et al., 1996a, J. Immunol. 156: 3911.
Toes et al., 1996b, Proc.Natl.Acad.Sci.U.S.A 93 : 7855.
Van der Burg SH et al, 1995, Hum Immunol. 44: 189.
Van der Burg SH et al., 1996, J. Immunol 156(9): 3308-14
Van der Burg SH et al, 2007, PNAS 104: 12087.
Viatte S, et al., 2006, Immunol Cell Biol 84:318-330.
Wang P, et al., 2008, PLoS Comput Biol 4: el000048.
World Health Organization. Hepatitis B. World Health Organization. Fact Sheet 204 (Updated July 2013). Accessible at:
http://www.who.int/mediacentre/factsheets/fs204/en/
Zeestraten et al, Int J Cancer. 2013 Apr 1; 132 (7): 1581-91
Zwaveling et al., 2002, J. Immunol. 169: 350.13.
Zoulim et al, 2012, B. J Hepatol 56 Suppl 1 :S112-S122.

Claims

1. A peptide comprising at least 70 predicted T-cell epitopes, and at least 3 proteasomal cleavage sites, wherein said peptide is derived from an HBV protein and wherein said peptide is from 15 to 100 amino acids in length.
2. A peptide according to claim 1, wherein said peptide comprises or consists of an amino acid sequence of any of the proteins selected from the group consisting of HBV polymerase, HBV core protein, HBV X-protein and HBV large surface protein.
3. A peptide according to claim 1 or 2, wherein said peptide comprises at least 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes and at least 1 predicted HLA class II-restricted CD4+ T-helper epitopes, preferably said peptide comprises a peptide selected from the group consisting of SEQ ID NO: 75, 51-74, 76-79, 1142- 1145 and 1468-1471.
4. A peptide according to any of the claims 1-3, wherein said peptide comprises at least 70 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes and at least 15 predicted HLA class II-restricted CD4+ T-helper epitopes, preferably said peptide comprises a peptide selected from the group consisting of SEQ ID NO: 75, 51-53, 55, 57, 60, 63, 64, 66, 68, 71, 72,74, 76-78, 1142, 1145, 1468-1471.
5. A peptide according to any of the claims 1 - 4, wherein said peptide comprises at least 95 predicted HLA class I-restricted CD8+ cytotoxic T-cell epitopes and at least 25 predicted HLA class II-restricted CD4+ T-helper epitopes, preferably said peptide comprises a peptide selected from the group consisting of SEQ ID NO: 75, 55, 60, 63, 64, 68, 71, 77, 1142 and 1469.
6. A peptide selected from the group consisting of SEQ ID NO: 75, 55, 60, 63, 64, 68, 71, 77, 1142 and 1469.
7. A peptide according to any one of claims 1 to 6, comprising a modified amino acid, a non-naturally occurring amino acid and/or a covalently linked functional group such as a fluorinated group, a human toll-like receptor ligand and/or agonist, an oligonucleotide conjugate, PSA, a sugar chain or glycan, a pam3cys and/or derivative thereof, preferably a pam3cys lipopeptide or variant or derivative thereof, CpG
oligodeoxynucleotides (CpG-ODNs), Cyclic dinucleotides (CDNs), 2-aminoisobutyric acid (Abu), Muramyl dipeptide (MDP), a DC pulse cassette, a tetanus toxin derived peptide.
8. A polynucleotide encoding a peptide according to any one of claims 1 to 7.
9. A composition comprising a peptide according to any one of claims 1 to 7 and/or a polynucleotide according to claim 8, and a pharmaceutically acceptable carrier.
10. A composition comprising at least two different peptides according to any one of claims 1 to 7 and/or at least two different polynucleotides according to claim 8, and a pharmaceutically acceptable carrier.
11. A composition according to claim 9 or 10, further comprising at least one adjuvant.
12. A peptide according to any one of claims 1 to 7 and/or a polynucleotide according to claim 8 and/or a composition according to any of one of claims 9-11, for use as a medicament.
13. A method for the prevention and/or treatment of an HBV related disease comprising administering to a subject an effective amount of a peptide according to any one of claims 1 to 7 and/or a polynucleotide according to claim 8 and/or a composition according to any one of claims 9-11.
14. A peptide according to any one of claims 1 to 7 and/or a polynucleotide according to claim 8 and/or a composition according to any one of claims 9-11, for the treatment and/or prevention of an HBV related disease or condition.
15. Use of a peptide according to any one of claims 1 to 7 and/or a polynucleotide according to claim 8 and/or a composition according to any one of claims 9-11, for the manufacturing of a medicament for the treatment and/or prevention of an HBV related disease or condition.
PCT/NL2015/050390 2014-06-02 2015-06-01 Synthetic long peptides (slp) for therapeutic vaccination against hepatitis b virus infection WO2015187009A1 (en)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9718848B2 (en) 2015-12-03 2017-08-01 Glaxosmithkline Intellectual Property Development Limited Compounds
WO2017220463A1 (en) 2016-06-20 2017-12-28 Isa Pharmaceuticals B.V. Formulation of a peptide vaccine
US10047115B2 (en) 2015-01-29 2018-08-14 Glaxosmithkline Intellectual Property Development Limited Cyclic dinucleotides useful for the treatment of inter alia cancer
WO2019122050A1 (en) 2017-12-22 2019-06-27 Isa Pharmaceuticals B.V. Methods of immunization
US10450341B2 (en) 2014-06-04 2019-10-22 Glaxosmithkline Intellectual Property Development Limited Cyclic di-nucleotides as modulators of STING
WO2021110919A1 (en) 2019-12-07 2021-06-10 Isa Pharmaceuticals Treatment of diseases related to hepatitis b virus
EP3762010A4 (en) * 2018-03-06 2022-04-06 Precigen, Inc. Hepatitis b vaccines and uses of the same
WO2023285412A1 (en) 2021-07-12 2023-01-19 Isa Pharmaceuticals B.V. Improved substance quantification in complex mixtures
WO2023143445A1 (en) * 2022-01-25 2023-08-03 厦门大学 Epitope peptide and antibody for treating hbv infection and related diseases
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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EP3879556A1 (en) * 2020-03-11 2021-09-15 ABB Schweiz AG Power component including a main component and a sensor and emitter unit and system with the power component

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061065A1 (en) 1998-05-23 1999-12-02 Leiden University Medical Center Cd40 binding molecules and ctl peptides for treating tumors
WO2002070006A2 (en) 2000-12-08 2002-09-12 Academisch Ziekenhuis Leiden Long peptides of 22-45 amino acid residues that induce and/or enhance antigen specific immune responses
WO2003084999A1 (en) 2002-04-04 2003-10-16 Leiden University Medical Center Induction of anti-tumor ctl immunity through in vivo triggering of 4-1bb and/or cd40
WO2006034545A1 (en) * 2004-09-28 2006-04-06 Melbourne Health Variants of hepatitis b virus with resistance to anti-viral nucleoside agents and applications thereof
WO2006097285A1 (en) * 2005-03-15 2006-09-21 Innogenetics N.V. Hepatitis-b viral variants with reduced susceptibility to nucleoside analogs and uses thereof
US20090311283A1 (en) * 1992-01-29 2009-12-17 Pharmexa Inc. Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions
WO2010017209A2 (en) * 2008-08-04 2010-02-11 The Government Of The Usa As Represented By The Secretary Of The Department Of Health And Human Services Membrane proximal region of hiv gp41 anchored to the lipid layer of a virus-like particle vaccine
US20100068228A1 (en) * 1992-01-29 2010-03-18 Pharmexa Inc. Inducing Cellular Immune Responses to Hepatitis B Virus Using Peptide and Nucleic Acid Compositions
US20120149120A1 (en) * 2009-03-06 2012-06-14 Korea University Research And Business Foundation Chimeric protein, method for manufacturing the same, nano-sensor in which the chimeric protein is fixed, and application thereof
WO2013051936A1 (en) 2011-10-05 2013-04-11 Academisch Ziekenhuis Leiden Lumc Adjuvant compound

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6607727B1 (en) * 1991-08-26 2003-08-19 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitus B virus
ATE227118T1 (en) * 1993-08-02 2002-11-15 Scripps Research Inst PEPTIDES FOR INDURING A CYTOTOXIC T-LYMPHOCYTE RESPONSE DIRECTED AGAINST HEPATITIS B VIRUS
WO2001085208A2 (en) * 2000-05-05 2001-11-15 Cytos Biotechnology Ag Molecular antigen arrays and vaccines
JP2006501827A (en) * 2002-10-03 2006-01-19 エピミューン インコーポレイテッド Optimized multi-epitope constructs and uses thereof
CN101361969B (en) * 2008-01-29 2011-05-11 广州市恺泰生物科技有限公司 Therapeutic hepatitis b vaccine and preparation method and use thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090311283A1 (en) * 1992-01-29 2009-12-17 Pharmexa Inc. Inducing cellular immune responses to hepatitis B virus using peptide and nucleic acid compositions
US20100068228A1 (en) * 1992-01-29 2010-03-18 Pharmexa Inc. Inducing Cellular Immune Responses to Hepatitis B Virus Using Peptide and Nucleic Acid Compositions
WO1999061065A1 (en) 1998-05-23 1999-12-02 Leiden University Medical Center Cd40 binding molecules and ctl peptides for treating tumors
WO2002070006A2 (en) 2000-12-08 2002-09-12 Academisch Ziekenhuis Leiden Long peptides of 22-45 amino acid residues that induce and/or enhance antigen specific immune responses
WO2003084999A1 (en) 2002-04-04 2003-10-16 Leiden University Medical Center Induction of anti-tumor ctl immunity through in vivo triggering of 4-1bb and/or cd40
WO2006034545A1 (en) * 2004-09-28 2006-04-06 Melbourne Health Variants of hepatitis b virus with resistance to anti-viral nucleoside agents and applications thereof
WO2006097285A1 (en) * 2005-03-15 2006-09-21 Innogenetics N.V. Hepatitis-b viral variants with reduced susceptibility to nucleoside analogs and uses thereof
WO2010017209A2 (en) * 2008-08-04 2010-02-11 The Government Of The Usa As Represented By The Secretary Of The Department Of Health And Human Services Membrane proximal region of hiv gp41 anchored to the lipid layer of a virus-like particle vaccine
US20120149120A1 (en) * 2009-03-06 2012-06-14 Korea University Research And Business Foundation Chimeric protein, method for manufacturing the same, nano-sensor in which the chimeric protein is fixed, and application thereof
WO2013051936A1 (en) 2011-10-05 2013-04-11 Academisch Ziekenhuis Leiden Lumc Adjuvant compound

Non-Patent Citations (51)

* Cited by examiner, † Cited by third party
Title
"Nucleic Acid Hybridization"
"Oligonucleotide Synthesis"
"Remington; The Science and Practice of Pharmacy", 2005
"Remington; The Science and Practice of Pharmacy", 2005, UNIVERSITY OF SCIENCES IN PHILADELPHIA
ATHERTON, E.; SHEPPARD, R.: "Solid Phase Peptide Synthesis: A Practical Approach", 1989, IRL PRESS
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", vol. 1, 2, 1994, CURRENT PROTOCOLS
BARANY, G.; MERRIFIELD, R: "The Peptides", vol. 2, 1979, ACADEMIC PRESS, pages: 1 - 284
BROWN: "Molecular Biology LabFax", vol. I, II, 1998, ACADEMIC PRESS
BUCKANOVICH RJ ET AL., NATURE MEDICINE, vol. 14, 2008, pages 28
BUI ET AL., BMC BIOINFORMATICS, vol. 7, 2006, pages 153
CHAPIRO J ET AL., J IMMUNOL, vol. 176, 2006, pages 1053 - 1061
CRAIU A ET AL., PROC NATL ACAD SCI U S A, vol. 94, 1997, pages 10850 - 10855
FIELDS, G.B, METHODS ENZYMOL., vol. 289, 1997
GANEM ET AL., N ENGL J MED, vol. 350, 2004, pages 1118 - 1129
GRIMM ET AL., CLIN SCI (LOND, vol. 124, 2013, pages 77 - 85
H. L. ZAAIJER ET AL: "Substitution rate of the hepatitis B virus surface gene", JOURNAL OF VIRAL HEPATITIS, vol. 15, no. 4, 1 April 2008 (2008-04-01), pages 239 - 245, XP055133720, ISSN: 1352-0504, DOI: 10.1111/j.1365-2893.2007.00938.x *
HEPATITIS B. WORLD HEALTH ORGANIZATION. FACT SHEET, vol. 204, July 2013 (2013-07-01), Retrieved from the Internet <URL:http://www.who.int/mediacentre/factsheets/fs204/en>
HUANG ET AL., CURR OPIN IMMUNOL, vol. 23, 2011, pages 237 - 243
ISHIKAWA K, PNAS, vol. 91, 1994, pages 4892
KATO ET AL., IMMUNITY, vol. 1, 2005, pages 19 - 28
KESSLER JH ET AL., HUM IMMUNOL, vol. 64, 2003, pages 245 - 255
LANZAVECCHIA, NATURE, vol. 393, 1998, pages 413
LOK AS, N ENGL J MED, vol. 346, 2002, pages 1682 - 1683
LUNDEGAARD C ET AL., IMMUNOLOGY, vol. 130, 2010, pages 309
MICHEL ML ET AL., J HEPATOL, vol. 54, 2011, pages 1286 - 1296
MICHEL ML ET AL., J. HEPATOL, vol. 34, 2001, pages 917 - 921
MO XY ET AL., J IMMUNOL, vol. 163, 1999, pages 5851 - 5859
MOREL S ET AL., IMMUNITY, vol. 12, 2000, pages 107 - 117
NIELSEN M ET AL., IMMUNOGENETICS, vol. 57, 2005, pages 33
NIELSEN M ET AL., IMMUNOME RES, vol. 6, 2010, pages 9
RIDGE ET AL., NATURE, vol. 393, 1998, pages 474
ROCK ET AL., NAT. IMMUNOL., vol. 5, 2004, pages 670
ROSALIA ET AL., EUR J IMMUNOL, vol. 43, 2013, pages 2554 - 2565
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK; RUSSELL: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SCHIRLE M ET AL., EUR J IMMUNOL, vol. 30, 2000, pages 2216 - 2225
SCHIRLE M ET AL., J IMMUNOL METHODS, vol. 257, 2001, pages 1 - 16
SCHOENBERGER ET AL., NATURE, vol. 393, 1998, pages 480
STOLTZE L ET AL., EUR J IMMUNOL, vol. 28, 1998, pages 4029 - 4036
SUN ET AL., NAT. IMMUNOL, vol. 5, 2004, pages 927
THIMME ET AL., J VIROL, vol. 77, 2003, pages 68 - 76
TOES ET AL., J. IMMUNOL., vol. 156, 1996, pages 3911
TOES ET AL., PROC.NATL.ACAD.SCI.U.S.A, vol. 93, 1996, pages 7855
VAN DER BURG SH ET AL., HUM IMMUNOL., vol. 44, 1995, pages 189
VAN DER BURG SH ET AL., J. IMMUNOL, vol. 156, no. 9, 1996, pages 3308 - 14
VAN DER BURG SH ET AL., PNAS, vol. 104, 2007, pages 12087
VIATTE S ET AL., IMMUNOL CELL BIOL, vol. 84, 2006, pages 318 - 330
WANG P ET AL., PLOS COMPUT BIOL, vol. 4, 2008, pages EL000048
ZEESTRATEN ET AL., INT J CANCER., vol. 132, no. 7, 1 April 2013 (2013-04-01), pages 1581 - 91
ZOULIM ET AL., B. J HEPATOL, vol. 56, no. 1, 2012, pages S112 - S122
ZWAVELING ET AL., J. IMMUNOL., vol. 169, 2002

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