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WO2025172435A1 - Formulation d'antigène du vhb pour le traitement de l'hépatite b - Google Patents

Formulation d'antigène du vhb pour le traitement de l'hépatite b

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Publication number
WO2025172435A1
WO2025172435A1 PCT/EP2025/053853 EP2025053853W WO2025172435A1 WO 2025172435 A1 WO2025172435 A1 WO 2025172435A1 EP 2025053853 W EP2025053853 W EP 2025053853W WO 2025172435 A1 WO2025172435 A1 WO 2025172435A1
Authority
WO
WIPO (PCT)
Prior art keywords
hbv
hbsag
dose
hbcag
amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/053853
Other languages
English (en)
Inventor
Ulrike Protzer
Anna KOSINSKA
Jinpeng SU
Frank Thiele
Marian WIEGAND
Carlos Guzman
Thomas Ebensen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Original Assignee
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Helmholtz Zentrum fuer Infektionsforschung HZI GmbH, Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH filed Critical Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Publication of WO2025172435A1 publication Critical patent/WO2025172435A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • 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
    • 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
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • 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

  • HBV antigen formulation for treating Hepatitis B HBV antigen formulation for treating Hepatitis B
  • the invention relates to methods of vaccination, comprising administering to a human subject a first dose of an HBcAg particle, an HBsAg, and c-di-AMP; a second dose of the HBcAg particle, the HBsAg, and c-di-AMP; and a dose of a vaccine vector.
  • the invention also relates pharmaceutical compositions, combinations of HBcAg particles, HBsAg and c-di-AMP, and kits for use in said methods.
  • chimpanzees were only able to cure an acute HBV infection, when functional CD4+ and CD8+ T-cell responses were restored after antibody-mediated depletion (cf. , e.g. Thimme et al., CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection, J Virol, 2003. 77(1): p. 68-76).
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an HBcAg particle of the disclosure, an HBsAg of the disclosure, and c-di-AMP, and optionally a pharmaceutically acceptable carrier or excipient.
  • the present invention also relates to a combination comprising an HBcAg particle of the disclosure, an HBsAg of the disclosure, and c-di-AMP, and optionally a pharmaceutically acceptable carrier or excipient.
  • the present invention also relates to a kit comprising a first pharmaceutical composition comprising a HBcAg particle of the disclosure, a second pharmaceutical composition comprising an HBsAg of the disclosure, and c-di-AMP.
  • the present invention also relates to an HBcAg particle of the disclosure, an HBsAg of the disclosure, c-di-AMP, and/or a vaccine vector of the disclosure, for use in a method of vaccination.
  • the present invention also relates to a use of an HBcAg particle of the disclosure, an HBsAg of the disclosure, c-di-AMP, and/or a vaccine vector of the disclosure, in the manufacture of a medicament, wherein the medicament is for vaccination.
  • HBV vaccines provide a better protection against HBV of the same (sub)genotype as the HBV antigens comprised in a given vaccine than to other (sub)genotypes.
  • the present invention is directed at an efficient HBV vaccination method using components that ensure protection against more than one HBV (sub-) genotype.
  • Hepatitis B Virus core antigen (HBcAg) particle of the disclosure is provided.
  • said novel therapeutic vaccination regime herein optionally also referred to as VacB (or VacB vaccination regime; cf. e.g. Figure 1)
  • VacB or VacB vaccination regime; cf. e.g. Figure 1
  • the present disclosure also provides a therapeutic vaccination comprising a novel (particulate) protein prime and a (recombinant) vaccine vector boost regimen.
  • HBsAg preferably adjuvanded with c-di-AMP may induce HBV core- or S-specific CD4+ helper T-cell in peripheral blood lymphocytes and/or CD4+ T cell cytokines in the blood.
  • These lymphocytes and/or cytokines may support CD8+ T-cells and neutralizing anti-HBs antibodies that complex circulating HBV antigens and thus, preferably help to counteract potential skewing of T-cell responses by (high) antigen levels.
  • the therapeutic vaccination regime according to the present invention relates to a promising novel approach, preferably with broad applicability and an acceptable risk of side effects.
  • the underlying concept of restoring endogenous immune functions by the therapeutic vaccination regime according to the present invention does preferably not only allow for a cure of an HBV infection, but may preferably also be favorable over costly and cumbersome long-term application of antiviral drugs, which aim at controlling rather than curing an HBV infection. Also, such a long-term application of antiviral drugs requires the patient’s compliance, thus bearing the risk of discontinuous uptake, developing resistance and/or undesired side effects.
  • the novel therapeutic vaccination regime according to the present invention may preferably pave the way to a new treatment strategy that, for the first time, allows to treat, and preferably also to cure, an HBV infection.
  • the HBcAg particle may comprise HBV core proteins from two, three, four or more genotypes.
  • the HBcAg particle may comprise HBV core proteins from at least two genotypes selected from the group consisting of A, B, C, D, E, F, G, H and I.
  • the HBcAg particle may comprise HBV core proteins from two, three, four or more genotypes selected from the group consisting of A, B, C, D, E, F, G, H and I.
  • the term “antigen” refers to a molecule which contains one or more epitopes that stimulate a host's immune system to make a cellular antigen-specific immune response, or a humoral antibody response.
  • Antigens may include proteins, polypeptides, antigenic protein fragments and the like.
  • the antigen can be derived from any known virus, bacterium, parasite, prion, plants, protozoans, or fungus and can be a whole organism.
  • the term also includes tumor antigens. Synthetic antigens such as polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens are also included in this application.
  • the antigen in the present invention is a polypeptide or protein.
  • HBVAg may thus relate herein to a full-length HBV core protein.
  • the term may relate herein to a truncated HBV core protein and thus, a fragment of an HBV core protein.
  • a fragment is C-terminally truncated, i.e. lacking at least one C-terminal amino acids, compared to a respective full-length HBV core protein, more preferably such a fragment is C-terminally truncated, i.e. lacking at least one C-terminal amino acid, compared to a respective full-length HBV core protein.
  • the HBV genotypes are selected from the group consisting of C and D.
  • the HBcAg particle may comprise HBV core proteins from at least two different HBV genotypes selected from the group consisting of C and D.
  • the HBcAg particle may comprise, or consist of, HBV core proteins from genotypes C and D. This may be particularly advantageous as thus broad immune response covering >95% of circulating HBV strains can be covered.
  • the HBcAg particle may comprise HBV core proteins of in total two genotypes, wherein a first portion of the HBV core proteins of one of the two genotypes is truncated and a second portion of the HBV core proteins of the other of the two genotypes is truncated.
  • the HBcAg particle may comprise HBV core proteins of more than two genotypes, wherein a first portion of the HBV core proteins of one of the genotypes is truncated and the HBV core proteins of the other of the genotypes are truncated.
  • the HBcAg particle may comprise HBV core proteins of two or more genotypes, wherein a first portion of the HBV core proteins of one of the genotypes is truncated and the HBV core proteins of the other of the genotypes are full length.
  • the HBcAg particle may comprise HBV core proteins of two or more genotypes, wherein all HBV core proteins are full length.
  • the HBcAg particle may comprise full-length and truncated HBV core proteins.
  • the HBcAg particle may not comprise full-length HBV core proteins or may consist of truncated HBV core proteins.
  • the HBcAg particle comprises truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle may comprise HBV core proteins, wherein at least a portion of the HBV core proteins of HBV genotype C are truncated HBV core proteins and wherein HBV core proteins of HBV genotype D are full-length HBV core proteins.
  • the truncated HBV core proteins from HBV genotype C refer to a deletion of the C-terminal 20aa. Thus, no antigenic epitope is lost by the deletion of the 20aa in genotype C monomers compared to full length genotype D monomers.
  • a truncation is preferably chosen to not impair the immunogenicity of the HBcAg particle compared to an HBcAg particle comprising no truncated but only full-length HBV core proteins of the same HBV genotypes.
  • the HBcAg particle does not comprise full-length HBV core proteins from HBV genotype C but truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle thus consists of truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle comprises truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D (or less preferred vice versa).
  • the HBV core proteins from said HBV genotypes are preferably in a ratio from about 10:90 to about 90:10, about 20:80 to about 80:20, about 30:70 to about 70:30, about 40:60 to about 60:40, or in an approximately equimolar ratio.
  • HBV core proteins comprised in the HBcAg particle may be observed in an approximate ratio of about 30:70 to about 70:30, about 40:60 to about 60:40, or about 50:50 as regards the genotypes C and D.
  • the HBcAg particle is preferably a self-assembling particulate capsid that may comprise about 50 to 200 dimers of HBV core proteins.
  • the HBcAg particle preferably comprises - or consists of - dimers of HBV core proteins, wherein said dimers are assembled from i) HBV core proteins from HBV genotype C, ii) HBV core proteins from HBV genotype C and HBV core proteins from HBV genotype D, and/or iii) HBV core proteins from HBV genotype D.
  • the HBcAg particle preferably comprises dimers of HBV core proteins, wherein said dimers are assembled from i) truncated HBV core proteins from HBV genotype C, ii) truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D, and/or iii) full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle even consists of dimers of HBV core proteins, wherein said dimers are assembled from i) truncated HBV core proteins from HBV genotype C, ii) truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D, and/or iii) full-length HBV core proteins from HBV genotype D.
  • said HBV genotypes are in an approximately equimolar ratio, thus enabling the HBcAg particle to induce an, preferably comparablen immune response against HBV core proteins of both genotypes C and D while allowing to distinguish the two HBV core protein types and thus, to assess the HBcAg particle's HBV core protein composition analytically.
  • said proteins are preferably defined based on their sequence identity to a given reference sequence.
  • Techniques for determining sequence identity between two sequences of nucleic acids or amino acids are well known and established in the art. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • Percent (%)sequence identity with respect to antigens, epitopes and/or proteins described herein is preferably defined on amino acid level and thus, as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the respectively specified reference sequence (i.e. the antigen from which it is derived and/or to which it is compared), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The same is applicable to nucleotide sequences, mutatis mutandis.
  • nucleic acid sequences are provided by the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2: 482-489. This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6): 6745-6763. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application.
  • BLAST BLAST
  • Another alignment program is BLAST, used with default parameters.
  • the HBV core proteins from HBV genotype C have preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13.
  • the HBV core proteins from HBV genotype D have at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 14.
  • the HBV core proteins from HBV genotype C have preferably at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or HBV core proteins from HBV genotype D have at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 14.
  • the HBcAg particle of the disclosure comprises or consists of i) HBV core proteins from HBV genotype C having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or ii) HBV core proteins from HBV genotype D having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 14.
  • a truncated HBV core protein from HBV genotype C is a truncated HBV core protein that consists of amino acids 1 to 149 or 1 to 163 of the respective full-length HBV core protein set forth in SEQ ID NO: 8, thus having the sequence set forth in SEQ ID NO: 1 or 13.
  • the HBcAg particle of the present disclosure comprises preferably immunogenic HBV core proteins, or fragments thereof, of at least two different HBV genotypes.
  • the HBcAg particle preferably induces an immune response against the comprised antigenic HBV core proteins of multiple HBV genotypes.
  • the HBcAg particle is preferably capable of inducing i) an immune response against the HBV core proteins it is composed of and/or ii) an antigen-specific adaptive immune response.
  • said immune response is associated with i) anti-HBcAg antibody induction and/or with ii) HBcAg- specific CD4+/CD8+ T-cell induction.
  • strong polyclonal and multi-specific CD8+ and CD4+ T-cell responses can preferably be induced.
  • Adaptive immune responses are the basis for effective immunization against infectious diseases like HBV with cells of the adaptive immune system including B cells, which differentiate into plasma cells to produce antibodies, and antigen-specific T cells. Said T cells, ca n be stimulated to differentiate, e.g., into cytotoxic CD8+ cells or CD4+ T-helper cells. CD8+ cytotoxic T cells are primarily involved in the destruction of cells infected by foreign agents, such as viruses. Upon resolution of the infection, few of these stimulated and differentiated CD8+ cells are retained as memory cells that can quickly differentiate into effector cells upon subsequent encounters with the same antigen.
  • the present disclosure relates also to a pharmaceutical composition
  • a pharmaceutical composition comprising the HBcAg particle as disclosed herein above and optionally a pharmaceutically acceptable carrier or excipient.
  • additional factors and/or agents may be included in the pharmaceutical composition comprising the HBcAg particle as disclosed herein to preferably produce a synergistic effect and/or minimize side-effects.
  • the pharmaceutical composition may comprise one or more excipient and/or one or more pharmaceutically acceptable and/or approved carrier as additive, optionally also one or more selected from the group consisting of an adjuvant, a preservative, an antibiotic, a diluent, peptides and/or a stabilizing excipient.
  • Such auxiliary substances can be, e.g., water, saline, glycerol, ethanol, wetting or emulsifying agents, a detergent, an amino acid, a sugar, a surfactant, such as a kolliphor, pH buffering substances, or the like.
  • the term "pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the HBcAg particle according to the present disclosure but rather stabilizes it against environmental stress.
  • the characteristics of the carrier will depend on the route of administration and whether the vaccine antigens are lyophilized or used in solution.
  • the pharmaceutical composition may further contain other agents which either enhance the activity or use in treatment.
  • Suitable pharmaceutically acceptable carriers and/or excipients are typically large, slowly metabolized molecules such as modified nucleic acids, proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.
  • the pharmaceutical composition comprising the HBcAg particle as disclosed further comprises an HBV surface antigen (HBsAg), wherein the HBsAg is preferably an particulate HBsAg.
  • HBV surface antigen HBsAg
  • the pharmaceutical composition can be considered an efficient component of an effective HBV infection therapy.
  • HBsAg and HBcAg particle are preferably comprised both in the pharmaceutical composition, it may also be envisioned that the HBcAg particle is comprised in a first pharmaceutical composition and the HBsAg in a second pharmaceutical composition, wherein said first and said second pharmaceutical composition can be administered separately, approximately simultaneously or simultaneously.
  • said first and said second pharmaceutical compositions are mixed together before, preferably directly before, administration, e.g. via intramuscular injection.
  • a “HBV surface antigen” or “HBsAg” refers to a transmembrane protein of HBV that forms the viral envelop, which comprises a cell-derived lipid bilayer with embedded HBV surface (HBs) proteins, or a fragment thereof.
  • HBsAg may comprise the S protein only, but may also comprise one or more pre-S regions, such as pre-S1 region and/or pre- S2 region.
  • HBsAg may contain the small (S) as well as the middle (M) and large (L) envelope proteins.
  • HBV surface proteins confer the binding of the HBV virion to their respective receptors, NTCP, e.g., on hepatocytes.
  • HBV surface antigens represent the antigenic component of all prophylactic HBV vaccines to date. More specifically, an HBV surface protein may relate on any one of the three variants, the small (S), middle (M), and large (L) surface protein, which are translated from distinct mRNAs. Common to all three variants, is S protein containing the “a” determinant that is located at codon positions 124 to 147 within the major hydrophilic region (MHR) of the S gene.
  • MHR major hydrophilic region
  • the pre-S/S gene has three in-frame initiation codons and encodes the small (S) as well as the middle (M) and large (L) envelope proteins, which contain pre-S2 and pre-S (pre-S1 and pre-S2) sequences, respectively.
  • the M protein is an extension of the S protein, with an additional 55 amino acids (i.e., pre-S2 region)
  • the L protein is an extension of the M protein, with an additional 108-119 amino acids depending on the genotype (i.e. pre-S1 region).
  • the amino acids sequence at the C terminus of the L and M protein, respectively, is identical to the S protein and is referred to as the S region.
  • the pre-S (pre-S1 and pre-S2) region of the L protein may be crucial for viral replication.
  • An HBsAg of the disclosure is preferably an antigen composed of HBV surface protein monomers or dimers, or a, preferably immunogenic, fragment thereof.
  • the HBsAg is preferably a particulate antigen.
  • A, preferably immunogenic, fragment of a surface protein relates to proteins or peptides derived from any full-length surface protein of any HBV serotype or genotype that is N-terminally and/or C-terminally shortened, i.e. lacking at least one of the N- terminal and/or C-terminal amino acids.
  • the CpG adjuvant CpG-1018 is an unmethylated cytosine phosphoguanosine (CpG) enriched oligodeoxynucleotide (ODN) immunostimulatory adjuvant that mediates its effect by binding to TLR9.
  • CpG-1018 may have the advantage of being comprised for example in the commercially available HEPLISAV-B® and thus, represents a well- studied adjuvant for a HBsAg.
  • the present disclosure relates also to a container comprising one or more doses of the pharmaceutical composition comprising the HBcAg particle as disclosed herein above.
  • said container may represent a packing unit of the pharmaceutical composition as described herein above.
  • a “dose”, and more specifically an “effective dose” or even more specifically, a “therapeutically effective dose”, refers herein to that amount of a given compound, ingredient and/or therapeutic agent that is sufficient to result in amelioration of symptoms, e.g. treatment, healing, prevention or amelioration of a given condition like an HBV infection.
  • an amount of a given compound, ingredient and/or therapeutic agent is an amount sufficient to effect beneficial or desired effects of a treatment.
  • said doses may preferably refer to a therapeutic effect of a given compound or ingredient like an HBcAg particle or an HBsAg, wherein said therapeutic effect is preferably a curative effect.
  • a prophylactic effect may be encompassed.
  • Effective doses affecting the immune response vary depending upon many different factors, including the type of antigen or vaccine, means of administration, addition of adjuvant, target site, whether the subjects human or an animal, and whether treatment is prophylactic or curative. However, the skilled person is aware of suitable techniques to assess therapeutically effective doses for a given combination of component, route of administration etc. Preferred doses of the HBcAg particle or the HBsAg are disclosed herein further below.
  • An effective dose can be administered in one or more individual administrations like intramuscular injections. Furthermore, a dose can be administered alone with one agent or in combination with one or more additional agents.
  • the present invention relates also to a kit comprising the pharmaceutical composition comprising the HBcAg particle as disclosed herein above and a second pharmaceutical composition comprising an HBsAg, and c-di-AMP, wherein the c-di-AMP can be comprised in a third pharmaceutical composition and/or in the first and/or second pharmaceutical composition. More specifically, in case of said kit, the pharmaceutical composition comprising the HBcAg particle as disclosed herein above does preferably not further comprise an HBsAg.
  • the second pharmaceutical composition further comprises an adjuvant.
  • said adjuvant is preferably a cyclic dinucleotide.
  • Said adjuvant is particularly preferred c-di-AMP.
  • the kit may comprise a first container comprising one or more doses of the pharmaceutical composition comprising the HBcAg particle and a second container comprising one or more doses of the second pharmaceutical composition.
  • the kit may comprise a first container comprising one or more doses of a pharmaceutical composition comprising both, an HBsAg and an HBcAg as disclosed herein.
  • the kit may optionally comprise a third container comprising an adjuvant.
  • said kit may represent another version of a packing unit, wherein the HBcAg particle, the HBsAg and the adjuvant are comprised in different containers and wherein it is envisioned that said components can be administered, e.g., separately, approximately simultaneously or simultaneously.
  • said pharmaceutical compositions are either lyophilized or formulated together or mixed together before, preferably directly before, administration, e.g. via intramuscular injection.
  • the adjuvant comprised in the second pharmaceutical composition and/or the adjuvant comprised in the third container is a cyclic dinucleotide, preferably c-di-AMP.
  • the adjuvant preferably the same applies as stated above herein in the context of the pharmaceutical composition comprising the HBcAg particle disclosed herein.
  • a dose of the pharmaceutical composition comprises the HBcAg particle in an amount from about 10pg to about 100pg, preferably in an amount from about 30pg to about 90pg, more preferably in an amount from about 40pg to about 75pg, most preferably in an amount of about 40pg or of about 75pg.
  • a dose of the pharmaceutical composition in case of the container and/or a dose of the second pharmaceutical composition in case of the kit comprises HBsAg, wherein the HBsAg is preferably a particulate HBsAg.
  • a dose of the pharmaceutical composition comprises HBsAg in an amount from about 10pg to about 100pg, preferably from about 20pg to about 80pg, preferably in an amount from about 30pg to about 60pg, more preferably in an amount from 30pg or of about 60pg.
  • kit and container may be understood as referring to different packaging units of the pharmaceutical composition comprising the HBcAg of the present invention, the same applies mutatis mutandis in case of the kit.
  • a dose of the second pharmaceutical composition preferably comprises HBsAg in an amount from about 1Opg to about 1OOpg, preferably from about 20pg to about 80pg, preferably in an amount from about 30pg to about 60pg, more preferably in an amount from 30pg or of about 60pg.
  • a dose of c-di-AMP is preferably in an amount from about 10pg to about 100pg, preferably in an amount from about 20pg to about 80pg, more preferably in an amount from about 30pg to about 60pg, most preferably in an amount of about 30pg or of about 60pg, or in an amount from about 10pg to about 50 pg, preferably in an amount from about 10 pg to about 30pg, preferably in an amount from about 10 pg to about 20pg, most preferably in an amount of about 15pg.
  • a dose of the pharmaceutical composition preferably comprises the HBcAg particle in an amount of about 40pg or of about 75pg and a dose of the pharmaceutical composition comprises, preferably particulate, HBsAg in an amount of about 30pg or of about 60pg, and a dose of the pharpharmaceutical composition preferably comprises c-di- AMP in an amount of about 15pg, of about about 30pg or of about 60pg, preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 40pg and a dose of the pharmaceutical composition comprises, preferably particulate, HBsAg in an amount of about 30pg, and a dose of the pharmaceutical composition comprises c-di-AMP in an amount of 30pg; even more preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 75pg and a dose of the pharmaceutical composition comprises, preferably particulate, HBsAg in an
  • a dose of the pharmaceutical composition preferably comprises the HBcAg particle in an amount of about 40pg or of about 75pg and a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 30pg or of about 60pg, and a dose of the pharpharmaceutical composition preferably comprises c-di-AMP in an amount of about 15pg, or about 30pg or of about 60pg; preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 40pg and a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 30pg, and a dose of the pharmaceutical composition comprises c-di-AMP in an amount of 30pg; even more preferably, a dose of the pharmaceutical composition comprises the HBcAg
  • the present invention relates also to the disclosed HBcAg particle or the disclosed pharmaceutical composition comprising the HBcAg particle, or comprised in the disclosed container or in the disclosed kit, for use in therapy. More specifically, said components are preferably used in therapy and/or vaccination, preferably in therapeutic vaccination, preferably against HBV. Furthermore, it is to be noted that herein “therapy” and “therapeutic” may encompass both “cure” and “curative” as well as “prevention” and “preventive”. Thus, while the disclosed HBcAg particle or the disclosed pharmaceutical composition comprising the HBcAg particle, or comprised in the disclosed container or in the disclosed kit may be useful for preventing an HBV infection, preferably said components are preferably useful for curing an HBV infection.
  • the use is preferably in an immune stimulation method and/or in a vaccination method, preferably in a therapeutic vaccination method.
  • the HBcAg particle is preferably capable of inducing an immune response against HBV core proteins it is composed of such as HBV core proteins of two or more genotypes like C and D.
  • the disclosed HBcAg particle or the disclosed pharmaceutical composition comprising the HBcAg particle, or comprised in the disclosed container or in the disclosed kit are preferably well suited for use in an immune stimulation method and/or vaccination.
  • Immune stimulation can be measured by determining anti-HBs antibodies, for example using ELISA-based immunoassays.
  • the invention relates also to the disclosed HBcAg particle, the disclosed HBsAg and/or c-di-AMP or the disclosed pharmaceutical composition comprising the HBcAg particle, the disclosed HBsAg and/or c-di- AMP, optionally comprised in the disclosed container or in the disclosed kit, for use in therapy, weherin the use is in a therapeutic immune stimulation method, preferably in a therapeutic vaccination method, most preferably in a curative vaccination method.
  • the present invention relates also to the disclosed HBcAg particle, the disclosed HBsAg and/or c-di-AMP or the disclosed pharmaceutical composition comprising the HBcAg particle, the disclosed HBsAg and/or c-di-AMP, or comprised in the disclosed container or in the disclosed kit, for use in treating an HBV infection.
  • the innovative components may be especially advantageous for treating an HBV infection by stimulating an, preferably adaptive, immune response directed against the HBV core proteins and HBV surface proteins comprised therein.
  • treat refers to clinical intervention designed to alter the natural course of the subject being treated during the course of a physiological condition or disorder or clinical pathology.
  • a treatment may be a therapeutic treatment and/or a prophylactic or preventative measure, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development or spread of a hyperproliferative condition, such as cancer.
  • Desired effects of treatment include, but not limited to, decreasing the rate of disease progression, ameliorating or palliating the disease state, alleviating symptoms, stabilizing or not worsening the disease state, and remission of improved prognosis, whether detectable or undetectable.
  • the use preferably comprises inducing anti-HBcAg antibodies and/or inducing HBcAg-specific CD4+/CD8+ T-cells. Further, the use preferably enhances the induction of anti-HBs antibodies and HBsAg-specific CD4+/CD8+ T cells by infrastructural help.
  • such a vector system may comprise for example two (expression) vectors, wherein one of the two (expression) vectors encodes a full-length HBV core protein from HBV genotype D, and the other one of the two (expression) vectors encodes a truncated HBV core protein of HBV genotype C.
  • both (expression) vectors are introduced into a bacterial cell like an E. coli cell, the two HBV core proteins may be expressed and selfassemble into a (mosaic) HBcAg particle that can be isolated from the cell.
  • a multicistronic, e.g. expression, vector may be used that encodes for all different HBV core proteins that shall be comprised in the HBcAg particle of the disclosure.
  • a bicistronic plasmid may be used for generating a HBcAg particle comprising HBV core proteins from genotypes C and D.
  • a multicistronic vector may be advantageous for obtaining an approximately defined ratio of HBV core proteins like an approximately equimolar ratio of HBV core proteins from genotypes C and D in case of a bicistronic (expression) vector like a plasmid.
  • an expression cassette may be used, wherein the sequences encoding the different HBV core proteins are under the control of the same promoter.
  • expression cassette encompasses DNA as well as RNA sequences which are preferably capable of directing expression of a particular nucleotide sequence in an appropriate host cell like E. coli. In general, it comprises a promoter operably linked to a polynucleotide of interest, which is optionally operably linked to a termination signal and/or other regulatory elements.
  • the expression cassette may comprise a transcription regulating nucleotide sequence.
  • An expression cassette may also comprise sequences required for proper translation of the nucleotide sequence.
  • the expression cassette may be one, which is naturally occurring but has preferably been obtained in a recombinant form useful for heterologous expression.
  • the coding region usually codes for a protein of interest.
  • the expression cassette comprising the polynucleotide sequence of interest may also be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette is preferably capable of inducing transcription in respective host cells.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • Nucleic acid sequences disclosed herein and thus, encoding any HBV antigen, are preferably codon optimized.
  • a "codon-optimized" nucleic acid sequence refers to a nucleic acid sequence containing codons that are replaced by codons preferred by the desired host cell, preferably an E. coli and/or human host cell depending on the host.
  • a nucleic acid sequence is converted into a codon-optimized nucleic acid sequence having an identical translated polypeptide sequence, but with alternative codon usage, in particular using the most frequently codons of the host organism.
  • the method of creating a codon-optimized nucleic acid sequence of an antigen generally includes identifying codons in the naturally occurring sequence of an antigen that are commonly not associated with high expressing genes in the host and replacing them with codons that are known to be widely used in gene expression of the host.
  • a codon-optimized nucleic acid sequence may show improved expression over the naturally occurring sequence in the desired host cell. Whether a codon optimized sequence will induce an improvement in the protein production over the non-optimized sequence can be examined by a skilled person. Furthermore, also respective techniques for codon optimization are known in the art.
  • the present disclosure relates also to an expression cassette, an mRNA, or a cDNA, encoding an HBV core protein from HBV genotype C and an HBV core protein from HBV genotype D, wherein the expression cassette, mRNA, or cDNA, only comprises coding sequences for two or more HBV core proteins.
  • Such an expression cassette may be highly advantageous for the generation of a HBcAg particle of the present disclosure, wherein said particle comprises HBV core proteins from HBV genotypes C and D, preferably in a ratio where each core protein is represented by at least about 25%, perferably in a ratio where each core protein is represented by at least about 30%, preferably in an approximately equimolar ratio.
  • the expression cassette, mRNA, or cDNA preferably encodes an HBV core protein from HBV genotype C comprising a sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13.
  • the expression cassette, mRNA, or cDNA encodes an HBV core protein from HBV genotype D comprising a sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 14.
  • the expression cassette, mRNA, or cDNA preferably encodes i) an HBV core protein from HBV genotype C comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or ii) an HBV core protein from HBV genotype D comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 14.
  • the expression cassette, mRNA, or cDNA encodes i) an HBV core protein from HBV genotype C comprising a sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or ii) an HBV core protein from HBV genotype D comprising a sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 14.
  • the expression cassette, mRNA, or cDNA preferably comprises a first nucleotide sequence encoding an HBV core protein from HBV genotype C, wherein said first nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 or 15.
  • the expression cassette, mRNA, or cDNA comprises a second nucleotide sequence encoding an HBV core protein from HBV genotype D, wherein said second nucleotide sequence has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 16.
  • the expression cassette, mRNA, or cDNA preferably comprises of i) a first nucleotide sequence encoding an HBV core protein from HBV genotype C, wherein said first nucleotide sequence has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 or 15, and/or ii) a second nucleotide sequence encoding an HBV core protein from HBV genotype D, wherein said second nucleotide sequence has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 16.
  • the expression cassette, mRNA, or cDNA comprises i) a first nucleotide sequence encoding an HBV core protein from HBV genotype C, wherein said first nucleotide sequence has at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 or 15, and/or ii) a second nucleotide sequence encoding an HBV core protein from HBV genotype D, wherein said second nucleotide sequence has at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 16.
  • the expression cassette, mRNA, or cDNA comprises i) a first nucleotide sequence encoding an HBV core protein from HBV genotype C, wherein said first nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 3 or 15, and/or ii) a second nucleotide sequence encoding an HBV core protein from HBV genotype D, wherein said second nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 4 or 16.
  • the present disclosure relates also to a nucleic acid molecule comprising the disclosed expression cassette, mRNA, or cDNA.
  • said nucleic acid molecule may be a DNA molecule like a DNA based vector comprising the expression cassette, mRNA, or cDNA disclosed herein above. This may have advantages for introducing genetic information for generating the disclosed HBcAg particle in an expression system disclosed herein, such as E. coli.
  • said nucleic acid molecule may be an RNA molecule, preferably an mRNA molecule. Such an RNA molecule may be advantageous for a temporarily restricted generation of the disclosed HBcAg particle.
  • RNA molecule may relate to an mRNA molecule and thus, a transcript of an, e.g., DNA based vector comprising the expression cassette or cDNA disclosed herein. This may be the case, e.g., when splicing sites are encoded in the expression cassette between the HBV core protein encoding sequences comprised therein.
  • it may refer to a RNA, preferably an mRNA molecule that can be introduced into a cell in a manner comparable to RNA based vaccinations. This has may have advantage that genetic information is not permanently transferred into the target cell.
  • the nucleic acid molecule comprising the expression cassette is a DNA molecule, which is preferably transferred into a host cell, such as E. coli, for the generation of HBV core proteins that can self-assemble into particulate HBcAg particles that can be isolated as HBcAg particles according to the disclosure.
  • the present disclosure relates also to an expression vector comprising the disclosed expression cassette, the disclosed cDNA, or the disclosed nucleic acid molecule.
  • the expression vector is a recombinant vector.
  • This may be advantageous for using a well-established and well-characterized expression vector like a plasmid for efficient generation of HBcAg particles comprising HBV core proteins from genotypes C and D by recombinantly introducing the respective expression cassette, cDNA, or nucleic acid molecule into the vector backbone.
  • HBcAg particle generation may be optimized, e.g. also in view of the chosen expression system, and/or host cell. Suitable expression systems and/or host cells are disclosed herein.
  • the expression vector comprises a sequence that has the nucleotide sequence set forth in SEQ ID NO: 10.
  • the expression vector may be a bicistronic plasmid for generating a (mosaic) HBcAg particle comprising HBV core proteins from genotypes C and D.
  • the expression vector is a recombinant vector and/or a plasmid.
  • the present disclosure relates also to a host cell comprising the disclosed expression cassette, mRNA, cDNA, optionally comprised in the disclosed nucleic acid molecule and/or the disclosed expression vector, or the disclosed expression vector encoding an HBV core protein from HBV genotype C and an HBV core protein from HBV genotype D.
  • the host cell may be prokaryotic or eukaryotic, such as a bacterial cell, an insect cell, a yeast cell, or a mammalian cell.
  • the host cell is an E. coli cell or a Spodoptera frugiperda cell.
  • the present disclosure relates also to a vaccine vector, wherein the vaccine vector is preferably a modified vaccinia virus Ankara (MVA) viral vector, and wherein the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
  • a " vaccine vector” as used herein refers to a bacterial or viral vaccine vector, preferably a viral vaccine vector, which can be an attenuated MVA virus.
  • vector refers preferably to a virus and in particular to an MVA virus used as the carrier. Typically, this attenuated virus is used to introduce nucleic acid encoding for antigens to cells of the subject.
  • a vaccine vector of the disclosure may be a viral vector that may be a viral particle having infectivity, which is also a carrier for introducing a gene into a cell. Viral vaccine vectors are familiar to the person skilled in the art.
  • a (recombinant) MVA virus may be an MVA virus that is produced by standard genetic engineering methods.
  • MVA is particularly well-suited as vector system.
  • MVA is related to vaccinia virus, a member of the genera Orthopoxvirus, in the family of Poxviridae.
  • MVA was generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for review see Mayr, A., et al. Infection 3, 6-14 (1975)).
  • CVA Ankara strain of vaccinia virus
  • the genome of the resulting MVA virus had about 31 kilobases of its genomic sequence deleted and, therefore, was described as highly host cell restricted for replication to avian cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 (1991)).
  • MVA-F6 Primary Chicken Embryo Fibroblast
  • MVA The restricted host range of MVA may explain the non-virulent phenotype observed in vivo in a wide range of mammalian species including humans. Therefore, this MVA strain has been tested in clinical trials as a vaccine to immunize against the human smallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 (1987); Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 (1974)). These studies involved over 120,000 humans, including high-risk patients, and proved that, compared to vaccinia-based vaccines, MVA had diminished virulence and was well tolerated, while it still induced a good specific immune response.
  • MVA is a well suited, and herein preferred, vector system that is a safe, well tolerated and immunogenic vaccine platform preferably capable of inducing a multimodal humoral and cell-based immunological antigen response.
  • the expression cassette is the expression cassette encoding one or more HBV core proteins disclosed herein.
  • the expression cassette is not naturally occurring (i.e., heterologous or exogenous or foreign) in the MVA viral vector, though preferably capable of inducing transcription in respective host cells.
  • said expression cassette is typically generated by means of recombination, resulting in a recombinant MVA viral vector.
  • the promoter is preferably a poxviral promoter.
  • Such a poxviral promoter may be a natural occurring promoter or a synthetic promoter.
  • the poxvirus promoter may be a Pr7.5 promoter, a hybrid early/late promoter, a PrS promoter, a synthetic or natural early or late promoter such as one of the promoters described in WO 2010/102822 or in WO 2005/054484, or cowpox virus ATI promoter.
  • a preferred promoter is, e.g., the promoter PH5 as described in US 2011/0064769.
  • the expression cassette preferably comprises a. a nucleotide sequence encoding an HBsAg from HBV genotype A, which preferably comprises a sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 , b. a nucleotide sequence encoding an HBcAg from HBV genotype D, which preferably comprises a sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 12, c.
  • the expression cassette comprised in the vaccine vector preferably encodes for, and is thus preferably capable of expressing, two HBV surface proteins, two HBV core proteins as well as a polymerase from HBV or at least a RT domain of the HBV polymerase.
  • surface protein recited in a. may for example be an surface protein of HBV serotype adw, such as of HBV genotype A serotype adw, such as of HBV genotype A2 serotype adw2.
  • the core protein recited in b. may for example be a core protein of HBV serotype ayw, such as of HBV genotype D serotype ayw.
  • the disclosed expression cassette encodes for two HBsAg and two HBcAg with regions of high sequence similarity being naturally present between the two HBsAg encoding sequences as well as the two HBcAg encoding sequences.
  • the expression cassette sequence disclosed herein was modified via codon optimization specifically in view of sequence regions of high similarity. Sequence integrity was assessed over several, e.g. 6 to 7, serial MVA viral passages without any detection of a mutation including any recombination event within the expression cassette.
  • the disclosed sequence preferably has the advantage of ensuring equimolar expression of several HBV antigens of different HBV genotypes using a stable MVA vector with limited risk of homologous recombination within the expression cassette.
  • the term “MHBVac” refers to a vaccine vector with said vector being an MVA viral vector and having a genome set forth in SEQ ID NO: 6.
  • MHBVac comprises as an insert the nucleotide sequence set forth in SEQ ID NO: 5.
  • the MHBVac is preferably capable of expressing two different HBsAg, two different HBcAg and a RT domain of an HBV polymerase covering in total genotypes A, C, and D.
  • the MHBVac is particularly well suited for inducing and/or strengthening an immune response against multiple HBV antigens of different HBV genotypes.
  • the MHBVac refers preferably to a vaccine vector as disclosed herein.
  • the vaccine vector of the disclosure comprises a nucleic acid molecule comprising a nucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence that has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence that has the nucleotide sequence set forth in SEQ ID NO: 6.
  • the vaccine vector disclosed herein comprises preferably an expression cassette encoding multiple HBV antigens of different HBV genotypes. Moreover, it is envisioned that all of said HBV antigens are preferably translated as one single polypeptide chain comprising said HBV antigens as schematically depicted in Figure 2. On the polypeptide chain, antigen sequences are preferably separated by self-cleavage site sequences such as P2A or T2A. Thus, the polypeptide chain comprising several antigens will be post-translationally cleaved to multiple polypeptide chains, wherein each of the multiple polypeptide chains may comprise a single HBV antigen.
  • FIG. 3 Shown in Figure 3 is a preferred nucleotide sequence arrangement encoding a polypeptide chain comprising several antigens from N-terminus to C-terminus: a HBsAg from HBV genotype A/adw, a P2A site, a HBcAg from HBV genotype D/ayw, a P2A site, an immunogenic RT domain of a polymerase from HBV, a T2A site, an immunogenic HBsAg from HBV, a T2A site, and an immunogenic HBcAg from HBV.
  • the two different HBsAg will be located in a cellular membrane and may be secreted as subviral particles. These subviral particles may comprise both HBsAg that are from different HBV genotypes and may be taken up by antigen-presenting cells, which may increase the induced immune response.
  • the HBcAg may form particles and more specifically particulate capsids, wherein the capsids may be empty and may similarly comprise HBcAg from different HBV genotypes. Such a mosaic HBcAg particle will trigger an immune response against multiple HBV genotypes.
  • the polymerase will be degraded in the proteasome and presented by in an H LA context.
  • the disclosed arrangement may further have the advantage that most of the only partially processed proteins, i.e. proteins where a self-cleaving site has not been cleaved for example, will be incorporated into secreted particles, which are preferably capable of further increasing immune stimulation and/or of further enhancing and broadening the induced (adaptive) immune response
  • the present disclosure relates also to a pharmaceutical composition
  • a pharmaceutical composition comprising the vaccine vector disclosed herein above and optionally a pharmaceutically acceptable carrier or excipient.
  • said pharmaceutically acceptable carrier and/or excipients the same applies as stated herein above in the context of the pharmaceutical composition comprising the HBcAg particle disclosed herein above.
  • said pharmaceutical composition comprising the vaccine vector may comprise one or more excipient and/or one or more pharmaceutically acceptable and/or approved carrier as additive, optionally also one or more selected from the group consisting of an antibiotic, a preservative, an adjuvant, a diluent and/or a stabilizer.
  • Such auxiliary substances can be, e.g., water, saline, glycerol, ethanol, wetting or emulsifying agents, a detergent, an amino acid, a sugar, a surfactant, such as a kolliphor, pH buffering substances, or the like.
  • said pharmaceutically acceptable carrier or excipient preferably refer to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s), e.g. of the vaccine vector disclosed herein.
  • the characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition may further contain other agents which either enhance the activity or use in treatment.
  • Suitable pharmaceutically acceptable carriers and/or excipients are typically large, slowly metabolized molecules such as as modified nucleic acids, proteins, polysaccharides, polylactic acids, 33 dditionallyc acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. Such a pharmaceutically acceptable carrier or exciepient may be preferably advantageous in producing or supporting a synergistic effect and/or to minimize side-effects.
  • the present disclosure relates also to a container comprising one or more doses of the pharmaceutical composition comprising the disclosed vaccine vector, wherein a dose of said pharmaceutical composition comprises the vaccine vector in an amount from about 1x 10 A 7 infectious focus units (ifu) to about 1x 10 A 9 ifu, preferably in an amount from about 5x 10 A 7 ifu to about 1x 10 A 9 ifu, more preferably in an amount of about 6x 10 A 7 ifu or of about 3x 10 A 8 ifu or of about 5x 10 A 8 ifu, most preferably in an amount of about 3x 10 A 8 ifu. Determination of IFUs is well known to the skilled artisan, e.g.
  • the present disclosure relates also to the disclosed vaccine vector, or the disclosed pharmaceutical composition comprising the same, optionally comprised in the disclosed container, for use in therapy. More specifically, said components are preferably used in therapy and/or vaccination, preferably in therapeutic vaccination, preferably against HBV. As regards said therapy, the same applies as stated herein above in the context of the HBcAg particle and its respective pharmaceutical composition, optionally comprised in a disclosed kit or container.
  • the disclosed vaccine vector, or the disclosed pharmaceutical composition comprising the same, optionally comprised in the disclosed container may be advantageous for preventing and/or, even more, curing an HBV infection.
  • the disclosed vaccine vector, or the disclosed pharmaceutical composition comprising the same, optionally comprised in the disclosed container is preferably used in therapy and/or vaccination, preferably in therapeutic vaccination, preferably in therapeutic vaccination against HBV.
  • the use is preferably in an immune stimulation method and/or in a vaccination method, preferably in a therapeutic vaccination method.
  • the disclosed vaccine vector is preferably capable of inducing an immune response against HBV core proteins, HBV surface proteins and the RT domain of the HBV polymerase and thus, it is preferably capable of expressing antigens of different type and genotype.
  • the immune stimulation method and/or vaccination method preferably a therapeutic vaccination method, most preferably a curative vaccination method, the same applies as stated herein above in the context of the HBcAg particle and its respective pharmaceutical composition, optionally comprised in a kit or container.
  • the present disclosure relates also to the disclosed vaccine vector or the disclosed pharmaceutical composition comprising the vaccine vector, optionally comprised in the disclosed container, for use in treating an HBV infection.
  • the vaccine vector may be especially advantageous for treating an HBV infection by stimulating an, preferably adaptive, immune response directed against the HBV core proteins, HBV surface proteins and the RT domain of the HBV polymerase comprised therein, in particular an immune response against multiple HBV genotypes, which may be of particular relevance in some regions due to their (frequent) occurrence.
  • the use preferably comprises inducing anti-HBcAg antibodies and/or inducing HBcAg-specific CD4+/CD8+ T-cells, and also preferably comprises enhancing the induction of anti-HBsAg antibodies and HBsAg-specific CD4+/CD8+ T-cells, which may be caused by instrastructural help.
  • a dose of a vaccine vector wherein the vaccine vector expresses a. an HBsAg from HBV genotype A; b. an HBcAg from HBV genotype D; c. an HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; d. an HBcAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 17; and e. an RT comprising a sequence domain of a polymerase from HBV having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.
  • the HBcAg particle used in the method of vaccination of the invention is preferably the HBcAg particle disclosed herein.
  • said HbsAg preferably comprises HBV surface proteins of HBV subtype ayw or adw, genotype A, preferably serotype adw genotype A, more preferably serotype adw (genotype A2).
  • said HbsAg preferably comprises HBV surface proteins only of HBV genotype A, preferably of serotype adw genotype A, more preferably serotype adw (genotype A2).
  • said isolated HbsAg preferably comprises HBV surface proteins of HBV serotype adw, or only of HBV serotype adw.
  • the first dose of the HBcAg particle and of the HbsAg is preferably a first dose of a pharmaceutical composition disclosed herein, which comprises both, the HBcAg particle disclosed herein and the HbsAg disclosed hererin.
  • a pharmaceutical composition disclosed herein which comprises both, the HBcAg particle disclosed herein and the HbsAg disclosed hererin.
  • further comprised is c-di-AMP.
  • the first dose of the HBcAg particle and of the HbsAg may be a first dose of two individual pharmaceutical compositions of the disclosure, one comprising the HBcAg particle disclosed herein and the other comprising the HbsAg disclosed herein.
  • c-di-AMP may be comprised in the first and/or second pharmaceutical composition and/or in a third pharmaceutical composition.
  • the pharmaceutical composition(s) may be comprised in a container disclosed herein or in a kit disclosed herein.
  • the HbsAg comprises an amino acid sequence having at least 90% or 95%sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.
  • the HbsAg has the amino acid sequence set forth in SEQ ID NO: 11 .
  • the HBcAg is from HBV genotype D, preferably from HBV genotype D serotype ayw.
  • the HBcAg comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12.
  • the HBcAg has preferably an amino acid sequence having at least 90% or 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12.
  • the HBcAg has the amino acid sequence set forth in SEQ ID NO: 12.
  • the HbsAg comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7.
  • the HbsAg preferably comprises the amino acid sequence set forth in SEQ ID NO: 7.
  • said sequence represents a 400 amino acid long consensus sequence of a large surface protein of HBV genotype C.
  • SEQ ID NO: 7 is a consensus sequence of large surface proteins of genotype C strains, which was generated based on an alignment of 500 HBV sequences representing the worldwide distribution of HBV strains.
  • the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 100pg, the HBsAg in an amount from about 10pg to about 100pg, and c-di-AMP in an amount from about 10 pg to about 100 pg.
  • the first dose and the second dose each comprise the HBcAg particle in an amount from about 30pg to about 90pg, the HBsAg in an amount from about 20pg to about 80pg, and c-di-AMP in an amount from about 20 pg to about 80 pg or from about 10 pg to about 50 pg.
  • the dose of the vaccine vector comprises the vaccine vector in an amount from about 1x 10 A 7 ifu to about 1x 10 A 9 ifu, preferably in an amount from about 5x 10 A 7 ifu to about 5x 10 A 8 ifu, preferably in an amount from about 1x 10 A 8 ifu to about 5x 10 A 8 ifu, preferably in an amount from about 2x 10 A 8 ifu to about 4x 10 A 8 ifu, preferably in an amount of about 3x 10 A 8 ifu.
  • the disclosed vaccination method is preferably highly efficient in immune stimulation and/or in inducing a strong and broad immune response against multiple HBV antigens of different HBV geno- and/or serotypes that may be of particular relevance in some regions due to their (frequent) occurrence.
  • the method of vaccination is preferably inducing an immune response against the HBV antigens used for priming and boosting.
  • the method of vaccination is associated with induction of anti-HBs antibodies, which may be detectable by a decrease in HBsAg (in peripheral blood) of at least a factor of 2, preferably by >1log10 or preferably by HBsAg becoming undetectable in peripheral blood.
  • the method of vaccination is associated with a decrease in HBsAg (in peripheral blood) of at least a factor of 2, preferably by >1log10 or preferably by HBsAg becoming undetectable in peripheral blood.
  • a blood level decrease of HBsAg and I or the induction or alternatively an increase of anti-HBs antibodies can preferably be detected in serum or plasma of a given individual.
  • the present invention relates also to a use of an HBcAg particle, an HBsAg, c-di- AMP, and/or a vaccine vetor disclosed herein, for the manufacture of a medicament.
  • said medicament is a medicament for a method of vaccination, wherein said method preferably comprises administering to a human subject
  • the vaccine vector is preferably the vaccine vector disclosed herein.
  • the HBcAg particle is preferably the HBcAg particle disclosed herein.
  • the HBsAg is preferably the, preferably particulate, HBsAg disclosed herein, more preferably an HBsAg from HBV genotype A.
  • the method of vaccination is preferably the method of vaccination disclosed herein.
  • the HBcAg particle and optionally the HBsAg and/or the c-di- AMP, may be comprised in the respective disclosed pharmaceutical composition, optionally comprised in the respective disclosed container or in the respective disclosed kit.
  • the vaccine vector may be comprised in the respective disclosed pharmaceutical composition, optionally comprised in the respectively disclosed container.
  • the HBcAg particle is the HBcAg particle disclosed herein
  • the HBsAg is the HBsAg disclosed herein
  • the vaccine vector is the vaccine vector disclosed herein
  • the method of vaccination is the method of vaccination disclosed herein.
  • the present invention relates also to a use of a vaccine vector expressing a. an HBsAg from HBV genotype A; b. an HBcAg from HBV genotype D; c. an HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; d. an HBcAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 17; and e. an RT domain of a polymerase from HBV comprising a sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, for the manufacture of a medicament.
  • Said medicament is preferably a medicament for a method of vaccination, wherein said method preferably comprises administering to a human subject
  • the vaccine vector is preferably the vaccine vector disclosed herein.
  • the HBcAg particle is preferably the HBcAg particle disclosed herein.
  • the HBsAg is preferably the, preferably particulate, HBsAg disclosed herein, more preferably an HBsAg from HBV genotype A.
  • the method of vaccination is preferably the method of vaccination disclosed herein.
  • the HBcAg particle and optionally the HBsAg and/or c-di-AMP, may be comprised in the respective disclosed pharmaceutical composition, optionally comprised in the respective disclosed container or in the respective disclosed kit.
  • the vaccine vector may be comprised in the respective disclosed pharmaceutical composition, optionally comprised in the respectively disclosed container.
  • the HBcAg particle is the HBcAg particle disclosed herein
  • the HBsAg is the HBsAg disclosed herein
  • the vaccine vector is the vaccine vector disclosed herein
  • the method of vaccination is the method of vaccination disclosed herein.
  • the present invention relates also to a use of the expression cassette encoding an HBV core protein from HBV genotype C and an HBV core protein from HBV genotype D as disclosed herein, the nucleic acid sequence comprising the expression cassette as disclosed herein, the expression vector comprising the expression cassette as disclosed herein, and/or the expression vector encoding an HBV core protein from HBV genotype C and an HBV core protein from HBV genotype D as disclosed herein, for the manufacture of a medicament.
  • Said medicament is preferably a medicament for a method of vaccination, wherein said method preferably comprises administering to a human subject
  • a dose of a vaccine vector wherein the vaccine vector expresses a. an HBsAg from HBV genotype A; b. an HBcAg from HBV genotype D; c. an HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; d. an HBcAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 17; and e. an RT domain of a polymerase from HBV comprising a sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.
  • the vaccine vector is preferably the vaccine vector disclosed herein.
  • the HBcAg particle is preferably the HBcAg particle disclosed herein.
  • the HBsAg is preferably the, preferably particulate, HBsAg disclosed herein, more preferably an HBsAg from HBV genotype A.
  • the method of vaccination is preferably the method of vaccination disclosed herein.
  • the HBcAg particle and optionally the HBsAg and/or c-di-AMP, may be comprised in the respective disclosed pharmaceutical composition, optionally comprised in the respective disclosed container or in the respective disclosed kit.
  • the vaccine vector may be comprised in the respective disclosed pharmaceutical composition, optionally comprised in the respectively disclosed container.
  • the HBcAg particle is the HBcAg particle disclosed herein
  • the HBsAg is the HBsAg disclosed herein
  • the vaccine vector is the vaccine vector disclosed herein
  • the method of vaccination is the method of vaccination disclosed herein.
  • the invention is further characterized by the following items:
  • a method of vaccination comprising administering to a human subject
  • a dose of a vaccine vector wherein the vaccine vector expresses a. an HBsAg from HBV genotype A; b. an HBcAg from HBV genotype D; c. an HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; d. an HBcAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 17; and e. an RT domain of a polymerase from HBV comprising a sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.
  • first dose and the second dose each comprise the HBcAg particle in an amount from about 10 pg to about 100 pg, the HBsAg in an amount from about 10 pg to about 100 pg, and c-di-AMP in an amount from about 10 pg to about 100 pg.
  • first dose and the second dose each comprise the HBcAg particle in an amount from about 30 pg to about 90 pg, the HBsAg in an amount from about 20 pg to about 80 pg, and c-di-AMP in an amount from about 20 pg to about 80 pg or from about 10 pg to about 50 pg.
  • first dose and the second dose each comprise the HBcAg particle in an amount from about 60 pg to about 90 pg, the HBsAg in an amount from about 50 pg to about 70 pg, and c-di-AMP in an amount from about 50 pg to about 70 pg or from about 10 pg to about 20 pg.
  • first dose and the second dose each comprise the HBcAg particle in an amount of about 75 pg, the HBsAg in an amount of about 60 pg, and c-di-AMP in an amount of about 60 pg.
  • the dose of the vaccine vector comprises the vaccine vector in an amount from about 1x 10 A 8 ifu to about 5x 10 A 8 ifu.
  • the dose of the vaccine vector comprises the vaccine vector in an amount from about 2x 10 A 8 ifu to about 4x 10 A 8 ifu.
  • the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu.
  • the method of vaccination comprises administering the second dose about 1 week to about 8 weeks after the first dose.
  • the first dose and the second dose each comprise the HBcAg particle in an amount of about 75 pg, the HBsAg in an amount of about 60 pg, and c-di-AMP in an amount in an amount of about 60 pg
  • the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu
  • the second dose is administered about 4 weeks after the first dose
  • the dose of the vaccine vector is administered about 8 weeks after the first dose.
  • the first dose and the second dose each comprise the HBcAg particle in an amount of about 75 pg, the HBsAg in an amount of about 60 pg, and c-di-AMP in an amount in an amount of about 15 pg
  • the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu
  • the second dose is administered about 4 weeks after the first dose
  • the dose of the vaccine vector is administered about 8 weeks after the first dose.
  • the HBcAg particle comprises Hepatitis B Virus (HBV) core proteins from at least two different HBV genotypes.
  • HBV Hepatitis B Virus
  • the HBcAg particle is an isolated HBcAg particle.
  • the method of any one of the preceding items, wherein the HBcAg particle comprises HBV core proteins having HBV genotypes selected from the group consisting of A, B, C, and D.
  • the method of any one of the preceding items, wherein the HBcAg particle comprises HBV core proteins from the at least two different HBV genotypes that are in an approximately equimolar ratio.
  • the method of any one of the preceding items, wherein the HBcAg particle comprises HBV core proteins of not more than two different HBV genotypes.
  • the method of item 30, wherein the HBV genotypes are selected from the group consisting of C and D.
  • the HBcAg particle comprises truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle consists of truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle consists of truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D and wherein the HBV core proteins from said HBV genotypes are in a ratio of about 10:90 to about 90:10.
  • the method of any one of the preceding items, wherein the HBcAg particle is a selfassembling particulate capsid.
  • the method of any one of the preceding items, wherein the HBcAg particle comprises about 50 to 200 dimers of HBV core proteins and/or wherein the HBcAg particle has a size of about 20 to about 60 nm in diameter.
  • the HBcAg particle comprises about 80 to 100 dimers of HBV core proteins or about 110 to 130 dimers of HBV core proteins and/or wherein the HBcAg particle has a size of about 25 to about 50 nm, preferably a size of about 30 to about 34 nm in diameter.
  • the hydrodynamic radius of the HBcAg particle is about 40 nm to about 60 nm, preferably about 45 nm to about 55 nm, preferably about 48 nm to about 52 nm, preferably as measured by dynamic light scattering.
  • the HBcAg particle comprises about 90 dimers of HBV core proteins or about 120 dimers of HBV core proteins and/or wherein the HBcAg particle has a size of about 30 or about 34 nm in diameter.
  • the HBcAg particle comprises dimers of HBV core proteins, wherein said dimers are assembled from i) HBV core proteins from HBV genotype C, ii) HBV core proteins from HBV genotype C and HBV core proteins from HBV genotype D, and/or iii) HBV core proteins from HBV genotype D.
  • the HBcAg particle comprises dimers of HBV core proteins, wherein said dimers are assembled from i) truncated HBV core proteins from HBV genotype C, ii) truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D, and/or iii) full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle consists of dimers of HBV core proteins, wherein said dimers are assembled from i) truncated HBV core proteins from HBV genotype C, ii) truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D, and/or iii) full-length HBV core proteins from HBV genotype D.
  • the HBcAg particle is capable of inducing an immune response against the HBV core proteins it is composed of.
  • the method of any one of the preceding items, wherein the HBcAg particle is capable of inducing an antigen-specific adaptive immune response.
  • the method any one of the preceding items, wherein the HBsAg in (i) and/or (ii) is a particulate HBsAg.
  • the vaccine vector expresses in a. a HBsAg that is a small or large surface protein from HBV genotype A serotype adw.
  • a HBsAg that is a small surface protein from HBV genotype A serotype adw.
  • the vaccine vector expresses in c. a HBsAg that comprises the amino acid sequence set forth in SEQ ID NO: 7.
  • a HBcAg that comprises the amino acid sequence set forth in SEQ ID NO: 8 or 17.
  • the vaccine vector expresses in e. a RT domain that comprises the amino acid sequence set forth in SEQ ID NO: 9.
  • the vaccine vector is an MVA viral vector.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
  • the vaccine vector comprises a nucleic acid molecule comprising a.
  • nucleotide sequence encoding a HBsAg from HBV genotype A b. a nucleotide sequence encoding a HBcAg from HBV genotype D
  • a nucleotide sequence encoding a reverse transcriptase (RT) domain of a polymerase from HBV d. a nucleotide sequence encoding HBsAg from HBV genotype C, and e. a nucleotide sequence encoding HBcAg from HBV genotype C.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
  • the vaccine vector comprises a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 5.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence that has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6.
  • the vaccine vector comprises a nucleic acid molecule comprising a nucleotide sequence that has at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 6.
  • the vaccine vector comprises a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 6.
  • the first dose is administered to the human subject in a first injection, the second dose in a second injection, and/or the dose of the vaccine vector in a third injection, wherein the first, second and third injection are preferably intramuscular injections.
  • any one of the preceding items wherein the method is associated with a blood level increase of neutralizing and/or immune activating antibodies, wherein the antibodies are detectable in peripheral blood at a titer >10 lll/ml or wherein the blood level increase of neutralizing and/or immune activating antibodies results in a drop in HBsAg of at least about 11og10.
  • the method of any one of the preceding items, wherein the method is associated with a blood level increase of anti-HBsAg antibodies wherein the antibodies are detectable in peripheral blood at a titer of at least about 10 lll/ml or wherein the blood level increase of neutralizing and/or immune activating antibodies results in a drop in HBsAg of at least about 1 log 10.
  • any one of the preceding items wherein the method is associated with a reduction of HBsAg titers in peripheral blood by at least about 1 log 10 from start of the treatment, preferably as measured by chemiluminescent immunoassay (CLIA).
  • CLIA chemiluminescent immunoassay
  • a dose of the combination comprises the HBcAg particle in an amount of about 40 pg or about 75 pg, the HBsAg in an amount of about 30 pg or about 60 pg, and c-di-AMP in an amount of about 15 pg, about 30 pg or about 60 pg.
  • kits comprising a first pharmaceutical composition comprising a HBcAg particle preferably as defined in of any one of items 1-92, a second pharmaceutical composition comprising an HBsAg preferably as defined in any one of items 1-92, and c-di-AMP preferably as defined in any one of items 1-92.
  • a dose of the first pharmaceutical composition comprises the HBcAg particle in an amount from about 60 pg to about 90 pg
  • a dose of the second pharmaceutical composition comprises the HBsAg in an amount from about 50 pg to about 700 pg
  • the total amount of c-di-AMP that is comprised in a dose of the first pharmaceutical composition, the second pharmaceutical composition, and optionally the third pharmaceutical composition is from about 50 pg to about 70 pg or from about 10 pg to about 20 pg.
  • a dose of the first pharmaceutical composition comprises the HBcAg particle in an amount of about 75 pg
  • a dose of the second pharmaceutical composition comprises the HBsAg in an amount of about 60 pg
  • the total amount of c-di-AMP that is comprised in a dose of the first pharmaceutical composition, the second pharmaceutical composition, and optionally the third pharmaceutical composition is about 15 pg.
  • the pharmaceutical composition, the combination, or the kit for the use of item 125 wherein the use is in an immune stimulation method.
  • An HBcAg particle, an HBsAg, c-di-AMP, and/or a vaccine vector, wherein the HBcAg particle, the HBsAg, the c-di-AMP, and/or the vaccine vector is preferably defined according to any one of items 1-92, for use in a method of vaccination.
  • an HBcAg particle, an HBsAg, c-di-AMP, and/or a vaccine vector in the manufacture of a medicament wherein the HBcAg particle, the HbsAg, the c-di-AMP, and/or the vaccine vector is preferably defined according to any one of items 1-92, wherein the medicament is for vaccination.
  • the use of item 136 or 137, wherein the medicament is for use in a method of vaccination of any one of items 1-92.
  • FIG. 1 VacB as a novel therapeutic, preferably curative, vaccination regime for treating an HBV infection.
  • A Schematic representation of the VacB vaccination regime comprising 2x protein prime vaccinations (day 0 and approximately day 28) comprising a novel HBcAg particle and a HBsAg, followed by a vaccine vector boost vaccination (approximately day 56).
  • Mosaic core protein HBcAg particle disclosed herein comprising HBV core proteins from at least two different HBV genotypes; MVA-vector as well as MHBVac: vaccine vector disclosed herein expressing multiple HBV antigens from different HBV genotypes and being preferably an MVA vector like MHBVac.
  • FIG. 2 Multi-antigenic open reading frame. Depicted is the structure of the multi- antigenic polypeptide chain preferably comprised in the vaccine vector disclosed herein. Schematically depicted are the formation of subviral particles comprising HBV surface A/adw and C/ayw antigens, and the formation of empty capsids comprising HBV core D/ayw and C/ayw antigens. Partially unprocessed antigens are assumed to increase immune responses (especially enhance and broaden adaptive immune responses) due to incorporation into secreted virus-like particles.
  • Figure 4 Plasmid map of the vector used for production in E.coli of the disclosed HBcAg particle. The vector was generated for expression of both HBV core proteins from genotype C (truncated 1-163aa 1 18.54 kDa) and genotype D (full length 1-183aa / 21.12 kDa) that self-assemble into the disclosed HBcAg particle comprising HBV core proteins from two or more HBV genotypes.
  • Figure 5 Nucleotide sequence of the plasmid shown in Figure 4. Coding regions are indicated as follows: bold: nucleotide sequence encoding a truncated HBV core protein from HBV genotype C with the truncated HBV core protein consisting of 1-163aa, bold and underlined: nucleotide sequence encoding a full-length HBV core protein from HBV genotype D with the full- length HBV core protein consisting of 1-183aa.
  • said subunits do not relate to genotypes but in this Figure to asymmetric units which is a kind of “structural” unit within in an icosahedron (an icosahedron is usually composed of 60 so-called asymmetric units, e.g.
  • an asymmetric unit consists of 4 monomers (or 2 dimers), while in the subunits A, B, C, D each denote a monomer in the asymmetric unit shown in color).
  • the bases of the HBV core protein dimers form interdimer contacts, while the spikes of the HBV core protein dimers represent intradimer contacts. Residues with spectral perturbations attributed to mosaic assemblies are highlighted as black spheres, (b) Side view of the “AB” dimer.
  • FIG. 9 Comparison of homologous vs. heterologous prime I boost vaccination.
  • ICS intracellular cytokine staining
  • FIG. 13 VacB results in long-term HBV control in AAV-HBV mice.
  • mice were primed twice with mixture of HBsAg and the HBcAg particle adjuvanted with CpG-1018.
  • mice were boosted with MHBVac and afterwards monitored for 14 weeks.
  • HBV-specific CD4 + T-cell responses (upper panel) and CD8 + T-cell responses directed against immunodominant peptides from HBV S (peptide pool) protein (lower panel) were determined.
  • Active T cells were determined as IFNy + HBV-specific T cells by intracellular cytokine staining and flow cytometry, d: Days; TNFa: Tumor necrosis factor a; D: full-length HBcoreAg, genotype D; ACD: Mosaic HBcoreAg particles; IFNy: Interferon y; No Vac: non-vaccinated mice; ns: not significant.
  • Asterisks mark statistical significances: *P ⁇ 0.05; **P ⁇ 0.01 ; ***P ⁇ 0.001 ; ****P ⁇ 0.0001.
  • FIG. 18 TherVacB containing ACD mosaic HBcoreAg induces HBV-specific T-cell response against HBV genotype B.
  • mice No Vac - non-vaccinated mice; D: genotype D HBcoreAg (full-length core protein); ACD: mosaic HBcoreAg; IFNy: Interferon y; AAV-HBVgtB: AAV-HBV genotype B; pool: Overlapping peptide pools; ns: not significant. Asterisks mark statistical significances: *P ⁇ 0.05; **P ⁇ 0.01 ; ***P ⁇ 0.001.
  • HBcAg particle For generating a recombinant HBcAg particle, a bicistronic plasmid was generated for simultaneous expression of HBV core proteins from genotype C (truncated 1-163aa 1 18.54 kDa, “HBc gtC 1-163aa”, SEQ ID NO: 1) and genotype D (full length 1-183aa / 21.12 kDa, “HBc gtD 1- 183aa”, SEQ ID NO: 2). Sequence of HBc gtD 1-183aa was derived from GenBank Acc. No. V01460 and codon-optimized for expression in humans (GeneART, Regensburg/Germany).
  • a truncated monomer sequence from genotype C was chosen to have analytical means to proof the presence of monomers from both genotypes in a given HBcAg capsid.
  • the truncation and thus, the measurable difference in molecular mass provided hence options to clearly differentiate both monomer variants (genotype D vs. genotype C) by e.g. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after pulldown of capsids, cf.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • truncation was chosen to not impair the immunogenicity of the recombinant HBcAg particle, as the C-terminal deleted 20 amino acids do not contain any differences in the amino acid sequence between both genotypes C and D. Therefore, no antigenic epitope was lost by the deletion of the C-terminal 20 amino acids in genotype C monomers compared to full length genotype D monomers.
  • the open reading frame (ORF) sequence of HBc gtC 1-163aa was also synthesized by GeneART.
  • HBcAg insert sequences HBc gtC 1-163aa and HBc gtD 1-183aa, were initially cloned into pET28a2 (Novagen Inc.) for testing of functionality and protein expression. After verification of protein expression, both insert sequences were cloned into an intermediate donor plasmid, which contained a Tetracycline (Tet) repressor for conditional expression in mammalian cell culture. To enable large scale expression in bacteria like E.coli, the insert containing both ORFs of the HBV core antigens was cloned into a pRSF-Duet-1 plasmid via Agel/Pfol.
  • Tet Tetracycline
  • the obtained final plasmid “pRSF_T7-HBc163UP_T7-HBc183opt_noTetRep” was proven to be free of the Tet repressor. Presence of the correct insert sequences was verified by Sanger sequencing and measurement of protein expression in bacteria of both HBV core antigen variants was demonstrated by Western Blot. [00192]
  • the final plasmid ( Figure 4, Figure 5, SEQ ID NO: 10) had a size of 4825 bp and was generated for simultaneous expression of HBV core proteins from genotype C (truncated 1- 163aa I 18.54 kDa) and genotype D (full length 1-183aa I 21.12 kDa). The expression of both genotype sequences was induced by the identical T7 promotor to obtain equimolar expression and finally led to the assembly of “mosaic” HBcAg particle consisting of HBV core proteins from both genotypes.
  • Example 2 Obtaining a recombinant HBcAg particle and its pharmaceutical composition
  • the recombinant HBcAg particle was recombinantly produced in E.coli transduced with a plasmid described in Example 1 for IPTG (Isopropyl-p-D-thiogalactopyranosid) inducible expression of the HBV core proteins from genotype C (1-163aa) and genotype D (1-183).
  • IPTG Isopropyl-p-D-thiogalactopyranosid
  • the HBcAg particles were collected by cell lysis and afterwards processed by a cascaded downstream process (including ammonium sulfate (AMS) precipitation, two chromatography steps, diafiltration)
  • AMS ammonium sulfate
  • recombinant HBcAg particles were sterile filtered and aseptically filled in 2R glass vials (1.2 ml) to produce the investigational medicinal product (IMP) for clinical application.
  • the recombinant HBcAg particle obtained from Example 2 was characterized with regard to particulate structure, size, composition of both genotypes, and encapsidated nucleic acids.
  • TEM Transmission Electron Microscopy
  • Samples comprising recombinant HBcAg particles were diluted to 0.1 mg/ml, loaded onto copper grid and incubated. Grids were washed with HEPES buffer and stained with uranyl acetate. Images were taken using a TEM with 60.000x magnification (0.275 nm/pix). Scale bar was set to 200 nm.
  • TEM analysis showed a uniform pattern of all recombinant HBcAg particles concerning size, shape and integrity of the particles.
  • capsid species could be obtained: (A) capsids assembled of truncated genotype C HBV core proteins exclusively (18.54 kDa) - which would not be biotinylated, (B) capsids assembled of full-length genotype D HBV core proteins exclusively (24 kDa; the larger molecular mass of 24 kDa compared to the typical 21.16 kDa of genotype D HBV core proteins was due to the used biotin tag) - which would be biotinylated, and (C) capsids assembled of both genotype C and genotype D HBV core proteins (18.54 + 24 kDa) - which would be biotinylated.
  • a pull-down assay with Avidin-magnetic beats was performed after expression in E.coli to collect recombinant HBcAg particles comprising biotinylated genotype D HBV core proteins (i.e. species (B) and (C)) in the eluate, while recombinant HBcAg particles not comprising any biotinylated HBV core proteins (i.e. species (A)) were removed and collected in the flow-through.
  • the eluate as well as the flow-through weres analyzed by Western Blot (WB) with anti-HBc antibody recognizing the HBV core proteins from the two genotypes.
  • the flow-through contained HBV core proteins of 18 and 21 kDa, i.e. truncated HBV core proteins from genotype C and full-length HBV core proteins from genotype D.
  • HBV core proteins 18 and 21 kDa
  • truncated HBV core proteins from genotype C and full-length HBV core proteins from genotype D.
  • this did not reveal which kind of species (A) to (C) was present in the preparation.
  • the modified expression plasmid was used (i.e. expression with biotinylation of genotype D HBV core proteins)
  • the WB of the eluate revealed a strong 24 kDa as well as a 18 kDa band.
  • the recombinant HBcAg particles that were pulled-down were not only composed of the 24 kDa biotinylated HBV core proteins from genotype D but also consisted of HBV core proteins from genotype C.
  • HBV core proteins from genotype C HBV core proteins from genotype C.
  • This technique can probe the local environment around individual amino acid residues, such as the structural features of capsids assembled in the presence of a mixture of HBV core proteins from genotypes C and D.
  • three types of capsids could arise comprising HBV core proteins from both genotypes (truncated 1-163 aa genotype C and full-length 1-183 aa genotype D monomers; cf. Figure 7): (1) HBcAg particles consisting of heterodimers only, i.e. in which two HBV core proteins from genotype C and D form mixed dimers (cf. Figure 7 d), (2) HBcAg particles consisting of homodimers only , i.e.
  • HBV core protein dimers are formed from two HBV core proteins from the same genotype C or D, but with homodimers from both genotypes being comprised in the icosahedral assembly (cf. Figure 7 e), and (3) HBcAg particles consisting of both, homo- and heterodimers (1 and 2; cf. Figure 7 f).
  • HBV core proteins The biological function of HBV core proteins is to encapsidate the HBV genome. Therefore, the C-terminal region of the HBV core proteins possesses an arginine-rich domain that confers a positive charge in the capsid to enable/facilitate the binding and encapsidation of the RNA transcript of the viral genome (pgRNA) into the capsid. During maturation of the virions, the pgRNA is reverse transcribed into the viral genome (rcDNA).
  • HBV core proteins are prone to nucleic acid binding and when recombinant HBcAg particles are produced in E.coli cells (i.e. in the absence of pgRNA molecules), the biochemical properties and the inherent biological function provoking the binding of cellular RNA to the positively charged C-terminal domain and, thus, the encapsidation of host cell nucleic acids into the HBcAg (instead of the viral genome).
  • This encapsidation of E.coli RNAs is inherent to the generated recombinant HBcAg particle and cannot be prevented during assembly of capsids in expression cultures.
  • the binding of NAs into the capsid also stabilizes this particulate structure and is therefore necessary to obtain stable recombinant HBcAg particle preparations.
  • Recombinant HBcAg particles produced with the production process described herein in Example 1 was used for qualitative and quantitative analysis of the encapsidated NAs. Therefore, total RNA and total DNA was recombinant from batch 101764. Recombinant NAs were characterized by i) quantification using Qubit fluorometric quantification, ii) determination of fragment length using microchip electrophoresis, and iii) sequencing using cDNA-library (made from RNA) and shotgun library (made from DNA) using the Illumina technology. [00205] The encapsidated nucleic acid was composed of 99.59% RNA, while the amount of DNA made only 0.41% of the total nucleic acid content.
  • RNA made 9.44% w/w of the recombinant HBcAg particle sample (i.e. 94.4 ng RNA/pg protein).
  • the size of the recombinant NAs ranged for RNA between 100 and 3,000-4,000 nt and for DNA between 160 and 1 ,200 nt.
  • the RNA present in the HBcAg was constituted by mRNA (45.46%), rRNA (53.17%), tRNA (0.39%), tmRNA (0.73%) and ncRNA (0.24%).
  • Example 5 Optimizing the prime vaccination by combining protein antigens
  • Anti-HBc antibodies could only be detected in the group of mice receiving the vaccine formulation containing the recombinant HBcAg particle ( Figure 10 A).
  • the addition of the recombinant HBcAg particle to the vaccine formulation significantly improved anti-HBs responses ( Figure 10 B).
  • Example 6 Protein superior over DNA or RNA for prime vaccination
  • VacB immunogenicity and antiviral efficacy were compared in AAV-HBV mice after formulating HBsAg and the recombinant HBcAg particle with traditional Th2-activating adjuvant aluminum hydroxide (alum) or with CpG that also allows for inducing Th1 response.
  • alum Th2-activating adjuvant aluminum hydroxide
  • a GLP standard repeat-dose toxicity study was performed in Wistar rats to assess toxicity of the VacB vaccine components. The study was conducted according to “WHO guideline on nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines”.
  • a shortened but n+1 repeated dose vaccination regime with 3-fold protein prime on day 1 , 8 and 15 followed by a 2-fold vector boost on day 29 and 36 was used. According to the clinical application, all vaccine components were administered by i.m. injection. Blood samples were collected on day 2, 16 and at time point of final analysis, that was done on day 37 for the main group and day 50 for the recovery group (i.e. after a 14-day recovery period for assessment of possible findings).
  • Table 1 provides an overview of the study groups.
  • the maximum dose that was applied from each vaccine component corresponded to the highest planned full human dose (FHD) in the clinics, except for the CpG-1018 adjuvant, which is part of the HEPLISAV-B® formulation and provokes its adjuvant effect by binding on TLR9 receptors.
  • a single human dose of HEPLISAV-B® contains 20 pg HBsAg and 3,000 pg CpG-1018.
  • a two-fold dose of HEPLISAV-B® (corresponding to 40 pg HBsAg and 6,000 pg CpG-1018) is foreseen for study group B0.2.
  • a FHD of 3,000 pg 16,000 pg CpG-1018 is not applicable in rats due to differences in the expression pattern of TLR9 between humans and rodents with much broader expression of TLR9 in rodent tissues. Therefore, and in agreement with PEI, the maximum immunogenic dose of CpG-1018 (30 pg) was applied in case of the rats. However, applying an allometric scaling from human to rodents, the 30 pg dose corresponds to the foreseen two-fold dose of HEPLISAV-B® in humans. In addition, given that HEPLISAV-B® is already a marketed product (prophylactic vaccine for Hepatitis B), extensive safety data in humans exists.
  • the single vaccine components were provided by the respective GMP manufacturers. Ready-to-use formulations of the test items were prepared by mixing the vaccine components needed for the protein prime of the different groups in the respective concentration and volume (administration of 200 pl per animal). Test item formulations were labelled and shipped to the CRO of the GLP study (ATRC Aurigon Toxicological Research Center Ltd.) 24 - 72 hours prior to application.
  • VacB vaccine components were produced under GMP conditions, and preclinical GLP-conform safety I tolerability studies were completed in 2021.
  • the VacB vaccination approach was preclinically developed in C57BL/6 mice to select the most suitable vaccine components, vaccination schedule, dosage and application route.
  • the complete VacB vaccination regime will be applied to humans for the first time in the phase 1a clinical trial disclosed herein. More specifically, a single-center, open-label, first-in- human phase 1a clinical trial will be performed to evaluate an investigational medicinal product in healthy volunteers.
  • Table 2 Overview of study outline of phase 1a trial with N indicating number of healthy subjects to be enrolled per study arm. In total, 11 healthy subjects will be participating.
  • HBsAg will be used in the form of the commercially available and EMA and FDA approved HEPLISAV-B®.
  • Study arm A0 healthy subjects will receive 20 pg HEPLISAV-B® (0.5 mL) at two time points (day 0 and day 28) and subsequently 3 x 10 8 ifu MHBVac (in 0.5 mL; day 56).
  • Study arm B0.1 healthy subjects will receive 20 pg HEPLISAV-B® (0.5 mL) and 25 pg recombinant HBcAg particle (in 0.25 mL) into the same arm at two time points (day 0 and day 28) and subsequently 6 x 10 7 ifu MHBVac (in 0.5 mL; day 56).
  • Study arm B0.2 healthy subjects will receive 2 doses of 20 pg HEPLISAV-B® (0.5 mL) and 50 pg recombinant HBcAg particle (in 0.5 mL) into the same arm at two time points (day 0 and day 28) and subsequently 3 x 10 8 ifu MHBVac (in 0.5 mL; day 56).
  • Study arm C0.1 subjects will receive 60 pg HBsAg (in 0.3 mL) and 75 pg HBcoreAg (in 0.75 mL) into the same arm at two time points (day 0 and day 28) and subsequently 3 x 10 8 ifu MVA-HBVac (in 0.5 mL; day 56).
  • Study arm CO.2 subjects will receive 30 pg HBsAg adjuvanted with 30 pg c-di-AMP (in 0.3 mL) and 40 pg HBcoreAg (in 0.4 mL) into the same arm at two time points (day 0 and day 28) and subsequently 3 x 10 8 ifu MVA-HBVac (in 0.5 mL; day 56).
  • Study arm CO.3 subjects will receive 60 g HBsAg adjuvanted with 60 pg c-di-AMP (in 0.6 mL) or alternatively 15 pg c-di-AMP (in 15 mL) and 75 pg HBcoreAg (in 0.75 mL) into the same arm at two time points (day 0 and day 28) and subsequently 3 x 10 8 ifu MVA-HBVac (in 0.5 mL; day 56).
  • all healthy subjects will receive in total three intramuscular injections, namely two injections of protein ⁇ adjuvant on day 0 and day 28, and an MVA injection on day 56. All healthy subjects will be followed up until day 224 ⁇ 7.
  • the study will be conducted in a staggered approach in ascending order of the different study arms. Each study arm differs by a parameter like dose escalation or addition of a new vaccine component as indicated in Table 4 and Table 5, and will only be initiated after safety evaluation of the previous study arm.
  • Safety assessments cf. , e.g., Figure 14
  • AEs adverse events
  • SAEs serious adverse events
  • safety laboratory and vital signs laboratory and vital signs
  • Subjects will be vaccinated in all study arms in a staggered manner as indicated in Figure 15. There will be a minimum of 1 day for arm A0 and 2 days for arms B0.1 , B0.2, C0.1 , C0.2, and CO.3, respectively, between the vaccinations of the 1st subject and the 2nd and 3rd subject within a study arm.
  • the 2nd and 3rd subject within a study arm may be vaccinated on the same day.
  • BO.2, CO.2, and CO.3 the same applies for the 4th and 5th subject.
  • the 4 th and 5 th subject may be vaccinated on the same day, but min. 1 day after the 2nd and 3rd subject.
  • Participant may be on chronic or as needed medications if, in the opinion of the investigator, they pose no additional risk to participant safety or assessment of reactogenicity and immunogenicity and do not indicate worsening of a pre-existing medical condition.
  • Body mass index 18.5-32.0 kg/m2 and weight >50 kg at screening.
  • WOCBP Women of child-bearing potential
  • WOCBP who agree to comply with the applicable contraceptive requirements of the protocol from at least 14 days prior to vaccination until end of clinical trial or females who are permanently sterilized (at least 6 weeks post-sterilization). 9. The participant is co-operative and available for the entire clinical trial.
  • Any chronic or active neurologic disorder including seizures, and epilepsy, excluding a febrile seizure as a child and occasional migraine headaches.
  • Subjects with inflammatory, infectious and neuroinflammatory underlying disease which could cause an expected impairment of the blood brain barrier such as meningitis, multiple sclerosis, epilepsy, or Alzheimer’s disease.
  • Table 5 Overview of general properties of the HBcAg particles.
  • Table 7 Overview of general properties of the recombinant vaccine vector MHBVac.
  • Fever body temperature > 38.0°C
  • immunization may be postponed if screening window will not be exceeded.
  • subject does not receive day 28 vaccination, no ambulatory visit on day 35 will take place.
  • subject does not receive day 56 vaccination, no ambulatory visit on day 63 will take place.
  • a healthy subject discontinues the trial early (before 2nd and/or 3rd immunization) the subject will be replaced.
  • the amount of the recombinant vaccine vector used for the boost selected in this clinical trial will represent amounts with optimal immunogenicity and safety profile as observed in prior clinical trials using MVA-vector-based vaccines (Koch et al., Safety and immunogenicity of a modified vaccinia virus Ankara vector vaccine candidate for Middle East respiratory syndrome: an open-label, phase 1 trial. Lancet Infect Dis. 2020 Jul;20(7):827-838. doi: 10.1016/S1473- 3099(20)30248-6. Epub 2020 Apr 21. PMID: 32325037; PMCID: PMC7172913) and available MVA-based vaccines (e.g., Imvanex).
  • MVA-vector-based vaccines e.g., Imvanex
  • the amount of the heterologous protein prime selected in this clinical trial represent immunogenic amounts as observed in prior clinical trials.
  • 20 pg and 40 g of HBsAg are contained in commercially available vaccines (EngerixB Anlagen: 20 pg, HepVaxPRO: 40 pg; HEPLISAV B®: 20 pg; Fendrix: 20 pg; div. adjuvants).
  • the most common side effects are headache, pain, redness, swelling at the injection site and fatigue (tiredness).
  • 100 pg HBcAg combined with 100 pg HBsAg were applied in several hundered individuals in several smaller studies i.m.
  • the clinical trial is designed to investigate the safety and immunogenicity of the heterologous protein prime I MVA boost therapeutic HBV vaccination method according to the present invention with two ascending amount levels of the HBcAg particle and HBsAg adjuvanted with c-di-AMP in healthy subjects boosted with the vaccine vector.
  • Secondary endpoints will be assessed in view of an evaluation of an HBV-specific immunity with said secondary endpoints being collected and measured as followed:
  • hematology blood samples (2.7 mL) for hematology will be collected at different time points described and the following parameters will be assessed: hemoglobin, mean corpuscular hemoglobin concentration (MCHC), hematocrit, white blood cell (WBC) count (total and differential); red blood cells (RBC), neutrophils, mean corpuscular volume (MCV), lymphocytes, platelet count, monocytes, mean corpuscular hemoglobin (MCH), eosinophils, and basophils.
  • MCHC mean corpuscular hemoglobin concentration
  • WBC white blood cell
  • RBC red blood cells
  • neutrophils neutrophils
  • MCV mean corpuscular volume
  • lymphocytes platelet count
  • monocytes monocytes
  • MCH mean corpuscular hemoglobin
  • eosinophils basophils.
  • a safety urinalysis the following parameters will be analyzed in fresh midstream urine at different time points: pH, ketones, specific gravity, bilirubin, protein, blood, and glucose. Microscopic examination will be conducted if blood is detected during urinalysis. The microscopic examination will comprise of RBC, WBC, casts, and bacteria.
  • HIV testing HIV I and HIV II
  • HCV antibody screen HBV testing
  • HBsAg anti-HBc, anti-HBs
  • the information on the blood volume drawn for immunogenicity assays given below in Table 8 refers to maximum amounts.
  • Humoral and cellular immunogenicity assays may include, but are not limited to, those shown in Table 8.
  • Table 8 Humoral and cellular immunogenicity assays
  • the primary objective of the clinical trial will be the assessment of safety and reactogenicity following injection. Exposure to study medication will be summarized by number of injections and amounts injected using descriptive statistics. Demographics and baseline characteristics, as well as all primary end secondary endpoints will be presented by means of descriptive statistics. Continuous data will be summarized with number, mean or geometric mean, standard deviation as appropriate, as well as minimum, Q25, median, Q75 and maximum. Categorical data will be summarized by absolute and relative frequencies (number and percent). Related adverse events (classified as defined in the primary endpoints) and subjects suffering from those adverse events will be presented for each study arm and summarized according to system organ class and preferred term using MedDRA coding as well as severity. All safety information will be assessed by participant and by study arm. The baseline for calculation of change from baseline of safety laboratory measures will be defined as the time point closest but prior to the respective vaccination.
  • This clinical trial is designed to investigate the safety and immunogenicity of a heterologous protein prime/ MVA boost therapeutic hepatitis B vaccine candidate with ascending dose levels of the candidate vaccine adjuvanted HBsAg ⁇ HBcoreAg in chronic hepatitis infected subjects in two parts of the trial.
  • a novel nucleosidic adjuvant (c-di-AMP) will be added to protein antigen vaccinations in two ascending dose levels. All vaccinees will be boosted with an MVA-based vectored vaccine. The safety and tolerability will be assessed by collecting safety data (local and systemic reactogenicity, AEs and vital signs) at all study visits throughout the study. Efficacy of the vaccination will be assumed if there is a drop in HBsAg levels >1 Iog10 or loss of HBsAg two weeks, two and six months after the last vaccination. Details concerning analysis will be defined in the statistical analysis plan (SAP). The HBcAg, HBsAg, and modified vaccinia virus Ankara vector are the same as described in Example 10.
  • the aim of this clinical trial is to assess the safety, tolerability and immunogenicity of a heterologous protein prime/MVA boost therapeutic hepatitis B vaccine candidate in chronic hepatitis patients.
  • the primary endpoint concerns safety. Safety endpoints will be stratified by study arms, vaccine regimens and doses, following prime vaccination and for the entire prime/boost regimen. Primary safety endpoints include: • Frequencies and magnitudes of unsolicited adverse events
  • the secondary endpoints concern efficacy. Antiviral efficacy and immunogenicity endpoints will be assessed continuously throughout the trial, following prime vaccination and for the entire prime/boost regimen stratified by study arms, vaccine regimens and doses. The final efficacy and immunogenicity endpoints will apply two weeks, two and six months after completion of the vaccination regimen (last study visit).
  • This trial will be a multi-center, open-label, ascending dose phase 1 b/2a trial in 89 chronic hepatitis B infected subjects aged 18-70 years.
  • the intervention is a heterologous primeboost vaccine consisting of two protein-based primes (day 0 and day 28) and an MHBVac vector boost (day 56), all given as i.m. injections.
  • Phase 1b Part A (including study arms A1 , A2, A3 and A4) are considered first-in-CHB (Phase 1b), followed by dose consolidating arm A5 and A6 (Phase 2a)
  • Part B (including study arms B1 , B2 and B3) is a first-in-CHB (Phase 1b) priming exploration study, followed by a dose consolidating arm B4 (Phase 2a).
  • Phase 1b a first-in-CHB priming exploration study
  • B4 a dose consolidating arm B4
  • c-di-AMP an additional novel adjuvant, c-di-AMP is evaluated, to potentially allow increasing the dose of protein antigens for the prime vaccination as investigated in part A.
  • HBV infection (CHB) that fulfills the following criteria:
  • Subject may be on chronic or as needed medications if, in the opinion of the investigator, they pose no additional risk to subject safety or assessment of reactogenicity and immunogenicity and do not indicate worsening of a pre-existing medical condition.
  • Body mass index 18.5-32.0 kg/m2 and weight >50 kg at screening.
  • CLIA Chemiluminescent immunoassay
  • HB Hepatitis B
  • HBV Hepatitis B Virus
  • IFN laspasmodic factor
  • lgG immunoglobulin G
  • I L laspasmodic factor
  • LLOD lower limit of detection
  • LLOQ lower limit of quantification
  • PBMC peripheral blood mononuclear cell
  • S/CO signal cut off ratio
  • TNF Tumor Necrosis Factor
  • CLEIA chemiluminescent enzyme immunoassay
  • CMIA chemiluminescent microparticle immunoassay
  • Example 11 Comparing mosaic HBcoreAg with HBcoreAg of genotype D
  • ACD mosaic HBcoreAg particles were analyzed by ELISA. For this, 2.5 g of ACD HBcoreAg particles were stored at RT for an extended period of time and were assessed for their ability to bind monoclonal 8C9 antibodies at indicated time points ( Figure 17A). Further Activation of TCR-grafted human CD4+ T cells (2F2 TCR) that recognize a peptide from HBV genotype A-D was tested. TCR-grafted T cells were activated ex vivo by coculture with primary human dendritic cells supplemented with 10 pg of RIGA HBcAg (genotype D; D) or ACD HBcoreAg particles.
  • T-cell activation was determined via TNFa secretion measured by flow cytometry after intracellular cytokine staining (Figure 17B).
  • the final analysis was performed at week 5 after the first immunization (Figure 17C).
  • HBV-specific CD4+ T-cell responses upper panel
  • CD8+ T-cell responses directed against an immunodominant peptide from HBV S protein (lower panel) were determined.
  • Active T cells were determined as IFNy + HBV-specific T cells by intracellular cytokine staining and flow cytometry.
  • the ACD mosaic HBcoreAg induced stronger CD8+ T-cell responses against HBV S than HBcoreAg of genotype D ( Figure 17D).
  • Isolated lymphocytes were stimulated overnight with overlapping peptide pools covering HBV S protein or the Core protein (either genotype C or D) to determine HBV-specific effector T cells. Effector T cells were determined as IFNy + HBV-specific T cells by intracellular cytokine staining and flow cytometry. The mean of all mice is shown, and error bars indicate SEM. Statistical differences were calculated using unpaired t-tests. Overall, it can be derived that ACD mosaic HBcoreAg administered within TherVacB is superior in inducing HBV-S-specific T-cell responses (Figure 18B).

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Abstract

L'invention concerne des méthodes de vaccination, comprenant l'administration à un sujet humain d'une première dose d'une particule de HBcAg, d'une HBsAg et de c-di-AMP; d'une seconde dose de la particule de HBcAg, de HBsAg et de c-di-AMP; et d'une dose d'un vecteur de vaccin. L'invention concerne également des compositions pharmaceutiques, des combinaisons de particules de HBcAg, de HBsAg et de c-di-AMP, et des kits destinés à être utilisés dans lesdites méthodes.
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