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
  • A61K39/29—Hepatitis virus
  • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
• • 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
  • A61K2039/545—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
  • A61K2039/55505—Inorganic 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing 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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • 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
  • 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 aims at providing a, preferably curative, therapeutic vaccination of a (chronic) Hepatitis B Virus (HBV) infection.
• HBV Hepatitis B Virus
• the present invention relates to an Hepatitis B Virus core antigen (HBcAg) particle, comprising Hepatitis B Virus (HBV) core proteins from at least two different HBV genotypes.
• HBcAg Hepatitis B Virus core antigen
• HBV Hepatitis B Virus
• the present invention relates also to a pharmaceutical composition comprising the HBcAg particle disclosed herein and optionally a pharmaceutically acceptable carrier or excipient.
• the present invention relates also to a container comprising one or more doses of the pharmaceutical composition comprising the HBcAg particle disclosed herein.
• the present invention relates also to a kit comprising the pharmaceutical composition comprising the HBcAg particle disclosed herein and a second pharmaceutical composition comprising an HBsAg.
• the present invention relates also to the HBcAg particle disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein or in the kit disclosed herein, for use in therapy.
• the present invention relates also to the HBcAg particle disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein or in the kit disclosed herein, for use in treating an HBV infection.
• the present invention 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 only comprises coding sequences for two or more HBV core proteins.
• the present invention relates also to a nucleic acid molecule comprising the expression cassette, mRNA, or cDNA disclosed herein.
• the present invention relates also to an expression vector comprising the expression cassette or the cDNA disclosed herein or the nucleic acid molecule disclosed herein.
• the present invention relates also to an expression vector encoding an HBV core protein from HBV genotype C and an HBV core protein from HBV genotype D, wherein the expression vector has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10.
• the present invention relates also to a vaccine vector wherein the vaccine vector, preferably an 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 present invention also relates to an mRNA or cDNA comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
• the present invention relates also to the vaccine vector disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein, for use in therapy.
• the present invention relates also to the vaccine vector or mRNA disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein, for use in treating an HBV infection.
• the present invention relates also to a pharmaceutical composition comprising the vaccine vector disclosed herein and optionally a pharmaceutically acceptable carrier or excipient.
• the present invention relates also to a container comprising one or more doses of the pharmaceutical composition comprising the vaccine vector disclosed herein, wherein a dose of the pharmaceutical composition comprises the vaccine vector in an amount from about 1x 10 A 7 ifu to about 1x 10 A 9 ifu.
• the present invention relates also to a method of vaccination, comprising administering to a human subject (i) a first dose of an HBcAg particle and of an HBsAg, (ii) a second dose of the HBcAg particle and of the HBsAg, (iii) a dose of a vaccine vector wherein the vaccine vector expresses a 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 ; a 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; an HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; a 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 an RT
• the present invention relates also to an HBcAg particle, and optionally an HBsAg, for use in a method of vaccination.
• the present invention relates also to a vaccine vector expressing a HBsAg from HBV genotype A; a HBcAg from HBV genotype D; a HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; a 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 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 present invention relates also to a use of an HBcAg particle, and optionally an HBsAg, for the manufacture of a medicament.
• the present invention relates also to a use of a vaccine vector expressing a HBsAg from HBV genotype A; a HBcAg from HBV genotype D; a HBsAg comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; a 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 an RT domain of a polymerase from HBV having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, for the manufacture of a medicament.
• the present invention relates also to a use of the expression cassette, mRNA or cDNA disclosed herein, the nucleic acid sequence disclosed herein, the expression vector comprising the expression cassette 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 disclosed herein, for the manufacture of a medicament.
• Hepatitis B is an inflammatory disease of the liver and is caused by infection with the hepatitis B virus (HBV).
• HBV is a human pathogenic, hepatotropic DNA virus with transmission by blood-blood contact and vertically from mother to child.
• HBV carriers usually do not develop an efficient immune response against the virus (immune tolerance) and are therefore not able to fight the infection efficiently. More specifically, they neither produce a detectable amount of neutralizing antibodies directed against HBV surface antigen (HBsAg), nor a significant T cell response directed against HBV antigens.
• HBV is estimated to cause nearly 900,000 deaths annually with numbers predicted to rise in the absence of effective therapies.
• a prophylactic vaccine since 40 years, about 1/3 of all human beings still become infected with HBV at some point during their lifetime.
• HBV cure is not trivial as complete virus elimination can be rarely achieved. For practical reasons, endpoints are used that can be quantitatively evaluated. Thus, a functional HBV cure has been defined by suppression of viremia, normalization of alanine aminotransferase (ALT) levels and loss of HBsAg - ideally followed by anti-HBs seroconversion. Hence, a sustained therapeutic effect requires both antiviral suppression but in particular restoration of effector immune cell responses. Thus, a cure of an HBV infection should ideally be associated with virus-specific CD4+ and CD8+ T-cell responses.
• ALT alanine aminotransferase
• 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 essentials of T-cell responses to achieve an HBV cure is assumed to be based on their ability to eliminate HBV infected cells by their cytotoxic activity but also control HBV persistence, gene expression and replication in a non-cytolytic fashion (Guidotti.
• Approved therapies for an HBV infection comprise nucleoside and/or nucleotide analogues and interferon alpha.
• Nucleoside and/or nucleotide analogues are costly and have cure rates comparable to cure rates of spontaneous HBV elimination by the immune system, and the use of interferon alpha is declining due to severe side effects when used for treatment.
• Alternative approaches are in clinical development, which comprise, e.g., attempting to specifically suppress the replication of HBV and/or viral protein expression. Attempts are also being made to stimulate the innate immune system or to induce specific adaptive immune responses against HBV components through the administration of HBV antigens.
• current treatment options are of limited efficacy so far, thus failing to cure an HBV infection.
• the invention aims at providing a, preferably curative, therapeutic vaccination of a Hepatitis B Virus (HBV) infection.
• HBV Hepatitis B Virus
• the present invention relates to a Hepatitis B Virus core antigen (HBcAg) particle, comprising Hepatitis B Virus (HBV) core proteins from at least two different HBV genotypes, wherein the HBcAg particle is preferably an isolated HBcAg particle.
• the HBcAg particle is preferably a mosaic HBcAg particle.
• the present invention relates also to a pharmaceutical composition
• a pharmaceutical composition comprising the HBcAg particle disclosed herein and optionally a pharmaceutically acceptable carrier or excipient.
• the present invention relates also to a container comprising one or more doses of the pharmaceutical composition comprising the HBcAg particle disclosed herein.
• the present invention relates also to a kit comprising the pharmaceutical composition comprising the HBcAg particle disclosed herein and a second pharmaceutical composition comprising a HBsAg.
• the present invention relates also to the HBcAg particle disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein or in the kit disclosed herein, for use in therapy.
• the present invention relates also to the HBcAg particle disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein or in the kit disclosed herein, for use in treating an HBV infection.
• the present invention relates also to an expression cassette, mRNA, or cDNA encoding an HBV core protein from HBV genotype C and an HBV core protein from HBV genotype D, wherein the expression cassette only comprises coding sequences for two or more HBV core proteins.
• the present invention relates also to a nucleic acid molecule comprising the expression cassette, mRNA, or cDNA disclosed herein.
• the present invention relates also to an expression vector comprising the expression cassette or cDNA disclosed herein or the nucleic acid molecule disclosed herein.
• the present invention relates also to an expression vector or an mRNA encoding full-length or trincated HBV core proteins from two different HBV genotypes.
• the present invention relates also to an expression vector or an mRNA encoding an HBV core protein from HBV genotype C and ananan HBV core protein from HBV genotype D, wherein the expression vector has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 10.
• the present invention relates also to a vaccine vector, wherein the vaccine vector is preferably an 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 present invention relates also to the vaccine vector disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein, for use in therapy.
• the present invention relates also to the vaccine vector disclosed herein, optionally comprised in the pharmaceutical composition disclosed herein, optionally comprised in the container disclosed herein, for use in treating an HBV infection.
• the present invention relates also to a pharmaceutical composition
• a pharmaceutical composition comprising the vaccine vector or mRNA disclosed herein and optionally a pharmaceutically acceptable carrier or excipient.
• the present invention relates also to a container comprising one or more doses of the pharmaceutical composition comprising the vaccine vector disclosed herein, wherein a dose of the pharmaceutical composition comprises the vaccine vector in an amount from about 1x 10 A 7 ifu to about 1x 10 A 9 ifu.
• the present invention relates also to 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.
• the present invention relates also to an HBcAg particle, and optionally an HBsAg, for use in a method of vaccination.
• the present invention relates also to 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 use in a method of vaccination.
• the present invention relates also to the use of an HBcAg particle, and optionally an HBsAg, for the manufacture of a medicament.
• the present invention relates also to the 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; 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.
• the present invention relates also to the use of the expression cassette, mRNA, or cDNA disclosed herein, the nucleic acid molecule disclosed herein, the expression vector comprising the expression cassette or cDNA 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 disclosed herein, for the manufacture of a medicament.
• HBV Hepatitis B Virus
• HBV particles comprise an outer envelope and an inner core that encloses both viral DNA and a DNA polymerase with reverse transcriptase activity.
• the outer envelope comprises lipids and embedded proteins, which are involved in viral binding of, and entry into, susceptible cells like human cells.
• a protein embedded in the outer envelope of an HBV particle is also herein referred to as both an HBV surface protein (HBs).
• the inner core of an HBV particle is a particulate capsid, more specifically an icosahedral nucleocapsid.
• a structural protein of the inner core of an HBV particle, and thus of an HBV nucleocapsid is herein also referred to as an HBV core protein (HBc).
• Hepatitis B viruses are divided into four major serotypes (adr, adw, ayr, ayw) that can induce differential antibody responses based on antigenic epitopes present on the surface proteins, and into (at least) nine genotypes (A— I) according to overall nucleotide sequence variation of the HBV genome.
• HBV genotypes have a distinct geographical distribution and are used in tracing the evolution and transmission of the virus. Differences between genotypes affect disease severity, course and likelihood of complications, as well as response to treatment and possibly vaccination.
• HBV genotypes can be further divided into sub-genotypes, e.g. A1-5. For example, in Central Europe and the United States, the predominant sub-genotype is A2. However, only 1 % of the infected humans carry the A2 sub-genotype, while the majority of patients carry HBV of genotype B, C, or D. Thus, HBV genotypes and/or serotypes may regionally differ in their occurrence.
• 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.
• the present invention addresses the need for a therapy of an HBV infection that is associated with an induction of neutralizing antibodies and of a multi-specific T cell response directed to multiple HBV antigens and/or HBV genotypes by providing the embodiments as recited in the claims.
• the present invention relates to an Hepatitis B Virus core antigen (HBcAg) particle, comprising Hepatitis B Virus (HBV) core proteins from at least two different HBV genotypes.
• the HBcAg particle is preferably an isolated HBcAg particle.
• the HBcAg particle is preferably recombinant.
• the HBcAg particle is preferably a mosaic HBcAg particle.
• the present invention relates, inter alia, also to a MVA viral vaccine vector expressing multiple HBV antigens from different HBV genotypes.
• the present invention relates, inter alia, also to a method of vaccination comprising administering to a human subject a first and a second dose of both the novel HBcAg particle and an HBsAg as two protein priming vaccinations as well as a dose of a vaccine vector expressing multiple HBV antigens from different HBV genotypes as a boost vaccination.
• a novel HBcAg particle comprising HBV core proteins from multiple HBV genotypes can induce a broad immune response against the HBV core proteins comprised in said particle.
• the HBcAg particle may represent a powerful protein prime, in particular when combined with an, optionally adjuvanted, HBsAg.
• the HBsAg is preferably an isolated HBsAg.
• the HBsAg is preferably recombinant.
• balanced CD4+ TH1/TH2 T-cell responses for production of neutralizing anti-HBc and anti-HBs antibodies can preferably be induced.
• preclinical data suggests that priming with HBcAg also supports anti-HBs immune response.
• the inventors of the present application have surprisingly found that simultaneous priming with HBcAg and HBsAg have a synergistic effect.
• a strong activation of cytotoxic CD8+ T- cells for elimination of infected cells can preferably be observed.
• the novel HBcAg particle, in combination with an (adjuvanted) HBsAg, and the vaccine vector may preferably constitute key components of a novel therapeutic vaccination regime preferably has the potential to even cure HBV infections.
• 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 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.
• boosting with a vaccine vector such as a (recombinant) MVA vector, expressing HBsAg, HBcAg and the RT domain of the HBV polymerase may preferably be found to expand primed cytotoxic CD8+ T-cells that may lead to an elimination of infected hepatocytes in the liver.
• the present invention encompasses the use of a combination of antigens from different HBV genotypes and/or serotypes. This combination may preferably induce a broad immune response against multiple HBV strains and, moreover, also a stronger immune response against each HBV genotype compared to vaccines comprising only single antigens and/or antigens from a single HBV genotype.
• the vaccination regime disclosed herein preferably represents an efficient and powerful novel approach to induce immune control of an HBV infection by inducing a strong and polyclonal T-cell response and neutralizing antibodies against different HBV antigens from several HBV genotypes.
• the immune system can preferably combat and eliminate HBV.
• 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.
• 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 present invention also relates to an HBV core antigen (HBcAg) particle, comprising HBV core proteins from at least two different HBV genotypes.
• HBV core antigen (HBcAg) particle comprising HBV core proteins from at least two different HBV genotypes.
• said HBcAg particle comprises a combination of B-cell antigens and T-cell epitopes from HBV core proteins of different HBV genotypes.
• Such a combination may be advantageous to induce a broad immune response.
• an immune response against at least two and thus, multiple HBV genotypes can preferably be induced.
• such a combination is preferably capable of inducing a stronger immune response against each HBV genotype comprised in the combination compared to vaccines comprising only antigens from a single HBV genotype.
• 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.
• the term “antigen” refers to a (longer) sequence, in particular a (longer) amino acid sequence or protein sequence, whereas the phrase "antigenic epitope” or “an epitope of the antigen” encompasses a stretch of shorter sequence from the longer sequence.
• the term “antigen” thus encompasses epitopes.
• the term “antigen” also includes variants of proteins, polypeptides, and antigenic protein fragments as described herein.
• the term “antigen” encompasses sequences identical to the native sequence as well as modification to the native sequence, such as deletions, additions, insertions and substitutions.
• an antigen variant has at least about 50%, at least about 60% or 65%, at least about 70% or 75%, at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at least about 90%, 91%, 92%, 93%, or 94% and even more typically at least about 95%, 96%, 97%, 98% or 99%, most typically, at least about 99% amino acid identity with the reference antigen (i.e. the antigen from which it is derived).
• the reference antigen i.e. the antigen from which it is derived.
• an epitope forms part of the antigen that still elicit an immune response in a host.
• An epitope is, however, not limited to the exact sequence of the antigen from which it is derived.
• epitope encompasses sequences identical to the native sequence as well as modification to the native sequence, such as deletions, additions, insertions and substitutions.
• an epitope variant have at least about 50%, at least about 60% or 65%, at least about 70% or 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at least about 90%, 91%, 92%, 93%, or 94% and even more typically at least about 95%, 96%, 97%, 98% or 99%, most typically, at least about 99% amino acid identity with the reference epitope (i.e. the epitope from which it is derived).
• a “Hepatitis B Virus core antigen”, “HBV core antigen” or “HBcAg” refers to an HBV core protein or a fragment thereof.
• a full-length HBV core protein is 183 amino acids in length and consists of an (self-) assembly domain (amino acids 1 to 149) and a nucleic acid-binding domain (amino acids 150 to 183).
• the 34-residue long nucleic acidbinding domain is extremely basic, with 17 arginines, consistent with its function, thus conferring a positive charge inside the capsid that enables the binding of the viral genome.
• RNA transcript of the viral genome pgRNA
• the viral polymerase are selectively incorporated into the HBcAg capsid (stabilized by the positive charge), where the pgRNA becomes reverse transcribed into the viral genome (rcDNA).
• the viral genome containing capsids are surrounded by the viral envelop (containing HBsAg) and the so formed mature and infectious virions are secreted from the infected cells.
• a (recombinant) HBcAg particle is produced for example in E.
• an HBcAg particle “consisting of” HBV core protein subunits of at least two different HBV genotypes is preferably understood as referring to an HBcAg particle not comprising any other HBV core protein or fragment thereof except the specifically recited ones while it may nevertheless comprise non-viral nucleic acids enclosed.
• Such non-viral nucleic acids may be enclosed in an HBcAg particle may be advanategous as it can stabilize the HBcAg particle.
• 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.
• such a truncated HBV core protein comprises preferably at least 100, more preferably at least 125, even more preferably at least 149, most preferably at least 163 consecutive amino acids of a respective full-length HBV core protein.
• a fragment is usually, and preferably, immunogenic.
• such a truncated, preferably immunogenic, HBV core protein comprises at least amino acids 1 to 149, more preferably said HBV core protein comprises at least amino acids 1 to 163, or is a respective full length HBV core protein.
• a respective HBV core protein of genotype A may preferably comprise at least amino acids 1 to 163 or be a full-length HBV core protein of genotype A.
• an HBV core protein of genotype B may preferably comprise at least amino acids 1 to 163 or be a full-length HBV core protein of genotype B.
• an HBV core protein of genotype C may preferably comprise at least or consist of amino acids 1 to 163 or be a full-length HBV core protein of genotype C.
• an HBV core protein of genotype D may preferably comprise at least amino acids 1 to 163 or be an HBV core protein of genotype D.
• isolated HBcAg or HBsAg refers to an HBcAg or HBsAg (particle) that has been isolated from the environment in which it has been expressed.
• the isolated HBcAg or HBsAg (particle) comprises HBV core proteins or S-, M-, and/or L- proteins, which may have been expressed by a cell, such as a bacterial or mammalian host cell, or an in vitro translation system.
• an isolated HBcAg or HBsAg particle may refer to an HBcAg or HBsAg (particle) that has been partially or substantially purified from the environment in which it had been expressed and thus, e.g. from the cell (e.g. a cellular expression system) or the in vitro translation system that was used for HBV core or S-, M-, and/or L- protein expression.
• an isolated HBcAg particle may be substantially free of cellular material (optionally) and/or culture medium or in vitro translation medium components, or of chemical precursors or other chemicals when chemically synthesized (optionally) and may or may not contain host cell RNA.
• An isolated HBcAg or HBsAg particle is preferably ex vivo.
• immunogenic refers to the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human or animal.
• immunogenicity is the ability to induce a humoral and/or cell mediated immune response.
• the ability of an antigen to elicit immune responses is called immunogenicity, which can be humoral and/or cell-mediated immune responses.
• an immunogenic variant of a naturally occurring HBV surface antigen is an HBV surface antigen, in which the “a” determinant epitope has been replaced with the “a” determinant of an HBs-antigen of another serotype.
• an immunogenic variant of a naturally occurring HBV surface antigen, HBV core antigen or polymerase may have 90 % sequence identity with the amino acid sequence of the natural occurring HBV surface antigen, HBV core antigen or polymerase from HBV.
• the HBcAg of the present invention is in the form of a uniform or mosaic capsid particle, which is preferably a uniform or mosaic capsid.
• the HBcAg particle comprises HBV core proteins that form a particle such as a capsid.
• an HBV nucleocapsid comprises dimers of HBV core proteins. It is to be noted that not only full-length, but also truncated core proteins, comprising, e.g., only amino acids 1 to 149 or 1 to 163 of the respective full-length core protein, are preferably capable of forming capsids.
• dimers of HBV core antigens self-assemble into icosahedral (nucleo)capsids in solution comparable to HBV core proteins, forming preferably T3 particulate capsids comprising approximately 90 HBV core protein dimers or T4 particulate capsids comprising approximately 120 HBV core protein dimers.
• Said icosahedral particulate capsids have usually a size of about 30-34 nm in diameter.
• the isolated HBcAg particle has preferably a size of about 20 to about 60 nm, preferably about 25 to about 50 nm, such as about 30-34 nm in diameter.
• the isolated HbcAg particle preferably has a capsid-like structure comprising HBV core proteins from at least two different HBV genotypes filled with host cell RNA. Such a HbcAg particle may be advantageous to trigger an immune response against multiple HBV genotypes.
• the isolated HBcAg particle may have a hydrodynamic radius of about 20 nm to about 80 nm, about 30 nm to about 70 nm, about 40 nm to about 60 nm, preferably about 45 nm to about 55 nm, such as about 48 nm to about 52 nm, preferably when measured by dynamic light scattering.
• a “capsid” in the context of an (isolated) HbcAg particle refers to a capsid comprising, or consisting of, HBV core proteins and optionally nucleic acids like non-viral or viral RNA.
• the HbcAg particle may be a capsid comprising bacterial or mammalian RNA.
• the HbcAg particle is a self-assembling particulate capsid.
• the HbcAg particle is a self-assembling particulate mosaic capsid.
• the HBV genotypes are selected from the group consisting of A, B, C, and D.
• the HBcAg particle may comprise HBV core proteins from at least two different HBV genotypes selected from the group consisting of A, B, C, and D. This preferably has the advantage of inducing an immune response against A, B, C, and/or D HBV genotypes that may be of particular relevance in some regions due to their frequent occurrence.
• the HBcAg particle may comprise or consist of HBV core proteins from HBV genotypes i) A and B, ii) A and C, iii) A and D, iv) B and C, v) B and D or vi) C and D.
• Said at least two genotypes may be comprised in the isolated HbcAg particle with a given ratio.
• a ratio may be from about 10:90 to about 90:10, about 20:80 to about 80:20, about 30:70 to about 70:30, or about 40:60 to about 60:40.
• the ratio about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, about 80:20, or about 90:10.
• the HBV core proteins of the at least two different HBV genotypes are substantially balanced and thus, in case of two genotypes, for example, the HBcAg particle may comprise HBV core proteins from said two HBV genotypes in a ratio of, e.g. about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, or about 80:20, preferably of about 40:60, about 50:50, or about 60:40.
• Ratio specified herein are preferably molar ratios.
• the HBV core protein subunits from the at least two different HBV genotypes are in an approximately equimolar ratio.
• the HBV core proteins from the at least two different HBV genotypes may be in an approximately equimolar ratio.
• the isolated HBcAg particle may comprise HBV core proteins from at least two different genotypes, wherein said genotypes are in an approximately equimolar ratio. This may be advantageous to ensure a broad immune response against the HBV genotypes comprised in the HBcAg particle without any (substantial) bias towards a portion of the genotypes comprised in the HbcAg particle compared to the remaining genotypes comprised in said HbcAg particle.
• the HBcAg particle preferably induces a comparable strong immune response against the respecitve HBV genotypes that may be of particular relevance in some regions due to their (frequent) occurrence.
• the HBcAg is well suited for a regionally optimized HBV therapy.
• the terms “about” and/or “approximately” refer to a deviation of 20% or less, preferably of 15% or less, more preferably of 10% or less, even more preferably of 5% or less, most preferably of 1 % or less. Thus, said terms relate to a value that is within a deviation of, e.g., maximal 10% or 5% of a given value or range.
• the HBcAg particle comprises HBV core proteins of not more than two different HBV genotypes.
• said HBcAg particle may comprise HBV core proteins from at least two different HBV genotypes though of not more than two different HBV genotypes.
• said HBcAg particle may comprise, or consist of, HBV core proteins of two different HBV genotypes.
• 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 comprises HBV core proteins from HBV genotypes C and D.
• the HBV core proteins from said HBV genotypes are preferably in in an approximately equimolar ratio.
• the HBcAg particle may comprise or consist of HBV core proteins from genotypes C and D, wherein the HBV core proteins from said HBV genotypes are 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. This may be advantageous for inducing a broad immune response against the HBV genotypes C and D without any (substantial) bias towards one of the two genotypes.
• the HBV core proteins comprise at least amino acids 1 to 163 or are full-length HBV core proteins.
• the HBcAg particle may thus comprise HBV core proteins from at least two different HBV genotypes, wherein the HBV core proteins are full-length HBV core proteins.
• at least a portion of the HBV core proteins may be truncated HBV core proteins.
• the HBcAg particle may thus comprise HBV core proteins from at least two different HBV genotypes, wherein at least a portion of the HBV core proteins are truncated HBV core proteins.
• the HBcAg particle may comprise HBV core proteins of in total two genotypes, wherein HBV core proteins of one genotype are full-length and HBV core proteins of the other genotype are truncated.
• the HBcAg particle may comprise HBV core proteins of in total two genotypes, wherein both of the HBV core proteins are full length.
• the HBcAg particle may comprise HBV core proteins of in total two genotypes, wherein a portion of the HBV core proteins of one of the two genotypes is truncated.
• 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.
• HBcAg particle This may be advantageous especially from a technical point of view as different protein length allow for an easy and cheap analytical detection of HBV core proteins from different genotypes like genotypes C and D.
• presence and ratio of the different protein types comprised in an HBcAg particle can be shown, e.g. by a SDS- PAGE after HBcAg particle pull-down.
• the HBcAg particle may comprise a mixture of truncated HBV core proteins and full-length HBV core proteins from different HBV genotypes.
• the HbcAg particle comprises truncated HBV core proteins from one HBV genotype and full-length HBV core proteins from another HBV genotype.
• the genotypes can be individually from the group consisting of A, B, C, D, E, F, G, H and I, preferably from the group consisting of A, B, C, and D, preferably 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 (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 thus consists of truncated HBV core proteins from HBV genotype C and full-length HBV core proteins from HBV genotype D. wherein 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.
• 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.
• sequence identity denotes a property of sequences that measures their similarity or relationship.
• sequence identity means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a protein or polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the sequence specified as “reference” of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.
• BLAST Altschul et al., 1997)
• BLAST2 Altschul et al., 1990
• FASTA Pearson and Lipman, 1988
• GAP Needleman and Wunsch, 1970
• Smith-Waterman Smith and Waterman, 1981
• Wisconsin GCG Package for determining sequence identity using standard parameters.
• the percentage of sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5, November 16, 2002 (Altschul et al., 1997), calculating the percentage of numbers of “positives” (homologous amino acids) from the total number of amino acids selected for the alignment.
• 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.
• a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six).
• 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 invention 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.
• the HbcAg particle comprises HBV core proteins, wherein HBV core proteins from HBV genotype C have the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or HBV core proteins from HBV genotype D have the amino acid sequence set forth in SEQ ID NO: 2 or 14.
• the HbcAg particle consists of HBV core proteins, wherein HBV core proteins from HBV genotype C have the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or HBV core proteins from HBV genotype D have the amino acid sequence set forth in SEQ ID NO: 2 or 14.
• a preferred example of 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 invention 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.
• an adaptive immunity refers to an antigen-dependent and antigen-specific immune response.
• An adaptive immune response involves a lag time between exposure to an antigen and maximal response and has the capacity for memory, which enables a rapid and efficient immune response upon subsequent exposure to said antigen.
• Key functions of an adaptive immune response are the recognition of specific “non-self” antigens, their distinction from “self” antigens, the generation of pathogen-specific immunologic effector pathways that eliminate specific pathogens and/or pathogen-infected cells and the development of an immunologic memory that can quickly eliminate a specific pathogen should subsequent infections occur (cf., e.g., Bonilla and Oettgen, Adaptive immunity.
• induction of an immune response preferably an adaptive immune response, wherein the immune srepsonse is ideally associated with HBcAg- and HBsAg-specific CD4+/CD8+ T-cell induction and/or anti-HBsAg antibody production may be highly advantageous for an efficient therapy of an infection like HBV.
• the present invention 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 invention 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.
• Such a fragment comprises preferably at least 70, preferably at least 80, preferably at least 90, preferably at least 100, more preferably at least 125, most preferably at least 150 consecutive amino acids of the primary sequence of a surface protein and is usually immunogenic.
• a, preferably immunogenic, fragment comprises compared to the full-length protein at least amino acids 99 to 168 corresponding to the amino acid positons of the small surface protein.
• the pharmaceutical composition comprising the HBcAg particle as disclosed may further comprise a pharmaceutically acceptable carrier and/or excipient, such as a stabilizer and/or a stabilizing agent.
• a stabilizing agent may be phosphate buffered saline (PBS).
• PBS may be advantageous for stabilization of the HBsAg, which may be present in the pharmaceutical composition embedded in a particulate lipid bilayer.
• the pharmaceutical composition comprising the HBcAg particle as disclosed, optionally comprising an HBsAg, preferably comprises further an adjuvant, preferably a nucleosidic adjuvant, more preferably a CpG, most preferably CpG-1018.
• suitable adjuvants comprise adjuvants other than alumn, e.g. composite adjuvants containing MPL and QS21, poly-IC, polylC-LC, SD101 and toll-like, Rig-l-like or other pattern-recognition receptors.
• 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 preferably has 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 invention 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.
• the pharmaceutical composition comprising the HBcAg particle as disclosed herein above does preferably not further comprise an HBsAg.
• said kit may refer to a packing unit of a pharmaceutical composition comprising the HBcAg particle and a second pharmaceutical composition comprising an HBsAg, wherein said pharmaceutical compositions 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 second pharmaceutical composition further comprises an adjuvant.
• said adjuvant is preferably a nucleosidic adjuvant.
• Said adjuvant is particularly preferred a CpG, preferably CpG-1018.
• 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 nucleosidic adjuvant, preferably a CpG , even more preferably CpG-1018.
• a nucleosidic adjuvant preferably a CpG , even more preferably CpG-1018.
• said 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 10pg to about 75pg, more preferably in an amount from about 20pg to about 100pg, preferably to about 60pg, most preferably in an amount of about 25pg or of about 50pg.
• 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 5pg to about 100pg, preferably from about 5 g to about 75pg, preferably in an amount from about 10pg to about 50pg, more preferably in an amount from 20pg or of about 40pg.
• 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 5pg to about 1OOpg, preferably from about 5pg to about 75pg, preferably in an amount from about 1Opg to about 50pg, more preferably in an amount from 20pg or of about 40 g.
• HBsAg HBsAg produced in yeast that has been used in a number vaccines, like EngerixB, Fendrix, and HEPLISAV-B, at doses up of 20-40 pg for adults and thus, dosing of HBsAg using amounts from about 20pg to about 40pg has a very high and proven safety profile (cf. e.g., Halperin et al., Comparison of the safety and immunogenicity of hepatitis B virus surface antigen coadministered with an immunostimulatory phosphorothioate oligonucleotide and a licensed hepatitis B vaccine in healthy young adults, Vaccine.
• Halperin et al. Comparison of the safety and immunogenicity of hepatitis B virus surface antigen coadministered with an immunostimulatory phosphorothioate oligonucleotide and a licensed hepatitis B vaccine in healthy young adults, Vaccine.
• a dose of the pharmaceutical composition preferably comprises the HBcAg particle in an amount of about 25pg or of about 50pg and a dose of the pharmaceutical composition comprises, preferably particulate, HBsAg in an amount of about 20pg or of about 40 g, preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 25pg and a dose of the pharmaceutical composition comprises, preferably particulate, HBsAg in an amount of about 20pg; even more preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 50pg and a dose of the pharmaceutical composition comprises, preferably particulate, HBsAg in an amount of about 40pg.
• a dose of the pharmaceutical composition preferably comprises the HBcAg particle in an amount of about 25pg or of about 50pg and a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 20pg or of about 40pg; preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 25pg and a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 20 g; even more preferably, a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 50 g and a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 40pg.
• 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.
• Immune stimulation can be measured by determining hBcore- and/or HBs-specific CD8 T-cell responses, for example using fluorospot technique. Immune stimulation may be beneficial in cases that require support of the naturally occurring immune response without being limited to vaccination, especially in cases of an infection with HBV. Thus, anti- HBcAg and— by intrastructural help— anti-HBsAg antibody production can preferably be intensified and HBcAg-specific CD4+/CD8+ T-cell induction ensured. These aspects may also be beneficial in the context of vaccination which may be for example preventive or curative.
• the invention relates also to the disclosed HBcAg particle or the disclosed pharmaceutical composition comprising the HBcAg particle, 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 or the disclosed pharmaceutical composition comprising the HBcAg particle, 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.
• the innovative components and especially the HBcAg enables the induction of an immune response against multiple HBV genotypes like genotypes C and D that may be of particular relevance in some regions due to their (frequent) occurrence and provides an intrastructural help to induce HBsAg-directed CD4 T- and B-cell responses.
• 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.
• Desired effects of treatment also include prolonging survival as compared to expected survival if not receiving treatment.
• a subject in need of a treatment includes a subject already with the condition or disorder or prone to have the condition or disorder or a subject in which the condition or disorder is to be prevented.
• the subject may have a hepatitis B virus infection.
• the infection may be acute or chronic.
• the subject has a chronic hepatitis B virus infection.
• a “subject” is a vertebrate, preferably a mammal, more preferably a human.
• the term “mammal” is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples.
• the “mammal” used herein is human. Particularly preferred is a “subject” being a human.
• 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 intrastructural help.
• a vector system may be used for expression, e.g., in a cell-based expression system, such as a bacterial expression system, such as E. coli, or in a eukaryotic expression system, such as a baculoviral expression system.
• a cell-based expression system such as a bacterial expression system, such as E. coli
• a eukaryotic expression system such as a baculoviral expression system.
• GFP manufacturing practice
• a vector system can be generated comprising several, e.g. expression, vectors, wherein each of the vectors encodes an HBV core protein of an HBV genotype.
• 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 invention.
• 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.
• nucleic acid sequences encoding for different HBV core proteins may be comprised in individual expression cassettes, or all together in a single expression cassette.
• 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 H BV 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 invention 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.
• an expression cassette may be highly advantageous for the generation of a HBcAg particle of the present invention, 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 encodes i) an HBV core protein from HBV genotype C comprising a sequence having 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 the amino acid sequence set forth in SEQ ID NO: 2 or 14.
• the present invention 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.
• 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 vacciniabased 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.
• 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
• 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
• a nucleotide sequence encoding a reverse transcriptase (RT) domain of a polymerase from HBV which preferably comprises a sequence having at least 90 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, d.
• RT reverse transcriptase
• nucleotide sequence encoding HBsAg from HBV genotype C which preferably comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, and e. a nucleotide sequence encoding HBcAg from HBV genotype C, which preferably comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 17.
• 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 vaccine vector comprises an expression cassette encoding for a combination of immunogenic HBV antigens (core, surface and polymerase RT domain) from different HBV genotypes and/or serotypes.
• a combination may be advantageous for inducing a broad immune response against multiple HBV strains as well as at the same time a stronger immune response against each HBV genotype compared to vaccines comprising only antigens from a single HBV genotype.
• 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 vaccine vector comprises an expression cassette comprising a nucleotide sequence having at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
• the expression cassette comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5.
• the expression cassette has a coding sequence having the nucleotide sequence set forth in SEQ ID NO: 5.
• 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 invention 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 HLA 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 invention relates also to a pharmaceutical composition
• a pharmaceutical composition comprising the vaccine vector disclosed herein above and optionally a pharmaceutically acceptable carrier or excipient.
• 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, 37 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 invention 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 quantity of the vaccination vector defined herein is not limited by the quantification method used.
• dose the same preferably applies as stated herein above in the context of the kit and/or container comprising one or more doses of the disclosed pharmaceutical composition comprising the HBcAg particle.
• the vaccine vector refers preferably to that amount of the vaccine vector that is sufficient to result in a beneficial or desired effect of the treatment and thus, preferably to an amount that is sufficient to result in a therapeutic effect considering influencing factors such as means of administration and whether the treatment is intended to be prophylactic or curative.
• the present invention 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 disclosed vaccine vector, or the disclosed pharmaceutical composition comprising the same, optionally comprised in the disclosed container may be useful for preventing an HBV infection
• said disclosed vaccine vector, or disclosed pharmaceutical composition comprising the same, optionally comprised in the disclosed container is preferably useful for curing an HBV infection.
• the present invention 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.
• the present invention relates also to 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 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 vaccination method of the present invention herein optionally also referred to as “VacB” or “VacB vaccination regime”, represents a novel vaccination regime that is based on a pan-genotypic heterologous prime-boost therapeutic vaccine regime. It is particularly envisioned that preferably two priming steps are performed (cf. i and ii), followed by a boost (cf. iii). Priming is crucial for the immune stimulatory, and especially the therapeutic, success. Thus, for priming a, preferably adjuvanted, HBsAg and a particulate novel mosaic HBcAg particle are combined. Priming is followed by a boost using a vaccine vector boosting B and T cell responses.
• 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.
• 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.
• the pharmaceutical composition(s) may be comprised in a container disclosed herein or in a kit disclosed herein.
• the second dose of the HBcAg particle and of the HBsAg is preferably a second dose of the pharmaceutical composition disclosed herein which comprises both, the HBcAg particle disclosed herein and the HBsAg disclosed herein.
• the second dose of the HBcAg particle and of the HBsAg may be a second dose of two individual pharmaceutical compositions of the disclosure, one comprising the HBcAg particle disclosed herein and the other comprising the HBsAg disclosed herein.
• the pharmaceutical composition(s) may be comprised in a container disclosed herein or in a kit disclosed herei n.
• the same pharmaceutical composition(s) is/are used both in (i) and in (ii).
• the HBsAg is a particulate HBsAg.
• the HBsAg is an HBsAg from subtype ayw or adw and/or HBV genotype A.
• the HBsAg in (i) and/or (ii) is an HBsAg from HBV subtype adw.
• both in (i) and in (ii) the HBcAg particle comprises HBV core proteins from HBV genotypes C and D and/or the HBsAg is an HBsAg from HBV subtype adw.
• This may be particularly advantageous as priming is done in view of HBV core and surface antigens from HBV genotypes A, B, C, and D.
• a pan-genotypic priming may be ensured that is preferably well-suited especially for broad immune stimulation.
• the HBsAg is a small or large surface protein from HBV subtype adw, preferably a small or large surface protein from HBV subtype adw, even more preferably a small surface protein from HBV genotype A subtype adw, most preferably a small surface protein from HBV genotype A2 serotype adw.
• the HBsAg 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: 11.
• SEQ ID NO: 11 amino acid sequence
• 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 HBcAg 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: 8 or 17.
• the HBcAg preferably comprises the amino acid sequence set forth in SEQ ID NO: 8 or 17.
• SEQ ID NO: 8 represents a 183 amino acid long consensus sequence of an HBV core protein of HBV genotype C
• SEQ ID NO: 17 is a truncated version of SEQ ID NO:8 comprising amino acids 1 -149.
• SEQ ID NO: 8 is a consensus sequence of core proteins of genotype C strains that was generated based on alignment of 500 HBV-sequences representing the worldwide distribution of HBV strains.
• the RT domain 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: 9.
• the RT domain preferably comprises the amino acid sequence set forth in SEQ ID NO: 9.
• said sequence represents a 343 amino acid long consensus sequence across genotypes A, B, C and D of the reverse transcriptase (RT) domain of the HBV polymerase.
• SEQ ID NO: 9 is a consensus sequence of RT domains of genotype A, B, C, and D strains that was generated based on alignment of 500 HBV-sequences representing the worldwide distribution of HBV strains.
• the vaccine vector is the vaccine vector disclosed herein above, optionally comprised in the respective disclosed pharmaceutical composition comprising the same and/or in the respective disclosed container.
• the vaccine vector may be the vaccine vector disclosed herein above, comprised in the respective disclosed pharmaceutical composition or in the respective disclosed container.
• 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/or third injection are preferably intramuscular injections.
• the first dose, (ii) the second dose, and (iii) the dose of the vaccine vector are preferably administered intramuscularly.
• the first dose and/or (ii) the second dose of the HBcAg particle and of the HBsAg is preferably a dose of a pharmaceutical composition disclosed herein, which comprises both, the HBcAg particle disclosed herein and the HBsAg disclosed hererin.
• the first dose and/or (ii) the second dose of the HBcAg particle and of the HBsAg may be a dose of two individual pharmaceutical compositions of the disclosure, one comprising the HBcAg particle disclosed herein and the other comprising the HBsAg disclosed herein.
• the pharmaceutical composition(s) may be comprised in a container disclosed herein or in a kit disclosed herein.
• step (i) can encompass a single injection in case of a) and two or more injections in case of b).
• said two or more injections of (i) are done preferably in the same arm of the human subject.
• two or more injections of (i) are preferably done within less than 3 days, preferably within less than 3 days, 2 days or 1 day, even more preferably within less than 1 hour, most preferably within less than 30, 20 or 10 minutes. Particularly preferably, said two or more injections of (i) are done approximately simultaneously or simultaneously. Furthermore, the skilled artisan is aware that the same as stated herein as regards (i) applies mutatis mutandis as disclosed herein as regards (ii) and thus, the second dose of the HBcAg particle and of the HBsAg.
• the second dose preferably comprises the pharmaceutical composition comprising the HBcAg particle disclosed herein, optionally comprised in the respective disclosed container, or of the pharmaceutical composition of the HBcAg particle disclosed herein not comprising a particulate HBsAg but comprised in the respective disclosed kit.
• the injections of this paragraph are preferably intramuscular injections.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 100 g and the HBsAg in an amount from about 5 g to about 100pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 75pg and the HBsAg in an amount from about 5 g to about 75pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 20 g to about 100pg, preferably to about 60 g, and the HBsAg in an amount from about 5pg to about 75pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount of about 25 g or of about 50p and the HBsAg in an amount from about 5pg to about 75pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 100pg and the HBsAg in an amount from about 10 g to about 50pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 10 g to about 75pg and the HBsAg in an amount from about 10pg to about 50pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 20pg to about 100pg, preferably to 60pg, and the HBsAg in an amount from about 10 g to about 50pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount of about 25pg or of about 50p and the HBsAg in an amount from about 10pg to about 50pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 100pg and the HBsAg in an amount of about 20pg or of about 40pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 75pg and the HBsAg in an amount of about 20pg or of about 40 g.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 20 g to about 100pg, preferably to about 60pg, and the HBsAg in an amount of about 20pg or of about 40pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount of about 25pg or of about 50 g and the HBsAg in an amount of about 20pg or of about 40pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 25pg and the HBsAg in an amount of about 20pg.
• the first dose and the second dose each comprise the HBcAg particle in an amount from about 50pg and the HBsAg in an amount of about 40pg.
• Amounts of HBcAg and/or HBsAg are preferably determined by UV spectro photo metry .
• 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, 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 or of about 5x 10 A 8 ifu.
• the method of vaccination comprises administering the second dose about 1 week to about 8 weeks after the first dose, preferably about 2 weeks to about 6 weeks after the first dose, more preferably about 4 weeks after the first dose.
• the second dose is administered 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days after the first dose, preferably 25, 26, 27, 28, 29, 30, 31, or 32 days after the first dose, more preferably 27, 28 or 29 days after the first dose.
• This may be a particularly advantageous administration scheme as, e.g., also supported by other vaccination regimes as in the case of the commercially available HEPLISAV-B®.
• the latter is a CpG-adjuvanted recombinant yeast- derived HBsAg vaccine that has been shown to induce neutralizing antibody responses against different HBV genotypes after two intramuscular injections four weeks apart (Splawn et al., Heplisav-B vaccination for the prevention of hepatitis B virus infection in adults in the United States. Drugs Today (Bare). 2018 Jul;54(7):399-405. doi: 10.1358/dot.2018.54.7.2833984. PM ID: 30090877).
• the method of vaccination comprises administering the dose of the vaccine vector about 2 weeks to about 24 weeks after the first dose, preferably about 4 weeks to about 12 weeks after the first dose, more preferably about 8 weeks after the first dose.
• the vaccination method preferably comprises administering to a human subject a first dose of the HBcAg particle and of the HBsAg, a second dose of the HBcAg particle and of the HBsAg, and a dose of a vaccine vector as disclosed herein above, wherein a. the first and the second dose each comprise the HBcAg particle in an amount (1) from about 20pg to about 100pg, preferably to about 60pg, more preferably in an amount (2) of about 25pg or (3) of about 50pg, b.
• the first and the second dose each comprise the HBsAg in an amount (4) from about 1Opg to about 50 g, more preferably in an amount (5) of about 20pg or (6) of about 40pg, c.
• the dose of the vaccination vector comprises the vaccine vector in an amount (7) of about 6x 10 A 7 ifu or (8) of about 3x 10 A 8 ifu, d.
• the second dose is preferably administered (9) about 2 weeks to about 6, preferably (10) about 4 weeks after the first dose, and e. wherein the dose of the vaccine vector is preferably administered (11) about 4 weeks to about 12 weeks after the first dose, preferably (12) about 8 weeks after the first dose.
• the first dose and the second dose each comprise the HBcAg particle in an amount of about 25pg and the HBsAg in an amount of about 20pg
• the dose of the vaccine vector comprises the vaccine vector in an amount of about 6x 10 A 7 ifu, wherein the second dose is administered about 4 weeks after the first dose, and wherein 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 50pg and the HBsAg in an amount of about 40pg
• the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu, wherein the second dose is administered about 4 weeks after the first dose, and wherein 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 25pg and the HBsAg in an amount of about 20 g
• the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu, wherein the second dose is administered about 4 weeks after the first dose, and wherein the dose of the vaccine vector is administered about 8 weeks after the first dose.
• the greatest challenge for an HBV vaccine is that it is able to induce both a humoral as well as a cellular immune response and that this immune response is preferably directed to multiple genotypes and/or serotypes of HBV.
• the choice of the priming protein(s), the adjuvant, the vaccine vector, and/or the vaccination regime may all contribute to the effectiveness of the vaccination.
• 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 results obtained from the clinical study may show that the method of vaccination disclosed herein preferably induces a strong polyclonal and multispecific CD8+ and CD4+ T-cell responses.
• the disclosed vaccination method preferably relates to a therapeutic HBV vaccination with the potential to cure HBV infections.
• the method of vaccination is associated with a blood level increase of neutralizing and/or immune activating anti-HBs antibodies.
• the method of vaccination is associated with a blood level increase of neutralizing antibodies that neutralize HBsAg.
• the blood level increase may preferably be detectable by anti-HBs antibodies becoming detectable in peripheral blood at a titer >10 lU/ml or preferably at a titer >100 lU/ml.
• the method of vaccination is associated with a blood level increase of antibodies by a factor of at least about 2.
• 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 >1 Iog10 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 >1 log 10 or preferably by HBsAg becoming undetectable in peripheral blood.
• the method of vaccination is associated with a blood level increase of anti-HBcAg antibodies.
• the method of vaccination is associated with a blood level increase of anti-HBcAg antibodies by a factor of at least about 2.
• the method of vaccination results in a blood level increase of CD4+/CD8+ T-cells against HBsAg, HBcAg and/or a polymerase from HBV.
• Such an increase of CD4+/CD8+ T-cells against a given HBV antigen can be measured by various techniques known in the art such as EliSpot or FlouroSpot techniques or by flow cytometry following intracellular cytokine staining after antigen specific T-cell stimulation using peptide libraries or by staining of antigen-specific T-cell using HLA-multimers.
• said increase in measured using EliSpot or FlouroSpot techniques detecting cytokine-secreting T cells after antigen-spcific immune stimulation using peptide pools.
• the method of vaccination is associated with a blood level increase of HBsAg-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of HBsAg-specific CD4+ and/or CD8+ T-cells to at least about 10, preferably at least about 100 spots/500.000 peripheral blood mononuclear cells, as preferably measured by EliSpot technique.
• the method of vaccination is associated with a blood level increase of HBcAg-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of HBcAg-specific CD4+ and/or CD8+ T-cells to at least about 10, preferably at least about 100 spots/500.000 peripheral blood mononuclear cells, as preferably measured by EliSpot technique.
• the method of vaccination is associated with a blood level increase of RT-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of RT-specific CD4+ and/or CD8+ T-cells to at least about 10, preferably at least about 100 spots/500.000 peripheral blood mononuclear cells, as preferably measured by EliSpot technique.
• the method of vaccination is associated with a blood level increase of HBcAg- and HBsAg-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of HBcAg- and HBsAg-specific CD4+ and/or CD8+ T- cells at least about 10, preferably at least about 100 spots/500.000 peripheral blood mononuclear cells, as preferably measured by EliSpot technique.
• the method of vaccination is associated with a blood level increase of HBsAg-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of HBsAg-specific CD4+ and/or CD8+ T-cells by a factor of at least about 2 as detected by flow cytometry analysis after intracellular cytokine staining.
• the method of vaccination is associated with a blood level increase of HBcAg-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of HBsAg-specific CD4+ and/or CD8+ T-cells by a factor of at least about 2 as detected by flow cytometry analysis after intracellular cytokine staining.
• the method of vaccination is associated with a blood level increase of HBsAg- and HBcAg-specific CD4+ and/or CD8+ T-cells.
• the method of vaccination is associated with a blood level increase of HBsAg- and HBcAg-specific CD4+ and/or CD8+ T- cells by a factor of at least about 2 as detected by flow cytometry analysis after intracellular cytokine staining.
• the method of vaccination is not associated with dose limiting toxicity. More preferably, the method of vaccination is not associated with dose limiting toxicity with the first dose and the second dose each comprising the HBcAg particle in an amount from about 10pg to about 100pg and the HBsAg in an amount from about 5pg to about 100pg, and the dose of the vaccine vector comprising the vaccine vector in an amount from about 1x 10 A 7 ifu to about 1x 10 A 9 ifu.
• the method of vaccination is not associated with dose limiting toxicity with the first dose and the second dose each comprising the HBcAg particle in an amount of about 25pg or of about 50p and the HBsAg in an amount of about 20pg or of about 40pg, and the dose of the vaccine vector comprising the vaccine vector in an amount of about 6x 10 A 7 ifu or of about 3x 10 A 8 ifu.
• the method of vaccination may not be associated with dose limiting toxicity with the first dose and the second dose each comprising the HBcAg particle in an amount of about 25pg and the HBsAg in an amount of about 20pg, and the dose of the vaccine vector comprising the vaccine vector in an amount of about 6x 10 A 7 ifu.
• the method of vaccination may not be associated with dose limiting toxicity with the first dose and the second dose each comprising the HBcAg particle in an amount of about 50
• the method of vaccination is associated with a reduction of HBsAg titers in peripheral blood by at least about 1 log 10 from start of the treatment.
• the method of vaccination is associated with a reduction of HBsAg titers in peripheral blood below the limit of quantification.
• the method of vaccination is associated with a reduction of HBsAg titers in peripheral blood below the limit of detection.
• HBsAg titers are preferably measured by chemiluminescent immunoassay (CLIA).
• the method of vaccination is associated with an increased frequency of total HBV-specific, cytokine secreting T cells compared to pretreatment values, preferably as measured in a FluoroSpot assay.
• the increase is at least about two-fold.
• the increase is at least about five-fold.
• the method of vaccination is associated with increased number of cytokine secreting, HBV S-, core- and/or pol-specific T cells compared to pretreatment values, preferably as measured in an FluoroSpot assay or an intracellular cytokine staining (ICS) assay.
• the increase is at least about two-fold.
• the increase is at least about fivefold.
• the method of vaccination is associated with increased number of cytokine secreting, HBV S-specific T cells compared to pretreatment values, preferably as measured in an FluoroSpot assay or an intracellular cytokine staining (ICS) assay.
• ICS intracellular cytokine staining
• the method of vaccination is associated with increased number of cytokine secreting, core-specific T cells compared to pretreatment values, preferably as measured in an FuoroSpot assay or an intracellular cytokine staining (ICS) assay.
• FuoroSpot assay or an intracellular cytokine staining (ICS) assay.
• ICS intracellular cytokine staining
• the method of vaccination is associated with increased number of cytokine secreting, pol-specific T cells compared to pretreatment values, preferably as measured in an FuoroSpot assay or an intracellular cytokine staining (ICS) assay.
• FuoroSpot assay or an intracellular cytokine staining (ICS) assay.
• ICS intracellular cytokine staining
• the method of vaccination is associated with an at least about 20% reduction of HBcore-related antigen (HBcr-Ag) compared to pretreatment values or stable suppression (over >6 weeks), preferably as measured by chemiluminescent enzyme immunoassay (CLEIA).
• HBcr-Ag HBcore-related antigen
• CLIA chemiluminescent enzyme immunoassay
• the reduction of HBcr-Ag can even be higher, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%.
• the reduction of HBcr-Ag can be to below the limit of detection.
• the method of vaccination is a therapeutic vaccination method.
• the method of vaccination disclosed herein offers thus an alternative vaccination method that preferably has the advantage of providing a strong stimulation of the immune system, including preferably CD4+/CD8+ T-cell induction and/or anti-HBcAg antibody production.
• the disclosed method of vaccination is particularly well suited for applications in view of HBV and in particular in view of cases associated with an infection of an HBV genotype that may be of particular relevance in some regions due to their (frequent) occurrence.
• the method of vaccination is a vaccination method for treating an HBV infection.
• the HBV infection can be acute or chronic.
• the method of vaccination is a vaccination method for treating a chronic HBV infection.
• CD4+ TH1/TH2 T-cell responses can be induced for neutralizing anti-HBc antibody and anti-HBs antibody production by the two protein prime vaccinations and thus, a first and a second dose of both the HBcAg particle and an HBsAg.
• cytotoxic CD8+ T-cells can be activated for an efficient elimination of infected cells by the subsequent vaccine vector administration that serves as a further boost.
• the method of vaccination disclosed herein represents a novel therapeutic, preferably curative, vaccination regime for treating an HBV infection.
• the present invention relates also to an HBcAg particle and/or an HBsAg, for use in a method of vaccination.
• 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 and/or an, optionally nucleosidic, adjuvanted HBsAg, preferably a CpG, more preferably a CpG-1018, adjuvanted HBsAg.
• the method of vaccination is preferably the method of vaccination disclosed herein.
• the HBcAg particle, and optionally the HBsAg 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 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 use in a method of vaccination, said method preferably comprising 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 and/or an, optionally nucleosidic, adjuvanted HBsAg, preferably a CpG, more preferably a CpG-1018, adjuvanted HBsAg.
• the method of vaccination is preferably the method of vaccination disclosed herein.
• the HBcAg particle, and optionally the HBsAg 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 an HBcAg particle and/or an HBsAg, 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 method of vaccination is preferably the method of vaccination disclosed herein.
• the HBcAg particle, and optionally the HBsAg 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 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
• HBcAg particle of any one of the preceding items, wherein the HBcAg particle comprises HBV core proteins from HBV genotypes C and D and wherein the HBV core proteins from said HBV genotypes are in a ratio of about 10:90 to about 90:10.
• a container comprising one or more doses of the pharmaceutical composition of any one of items 28 to 33.
• a kit comprising the pharmaceutical composition of item 28 and a second pharmaceutical composition comprising an HBsAg, wherein the HBsAg is preferably an particulate HBsAg.
• the kit of item 35, wherein the second pharmaceutical composition further comprises an adjuvant.
• the kit of any one of items 35 to 37, wherein the adjuvant comprised in the second pharmaceutical composition and/or the adjuvant comprised in the third container is a nucleosidic adjuvant.
• the kit of any one of items 35 to 38, wherein the adjuvant comprised in the second pharmaceutical composition and/or the adjuvant comprised in the third container is a CpG.
• the kit of any one of items 35 to 39, wherein the adjuvant comprised in the second pharmaceutical composition and/or the adjuvant comprised in the third container is CpG- 1018.
• the container of item 34 or the kit of any one of items 35 to 40, wherein a dose of the pharmaceutical composition comprises the HBcAg particle in an amount from about 10pg to about 100pg.
• a dose of the pharmaceutical composition comprises the HBcAg particle in an amount from about 10pg to about 75 g.
• the container of item 34 or the kit of any one of items 35 to 42, wherein a dose of the pharmaceutical composition comprises the HBcAg particle in an amount from about 20pg to about 100pg, preferably to about 60pg.
• the container of item 34 or the kit of any one of items 35 to 43, wherein a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 25 g or of about 50 g.
• the container of item 34 or the kit of any one of items 35 to 44 wherein in case of the container a dose of the pharmaceutical composition or in case of the kit a dose of the second pharmaceutical composition comprises an HBsAg in an amount from about 5pg to about 1OOpg, preferably in an amount from about 5 g to about 75pg, wherein the HBsAg is preferably an particulate HBsAg.
• a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 25 g or of about 50pg and wherein in case of the container a dose of the pharmaceutical composition or in case of the kit a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 20pg or of about 40pg.
• a dose of the pharmaceutical composition comprises the HBcAg particle in an amount of about 25pg and wherein in case of the container a dose of the pharmaceutical composition or in case of the kit a dose of the second pharmaceutical composition comprises HBsAg in an amount of about 20 g.
• An expression cassette, an mRNA, or a cDNA wherein the expression cassette, mRNA, or cDNA, encodes 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.
• the expression cassette, mRNA, or cDNA, of item 58 wherein the expression cassette, mRNA, or cDNA, encodes an HBV core protein from HBV genotype C comprising a sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or an HBV core protein from HBV genotype D comprising a sequence that has 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, of item 58 or 59 wherein the expression cassette, mRNA, or cDNA, encodes an HBV core protein from HBV genotype C comprising a sequence that has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 13 and/or an HBV core protein from HBV genotype D comprising a sequence that has 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 of any one of items 58 to 61 , wherein the expression cassette, mRNA, or cDNA, comprises a first nucleotide sequence encoding an HBV core protein from HBV genotype C, wherein said first nucleotide sequence comprises a sequence that has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3 or 15, and/or wherein 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 comprises a sequence that has at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 16.
• nucleic acid molecule comprising the expression cassette, the mRNA, or the cDNA, of any one of items 58 to 64.
• the nucleic acid molecule of item 65 wherein the nucleic acid molecule is a DNA molecule.
• nucleic acid molecule of item 66 wherein the nucleic acid molecule is an mRNA molecule.
• An expression vector comprising the expression cassette or the cDNA of any one of items 58 to 64 or the nucleic acid molecule of item 65 or 66.
• the expression vector of item 68 wherein the expression vector is a recombinant vector.
• the expression vector of item 70 or 71 wherein the expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 10.
• a vaccine vector wherein the vaccine vector is preferably an 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 of item 74, wherein the nucleid acid molecule comprises a. a nucleotide sequence encoding a HBsAg from HBV genotype A, b. a nucleotide sequence encoding a HBcAg from HBV genotype D, c. a nucleotide sequence encoding a reverse transcriptase (RT) domain of a polymerase from HBV, d.
• RT reverse transcriptase
• nucleotide sequence encoding HBsAg from HBV genotype C, and e. a nucleotide sequence encoding HBcAg from HBV genotype C.
• a pharmaceutical composition comprising the vaccine vector of any one of items 74 to 80 and optionally a pharmaceutically acceptable carrier or excipient.
• a container comprising one or more doses of the pharmaceutical composition of item 81 , wherein a dose of the pharmaceutical composition comprises the vaccine vector in an amount from about 1x 10 A 7 ifu to about 1x 10 A 9 ifu.
• the container of item 82 wherein a dose of the pharmaceutical composition comprises the vaccine vector in an amount from about 5x 10 A 7 ifu to about 1x 10 A 9 ifu.
• the container of item 82 or 83, wherein a dose of the pharmaceutical composition comprises the vaccine vector 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.
• the container of any one of items 82 to 84, wherein a dose of the pharmaceutical composition comprises the vaccine vector in an amount of about 3x 10 A 8 ifu.
• the vaccine vector for the use of item 90, or the pharmaceutical composition for the use of item 90, or comprised in the container for the use of item 90, wherein the use comprises inducing anti-HBcAg antibodies.
• 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.
• the first dose of the HBcAg particle and of the HBsAg is a first dose of the pharmaceutical composition of any one of items 29 to 33, optionally comprised in the container of any one of items 34 or 41 to 50, or of the pharmaceutical composition of item 28 comprised in the kit of any one of items 35 to 50, wherein the HBsAg is preferably an particulate HBsAg.
• the method of vaccination of item 93 or 94 wherein in (ii) the second dose of the HBcAg and of the HBsAg is a second dose of the pharmaceutical composition of any one of items 29 to 33, optionally comprised in the container of any one of items 34 or 41 to 50, or of the pharmaceutical composition of item 28 comprised in the kit of any one of items 35 to 50, wherein the HBsAg is preferably an particulate HBsAg.
• the HBsAg is a small or large surface protein from HBV genotype A serotype adw.
• the HBsAg comprises the amino acid sequence set forth in SEQ ID NO: 7. .
• the method of vaccination of any one of items 93 to 104 wherein 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.
• the method of vaccination of any one of items 93 to 105 wherein the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 100pg and the HBsAg in an amount from about 5 g to about 100pg. .
• the method of vaccination of item 93 or 106 wherein the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 75 g and the HBsAg in an amount from about 5 g to about 75pg. .
• the method of vaccination of items 93 to 110 wherein the first dose and the second dose each comprise the HBcAg particle in an amount from about 10pg to about 75 g and the HBsAg in an amount from about 10 g to about 50pg. .
• the method of vaccination of items 93 to 111 wherein the first dose and the second dose each comprise the HBcAg particle in an amount from about 20pg to about 100pg, preferably to about 60pg, and the HBsAg in an amount from about 10pg to about 50pg..
• the method of vaccination of items 93 to 112 wherein the first dose and the second dose each comprise the HBcAg particle in an amount of about 25pg or of about 50p and the HBsAg in an amount from about 10 g to about 50pg. .
• the method of vaccination of any one of items 93 to 121 wherein the dose of the vaccine vector comprises the vaccine vector 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. .
• the method of vaccination of any one of items 93 to 124, wherein the method of vaccination comprises administering the second dose about 2 weeks to about 6 weeks after the first dose.
• the method of vaccination of any one of items 93 to 126, wherein the method of vaccination comprises administering the dose of the vaccine vector about 2 weeks to about 24 weeks after the first dose. .
• the method of vaccination of any one of items 93 to 127, wherein the method of vaccination comprises administering the dose of the vaccine vector about 4 weeks to about 12 weeks after the first dose. .
• the method of vaccination of any one of items 93 to 128, wherein the method of vaccination comprises administering the dose of the vaccine vector about 8 weeks after the first dose. .
• any one of items 93 to 129 wherein the first dose and the second dose each comprise the HBcAg particle in an amount of about 25pg and the HBsAg in an amount of about 20pg, wherein the dose of the vaccine vector comprises the vaccine vector in an amount of about 6x 10 A 7 ifu, wherein the second dose is administered about 4 weeks after the first dose, and wherein the dose of the vaccine vector is administered about 8 weeks after the first dose.
• any one of items 93 to 130 wherein the first dose and the second dose each comprise the HBcAg particle in an amount of about 50pg and the HBsAg in an amount of about 40pg, wherein the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu, wherein the second dose is administered about 4 weeks after the first dose, and wherein the dose of the vaccine vector is administered about 8 weeks after the first dose.
• the method of vaccination of any one of items 93 to 131 wherein the first dose and the second dose each comprise the HBcAg particle in an amount of about 25pg and the HBsAg in an amount of about 20pg, wherein the dose of the vaccine vector comprises the vaccine vector in an amount of about 3x 10 A 8 ifu, wherein the second dose is administered about 4 weeks after the first dose, and wherein the dose of the vaccine vector is administered about 8 weeks after the first dose. .
• the method of vaccination of any one of items 93 to 132 wherein the method of vaccination results in an induction of an immune response against the HBV antigens it is composed of. .
• the method of vaccination of any one of items 93 to 133 wherein the method of vaccination results in an induction of an antigen-specific adaptive immune response.
• the method of vaccination of any one of items 93 to 134 wherein the method of vaccination results in an induction of anti-HBcAg antibodies, anti-HBsAg antibodies and/or anti-RT antibodies.
• the method of vaccination of any one of items 93 to 136 wherein the method of vaccination 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 lU/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 vaccination of any one of items 93 to 141 wherein the method of vaccination is a vaccination method for treating a HBV infection.
• the method of any one of items 93 to 142 wherein the subject has a chronic hepatitis B virus infection.
• the method of any one of items 93 to 143, wherein the method is associated with a reduction of HBsAg titers in peripheral blood by at least about 1 Iog10 from start of the treatment, preferably as measured by chemiluminescent immunoassay (CLIA).
• CLIA chemiluminescent immunoassay
• the method of any one of items 93 to 144 wherein the method is associated with a reduction of HBsAg titers in peripheral blood below the limit of quantification or below the limit of detection.
• 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).
• 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.
• Figure 15 illustrates the immunization scheme of the Phase 1a clinical trial.
• Figure 16 schematically depicts the Phase 1b/2a clinical trial design.
• TCR-grafted T cells were activated ex vivo by co-culture with primary human dendritic cells supplemented with either 10 pg of RIGA HBcAg, full-length HBV core protein genotype D (D) or ACD HBcoreAg particles. T-cell activation was determined via TNFa secretion measured by flow cytometry after intracellular cytokine staining.
• 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.
• 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. VI. EXAMPLES
• Example 1 Generation of an (recombinant) HBcAg particle of the disclosure
• 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 I 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).
• Both HBcAg insert sequences 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.
• Tet Tetracycline
• the insert containing both ORFs of the HBV core antigens was cloned into a pRSF-Duet-1 plasmid via Agel/Pfol.
• 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 / 18.54 kDa) and genotype D (full length 1-183aa I 21.12 kDa).
• genotype C truncated 1- 163aa / 18.54 kDa
• 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.
• HBcAg particle indeed consists of HBV core proteins from both genotypes C and D
• a pull-down assay specific for genotype D HBV core proteins was performed as illustrated in Figure 6. Therefore, the expression plasmid was modified by adding a 15 aa AviTagTM -sequence to the N-terminal part of the sequence for the full-length genotype D HBV core protein.
• biotin ligases bound biotin to the AviTag peptide, thus leading to biotinylation of the HBV core proteins from genotype D, while HBV core proteins from genotype C were not labelled.
• 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).
• NAs encapsidated nucleic acids
• 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.
• 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).
• Example 6 Protein superior over DNA or RNA for prime vaccination
• Recombinant proteins are known to elicit strong antibody responses, but rather low CD8+ T-cell response, unless a potent Th1/Th2 adjuvant is used.
• alternative vaccines such as DNA and mRNA-based vaccines that do not require additional adjuvants, could improve the immunogenicity of VacB.
• mRNA- or DNA-based vaccines would allow the expression of several antigens at a time and avoid the complicated and expensive purification of recombinant proteins for clinical use.
• protein-, DNA- and RNA-based vaccines were compared for VacB priming followed by a boost with a recombinant vaccine vector like a recombinant MVA vaccine vector like MHBVac.
• mice were immunized twice with CpG-adjuvanted HBsAg and the recombinant HBcAg particle and for comparison with various doses of an mRNA vaccine, formulated in lipid nanoparticles, or a plasmid DNA vaccine. All demonstrated high levels of anti-HBc antibodies ( Figure 11 A), but priming with the different vaccines resulted in various levels of anti-HBs ( Figure 11 B).
• Adjuvanted protein HBsAg induced the strongest anti-HBs response, followed by a dose-dependent response to mRNA. Priming with DNA vaccine resulted in very low anti-HBs responses, independently of the dose used.
• Example 7 Defining the optimal adjuvant class for protein prime
• 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
• mice receiving VacB demonstrated a long-term, sustained decrease in serum HBsAg levels over the monitoring period ( Figure 13 A-B). Moreover, in all immunized mice serum HBeAg levels were significantly reduced until end of follow-up ( Figure 13 C). VacB performed equally well, independently of gender and type of clinical candidate Th1/Th2 adjuvant that were used for immunizations.
• 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 2 provides an overview of the study groups and Table 3 indicates the quality/batches of the vaccine components used for the GLP study.
• 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 / 6,000 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.
• Organ weight [00247] No test item formulation related organ weight differences compared with controls were noted for both sexes in main group animals at the end of the dosing period (Day 37) and in the recovery groups (Day 50).
• Topical steroids are permitted provided they are not required to be applied to injection site.
• HEPLISAV-B® comprises a HBsAg that is produced in yeast cells (Hansenula polymorpha) by recombinant DNA technology as well as the adjuvant CpG-1018 that is chemically synthesized.
• HEPLISAV-B® is provided as solution for intramuscular injection (0.5 mL) in pre-filled syringes without needle, wherein one syringe represents one dose.
• Table 8 Overview of general properties of HEPLISAV-B®.
• subjects may be withdrawn from vaccination, i.e. will not receive second and/or third immunizations if:
• 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 pg 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 / MVA boost therapeutic HBV vaccination method according to the present invention with two ascending amount levels of the HBcAg particle and adjuvanted HBsAg in healthy subjects, using for the latter HEPLISAV-B® including its adjuvant CpG-1018, 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 10 refers to maximum amounts.
• Humoral and cellular immunogenicity assays may include, but are not limited to, those shown in Table 10.
• 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.
• the adjuvant CpG1018 (an ingredient of HEPLISAV B®) 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 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 prime-boost vaccine consistingof 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).
• HBV infection (CHB) that fulfills the following criteria:
• Any chronic or active neurologic disorder including diagnosis of migraine, seizures and epilepsy. Exception: a febrile seizure as a child and occasional headaches.
• 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 pg 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 co-culture 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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