Hepatitis B immunization regimens and compositions

Abstract

The present invention relates to immunogenic compositions and combinations thereof that may find use in immunization regimens for the treatment of chronic hepatitis B. The immunogenic compositions include HBs, HBc, and a replication-deficient chimpanzee adenovirus (ChAd) vector containing a polynucleotide encoding the human invariant chain (hIi) fused to HBc. Another immunogenic composition includes a modified vaccinia virus Ankara (MVA) vector containing a polynucleotide encoding HBs and HBc. Another immunogenic composition includes recombinant HBs, a C-terminally truncated recombinant HBc, and an adjuvant containing MPL and QS-21. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to novel compositions that may find use in immunization regimens for the treatment of chronic hepatitis B. [Background technology]

[0002] Hepatitis B virus (HBV) infection is a major public health problem. Approximately 257 million people worldwide are infected with HBV [WHO, 2017]. The clinical course and outcome of HBV infection are primarily driven by the age at infection and the complex interplay between the virus and the host immune response [Ott, 2012; Maini, 2016]. Therefore, exposure to HBV can result in acute hepatitis that resolves spontaneously or can progress to various forms of chronic infection, such as an inactive hepatitis B surface antigen (HBsAg) carrier state, chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [Liaw, 2009]. HBsAg prevalence in the adult population is >2%, with rates of 5-8% in Southeast Asia and China and >8% in Africa. Between 15 and 40% of people with chronic hepatitis B infection (defined as serum HBsAg detectable for more than 6 months) will develop hepatic sequelae, of which liver cirrhosis (LC), hepatic decompensation, and HCC are the main complications.

[0003] Although routine prophylactic hepatitis B immunization in young children has been highly effective in reducing the incidence and prevalence of hepatitis B in many endemic countries, it has not yet resulted in a strong reduction in the prevalence of chronic hepatitis B infection (CHB) in adolescents and adults, and is not expected to impact HBV-related deaths until several decades after its introduction. In 2015, hepatitis B was responsible for 887,000 deaths, the majority due to cirrhosis and HCC [WHO, 2017].

[0004] Clinical management of chronic hepatitis B aims to improve survival and quality of life by preventing disease progression and, consequently, the development of HCC [Liaw, 2013]. Current treatment strategies are primarily based on long-term suppression of HBV DNA replication to achieve stabilization of HBV-induced liver disease and prevent progression. Serum HBV DNA levels are the underlying endpoint for all current treatment modalities. While the achievement of loss of (detectable) hepatitis B e antigen (HBeAg) is another valuable biomarker, loss of HBsAg, with or without anti-HBs seroconversion, is generally considered the optimal endpoint representing "functional cure," as it indicates significant suppression of HBV replication and viral protein expression [Block, 2017; Cornberg, 2017]. Currently, CHB patients have two main treatment options: pegylated interferon alpha (PegIFNα) or nucleotide (nucleoside) analogs (NAs) [EASL, 2017]. PegIFNα, which aims to induce long-term immune control through finite-duration treatment, can achieve sustained, off-treatment control, but durable virological responses and hepatitis B surface antigen (HBsAg) loss are limited to a small percentage of patients. Furthermore, its poor tolerability and long-term safety concerns make a significant number of patients ineligible for this type of treatment.

[0005] NAs act by suppressing DNA replication through the inhibition of HBV polymerase reverse transcriptase activity. NAs approved in Europe for HBV treatment include entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF), which are associated with a high barrier to HBV resistance, and lamivudine (LAM), adefovir dipivoxil (ADV), and telbivudine (TBV), which are associated with a low barrier to HBV resistance. The main advantages of treatment with potent NAs with a high barrier to resistance are their predictable, high, long-term antiviral efficacy, which leads to HBV DNA suppression in the majority of adherent patients, and their favorable safety profile. A disadvantage of NA treatment is its long-term treatment regimen, as NAs usually do not achieve HBV eradication and discontinuation of NAs can lead to HBV recurrence [Kranidioti, 2015]. HBsAg loss, representing functional cure, is currently the gold standard treatment endpoint in CHB [Block, 2017; Cornberg, 2017], but is rarely achieved with NA treatment [Zoutendijk, 2011].

[0006] Due to the low rate of HBsAg seroclearance [Zoutendijk, 2011] and the high risk of off-NA viral relapse [Kranidioti, 2015], most patients are maintained on long-term or even indefinite NA therapy, which may be associated with poor patient compliance with therapy, increased financial costs, and an increased risk of drug toxicity and drug resistance mutations due to long-term exposure [Terrault, 2015]. Therefore, new strategies to supplement NA therapy are needed to achieve a "functional cure" with a finite regimen.

[0007] New therapeutic strategies currently being explored include new antiviral strategies as well as novel immunotherapeutic strategies that boost HBV-specific adaptive immune responses or activate intrahepatic innate immunity [Durantel, 2016]. To date, none of these experimental treatments have been shown to be effective. Among the vaccination strategies evaluated, none have demonstrated robust polyfunctional CD8 expression against the HBV core antigen (HBcAg), which is crucial for restoring immune control against the virus. +However, previous efforts to develop recombinant vaccines based on HBV surface and / or pre-S antigens failed to induce T cell responses [Lau, 2002; Li, 2011; Liang, 2011; Bertoletti, 2012; Boni, 2012]. Early efforts at recombinant vaccines based on HBV surface and / or pre-S antigens induced preliminary antibody responses but failed to induce HBV-specific CD8+ T cell responses and provided no clinical or virological benefit [Jung, 2002; Vandepapeliere, 2007]. DNA vaccines expressing the HBV envelope failed to restore HBsAg- and HBcAg-specific T cell responses and therefore did not reduce the risk of relapse in patients after NA discontinuation [Fontaine, 2015]. Using a new delivery system, a DNA vaccine encoding S, pre-S1 / S2 (prime vaccine) and an MVA viral vector vaccine (boost vaccine) failed to induce T cells or reduce viremia, suggesting that HBV pre-S and surface antigens alone were not sufficient to cure patients [Cavenaugh, 2011]. More recently, vaccine strategies targeting multiple HBV antigens and new delivery systems have been investigated. A recombinant HBsAg / HBcAg vaccine reduced viral load to very low levels (i.e., approximately 50 IU / ml) in only half of patients [Al-Mahtab, 2013]. A DNA vaccine encoding S, pre-S1 / S2, core, polymerase, and X proteins, along with genetically adjuvanted IL-12, together with lamivudine, induced multispecific T cell responses and a >2 log reduction in viral load in half of patients. However, no change in quantitative detection of HBsAg, loss of HBsAg, or HBsAg seroconversion was observed in any patient [Yang, 2012]. GS-4774 vaccine, a yeast-based T cell vaccine expressing the large S, core, and X proteins of HBV, did not result in a significant reduction in HBsAg in virally suppressed CHB patients [Lok, 2016]. Summary of the Invention [Problem to be solved by the invention]

[0008] There remains an unmet need for treatments that can clear HBsAg, allowing patients to safely discontinue NA therapy without virologic or clinical relapse. [Means for solving the problem]

[0009] Summary of the Invention An immunogenic composition is provided, comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs), a nucleic acid encoding hepatitis B core antigen (HBc), and a nucleic acid encoding human invariant chain (hIi) fused to HBc. The composition may find use in a method for treating chronic hepatitis B by administering the composition in a prime-boost regimen with at least one other immunogenic composition.

[0010] In a further aspect, there is provided an immunogenic composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B core antigen (HBc). The composition may find use in a method for treating chronic hepatitis B by administering the composition in a prime-boost regimen with at least one other immunogenic composition.

[0011] In a further aspect, an immunogenic composition is provided comprising a recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B core antigen (HBc), and an adjuvant containing MPL (3-D monophosphoryl lipid A) and QS-21 (a triterpene glycoside purified from the bark of Quillaja saponaria). The composition may find use in a method for treating chronic hepatitis B by administering the composition in a prime-boost regimen with at least one other immunogenic composition.

[0012] In a further aspect, a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant. An immunogenic combination comprising:

[0013] The immunogenic combination may find use in a method for treating chronic hepatitis B (CHB) by administration of the composition in a prime-boost regimen.

[0014] The immunogenic combination may find use in a method for treating CHB in humans by sequential or simultaneous administration of the compositions. [Brief explanation of the drawings]

[0015] [Figure 1] HBc-specific (A) and HBs-specific (B) CD8+ T cell responses (individual animals (with median values) are represented) 14 days after primary immunization with ChAd155-HBV (with and without hIi) and 7 days after MVA-HBV booster immunization. [Figure 2] HBc-specific antibody responses (individual animals (with geometric mean titers (GMTs)) are presented) 14 days after primary immunization with ChAd155-HBV (with and without hIi) and 7 days after MVA-HBV booster immunization. [Figure 3] HBc- and HBs-specific CD4+ T cell responses 7 days after the third dose of HBc, HBs, or HBc-HBs formulated in NaCl, 50 μl of AS01B-4 (pools of 5 animals / group (with median values) are represented). [Figure 4]HBs-specific CD8+ T cell responses 7 days after the third dose of HBc, HBs, or HBc-HBs formulated in NaCl, 50 μl of AS01B-4 (pools of 5 animals / group (with median values) are represented). [Figure 5] Anti-HBc and anti-HBs antibody responses 14 days after the third dose of HBc, HBs, or HBc-HBs formulated in NaCl, 50 μl of AS01B-4 (individual animals (with geometric means and 95% CI) are presented). [Figure 6A-B] HBs(A) and HBc(B)-specific CD4+ T cell responses 7 days after the third administration of NaCl, HBc-HBs, HBc-HBs and Alum, HBc-HBs and AS01B-4, or HBc-HBs and AS01E-4 (pools of 5 animals / group (with median values) are represented). [Figure 6C] HBs-specific CD8+ (C) T cell responses 7 days after the third administration of NaCl, HBc-HBs, HBc-HBs and Alum, HBc-HBs and AS01B-4, or HBc-HBs and AS01E-4 (pools of 5 animals / group (with median values) are represented). [Figure 7] HBs(A) and HBc(B)-specific antibody responses 14 days after the third administration of NaCl, HBc-HBs, HBc-HBs and Alum, HBc-HBs and AS01B-4, or HBc-HBs and AS01E-4 (individual animals (with geometric means and 95% CI) are shown). [Figure 8A] HBc(A)-specific CD8+ T cell responses (individual animals (with median values)) 7 days after the second and fourth doses of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 8B] HBs(B)-specific CD8+ T cell responses (individual animals (with median values)) 7 days after the second and fourth doses of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 9]HBc(A)- or HBs(B)-specific CD4+ T cell responses (individual animals (with median values)) 7 days after the second and fourth doses of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 10] HBc- and HBs-specific CD4+ (A) and CD8+ (B) T cells among liver-infiltrating lymphocytes 7 days after the fourth dose of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein (pools of 3 or 4 animals (with median values)). [Figure 11] HBc-specific (A) and HBs-specific (B) antibody responses following a prime-boost vaccine regimen (individual animals (with geometric means) are represented). [Figure 12] mIi, HBc, and HBs-specific IFNγ ELISpot responses 2 weeks after the first and second injections of PBS or ChAd155-mIi-HBV vector (10 9 vp). [Figure 13] Anti-mIi antibody responses (ELISA) induced by two administrations of ChAd155-mIi-HBV (109 vp) in CB6F1 mice, 2 weeks after the first and second injections. [Figure 14] HBc-specific splenic (A) or liver (B) CD8+ T cells (individual animals (with medians)) 7 days after the second and 7 days after the fourth dose of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 15] HBc-specific splenic (A) or liver (B) CD4+ T cells (individual animals (with medians)) 7 days after the second and 7 days after the fourth dose of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 16]HBs-specific splenic (A) or liver (B) CD8+ T cells (individual animals (with medians)) 7 days after the second and 7 days after the fourth dose of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 17] HBs-specific splenic (A) or liver (B) CD4+ T cells (individual animals (with medians)) 7 days after the second and 7 days after the fourth dose of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein. [Figure 18] Anti-HBs (A) and anti-HBc (B) binding antibody responses on days 23, 65, and 93 (before administration of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein, 7 days after the second dose, and 7 days after the fourth dose). [Figure 19] AST (A) and ALT (B) levels measured in the serum of mice (groups 1, 2, 3, and 4) on days 38, 65, and 93 (7 days after the first, second, and fourth doses of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein; groups 1, 2, 3) or on day 93 (group 4). [Figure 20] HBs antigen levels in the serum of AAV2 / 8-HBV-injected mice before administration of NaCl, heterologous vector prime-boost with subsequent recombinant protein, or heterologous vector prime-boost with simultaneous recombinant protein, 7 days after the second dose, and 7 days after the fourth dose. [Figure 21] Structure of the HBc-2A-HBs construct. [Figure 22] Structure of the hIi-HBc-2A-HBs construct. [Figure 23] Frequency of HBc- and HBs-specific CD4+ T cells in leukocytes of CB6F1 mice 7 days after the second immunization with adjuvanted HBc, HBs, and various ratios of HBc / HBs. [Figure 24] Frequency of HBc- and HBs-specific CD4+ T cells in leukocytes of CB6F1 mice 7 days after the third immunization with adjuvanted HBc, HBs, and various ratios of HBc / HBs. [Figure 25] Frequency of HBs-specific CD8+ T cells in leukocytes of CB6F1 mice 7 days after the second and third immunizations with adjuvanted HBc, HBs, and various ratios of HBc / HBs. [Figure 26] Anti-HBc and HBs humoral responses induced in CB6F1 mice 14 days after the second immunization with adjuvanted HBc, HBs, and various ratios of HBc / HBs. [Figure 27] Anti-HBc and HBs humoral responses induced in CB6F1 mice 14 days after the third immunization with adjuvanted HBc, HBs, and various ratios of HBc / HBs. DETAILED DESCRIPTION OF THE INVENTION

[0016] Sequence Listing SEQ ID NO: 1: Amino acid sequence of HBs SEQ ID NO: 2: Amino acid sequence of HBc truncation SEQ ID NO: 3: Amino acid sequence of the spacer incorporating the 2A cleavage region of foot-and-mouth disease virus SEQ ID NO: 4: Nucleotide sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus SEQ ID NO: 5: Amino acid sequence of HBc-2A-HBs SEQ ID NO: 6: Nucleotide sequence encoding HBc-2A-HBs SEQ ID NO: 7: Amino acid sequence of hIi SEQ ID NO: 8: Nucleotide sequence encoding hIi SEQ ID NO: 9: Amino acid sequence of hIi-HBc-2A-HBs SEQ ID NO: 10: Nucleotide sequence encoding hIi-HBc-2A-HBs SEQ ID NO: 11: Amino acid sequence of HBc SEQ ID NO: 12: Amino acid sequence of hIi alternative variant SEQ ID NO: 13: Nucleotide sequence encoding hI alternative variant SEQ ID NO: 14: Alternative nucleic acid sequence of hIi-HBc-2A-HBs SEQ ID NO: 15: Alternative amino acid sequence of hIi-HBc-2A-HBs

[0017] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0018] Throughout this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0019] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate disclosure by virtue of prior invention. All definitions provided herein with respect to one aspect of the invention also apply to other aspects of the invention.

[0020] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length, co-translational modification, or post-translational modification. A fusion protein (or "chimeric protein") is a recombinant protein containing two or more peptide-linked proteins. Fusion proteins are made by joining two or more genes that originally encoded separate proteins. Translation of the fusion genes results in a single fusion protein. With respect to a protein or polypeptide, recombinant means that the protein is expressed from a recombinant polynucleotide.

[0021] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric macromolecule made from nucleotide monomers. Suitably, the polynucleotides of the present invention are recombinant. By recombinant, we mean that the polynucleotide is the product of at least one cloning, restriction, or ligation step, or other procedure that results in a polynucleotide that differs from a polynucleotide found in nature.

[0022] A heterologous nucleic acid sequence refers to any nucleic acid sequence that is not isolated from, derived from, or based on a naturally occurring nucleic acid sequence found in a host organism. "Naturally occurring" means a sequence that is found in nature and has not been synthetically prepared or modified. A sequence is "derived" from a source if it has been isolated from the source but has been modified (e.g., by deletion, substitution (mutation), insertion, or other modification), suitably so as not to disrupt the normal function of the source gene.

[0023] Suitably, the polynucleotide used in the present invention is isolated. An "isolated" polynucleotide is one that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated, for example, if it is cloned into a vector that is not part of its natural environment, or if it is contained in a cDNA.

[0024] The term "treating," as used herein with respect to chronic hepatitis B infection, refers to the administration of an appropriate composition with the intent of reducing the symptoms of CHB, preventing the progression of CHB, or reducing the level of one or more detectable markers of CHB. The term "treatment" should be interpreted accordingly. For example, preventing the progression of CHB may include preventing the onset of liver disease or stabilizing existing liver disease, as indicated by ALT (alanine transaminase) levels, liver fibrosis, or other suitable detectable markers. Other markers of CHB include serum HBV DNA levels, which are indicative of viral replication, and serum HBs antigen levels, which are indicative of viral load. Thus, treating CHB may include reducing serum HBsAg (e.g., as determined by a quantitative immunoassay) or HBV DNA (e.g., as determined by the Cobas® HBV Assay (Roche) or equivalent) levels to undetectable levels ("clearing" HBsAg or HBV DNA).

[0025] "Concurrent" administration, as used herein, refers to administration during the same ongoing immune response, and "concurrently" should be interpreted accordingly. Preferably, both components are administered simultaneously (e.g., co-administration of a vector-containing composition and a protein-containing composition), although one component can be administered within minutes (e.g., at the same medical appointment or physician's visit) or within hours of the other component. Such administration is also referred to as co-administration. Co-administration of separate components may be by the same route of administration, e.g., intramuscular injection. Alternatively, co-administration of separate components may be by different routes of administration, e.g., intramuscular and intradermal injection, intramuscular and intranasal administration, inhalation and subcutaneous administration, etc. In some embodiments, co-administration may refer to administration of an adenoviral vector and a protein component. In other embodiments, co-administration refers to administration of an adenoviral vector and another viral vector, e.g., a poxvirus, e.g., MVA. In other embodiments, co-administration refers to administration of an adenoviral vector and an adjuvanted protein component.

[0026] "Sequential" administration refers to the administration of a first composition followed by the administration of a second composition a significant time later. The period between two sequential administrations is 1 week to 12 months, e.g., 2 weeks to 12 weeks, e.g., 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks, 6 months, or 12 months. More specifically, it is 4 weeks to 8 weeks, e.g., the period between sequential administrations may be 4 weeks. Thus, sequential administration encompasses the first administration and subsequent administrations in a prime-boost setting, i.e., when the administration of the second composition is not performed during the ongoing immune response generated by the first administration.

[0027] An "immunogenic combination," as used herein, refers to multiple separately formulated immunogenic compositions that are administered sequentially and / or simultaneously in a single immunization regimen, e.g., a prime-boost regimen, where each separately formulated immunogenic composition is a component of the immunogenic combination.

[0028] With regard to percent homology, when looking at a pairwise alignment of two sequences, one can observe aligned identical residues ("identities") between the two sequences. The percent identity (or homology) can be calculated by (a) multiplying the quotient between the number of identities and the total length of the reference sequence by 100 (i.e., percent identity = (number of identities x 100) / length of reference sequence).

[0029] Compositions and Combinations In one aspect of the present invention, an immunogenic composition is provided comprising a ChAd vector selected from the group consisting of ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also referred to as C7), and Pan 9, particularly ChAd63 or ChAd155, comprising vector inserts encoding HBs and HBc. In one embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). In a specific embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding a spacer incorporating the 2A cleavage region of FMDV. In a specific embodiment, HBc is fused to hIi. In a specific embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs, e.g., an insert encoding a construct having the structure shown in Figure 22. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto) separated by a sequence encoding a spacer (e.g., SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto) incorporating the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) is fused to an hIi (e.g., SEQ ID NO: 7 or an amino acid sequence at least 98% homologous thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 12). In one specific embodiment, the vector is a ChAd155 vector. In one embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 9. In one embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 10. In one embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 10. In one embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 15. In one embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 14.

[0030] In one aspect of the present invention, an immunogenic composition is provided, comprising an MVA vector containing a vector insert encoding HBc and HBs, separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). In a specific embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding a spacer incorporating the 2A cleavage region of FMDV. In a specific embodiment, the composition comprises an MVA vector containing a polynucleotide vector insert encoding HBc, 2A, and HBs, e.g., an insert encoding a construct having the structure shown in Figure 21. In a specific embodiment, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% identical thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% identical thereto), separated by a sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus (FMDV). In one embodiment, the composition comprises an MVA vector containing a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition comprises an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO:6.

[0031] In one aspect of the present invention, an immunogenic composition is provided comprising a full-length recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant comprising MPL and QS-21. In a specific embodiment, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of the wild-type core antigen protein, e.g., amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of the wild-type hepatitis B core antigen protein. In one embodiment, the composition comprises a full-length recombinant HBs, amino acids 1-149 of HBc, and an adjuvant comprising MPL and QS-21. In a specific embodiment, the recombinant protein HBs and HBc antigen are in the form of virus-like particles. In one embodiment, the composition comprises recombinant HBc and recombinant HBs in a 1:1 ratio. In another embodiment, the ratio of HBc to HBs in the composition is greater than 1, e.g., the ratio of HBc to HBs may be 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1 or greater, particularly 3:1 to 5:1, e.g., 3:1, 4:1, or 5:1, particularly 4:1. In a specific embodiment, the composition comprises recombinant HBc and recombinant HBs at a ratio of 4:1 or greater. In a specific embodiment, the composition comprises full-length recombinant hepatitis B surface antigen (HBs) (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant comprising MPL and QS-21. In certain embodiments, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., amino acids 1-149 of HBc (e.g., SEQ ID NO: 2 or an amino acid sequence at least 98% homologous thereto). For example, a composition may comprise full-length recombinant HBs (SEQ ID NO: 1), amino acids 1-149 of HBc (SEQ ID NO: 2), and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in phosphate buffered saline.

[0032] In a further aspect, a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant. An immunogenic combination comprising:

[0033] In one embodiment, the composition of element a) of the immunogenic combination comprises a ChAd vector selected from the group consisting of ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also referred to as C7), and Pan 9, particularly ChAd63 or ChAd155, comprising vector inserts encoding HBc and HBs. In one embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). In a specific embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding a spacer incorporating the 2A cleavage region of FMDV. In a specific embodiment, HBc is fused to hIi. In a specific embodiment, the composition of element a) of the immunogenic combination comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs, e.g., an insert encoding a construct having the structure shown in Figure 22. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto) separated by a sequence encoding a spacer (e.g., SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto) incorporating the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) is fused to an hIi (e.g., SEQ ID NO: 7 or an amino acid sequence at least 98% homologous thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 12). In one specific embodiment, the vector is a ChAd155 vector. In one embodiment, the composition of element a) of the immunogenic combination comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:9.In one embodiment, the composition of element a) of the immunogenic combination comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 10. In one embodiment, the composition of element a) of the immunogenic combination comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 15. In one embodiment, the composition of element a) of the immunogenic combination comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 14.

[0034] In one embodiment, the composition of element b) of the immunogenic combination comprises an MVA vector comprising vector inserts encoding HBc and HBs, separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). In a specific embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding a spacer incorporating the 2A cleavage region of FMDV. In a specific embodiment, the composition of element b) of the immunogenic combination comprises an MVA vector comprising polynucleotide vector inserts encoding HBc, 2A, and HBs, e.g., an insert encoding a construct having the structure shown in Figure 21. In a specific embodiment, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto), separated by a sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus (FMDV). In one embodiment, the composition of element b) of the immunogenic combination comprises an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition of element b) of the immunogenic combination comprises an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 6.

[0035] In one embodiment, the composition of element c) of the immunogenic combination comprises full-length recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant comprising MPL and QS-21. In a specific embodiment, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., comprising 145-149 amino acids of the assembly domain of wild-type core antigen protein, e.g., amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of wild-type hepatitis B core antigen protein. In one embodiment, the composition of element c) of the immunogenic combination comprises full-length recombinant HBs, amino acids 1-149 of HBc, and an adjuvant comprising MPL and QS-21. In a specific embodiment, the recombinant protein HBs and HBc antigen are in the form of virus-like particles. In one embodiment, the composition comprises recombinant HBc and recombinant HBs in a 1:1 ratio. In another embodiment, the ratio of HBc to HBs in the composition is greater than 1, e.g., the ratio of HBc to HBs may be 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1 or greater, particularly 3:1 to 5:1, e.g., 3:1, 4:1, or 5:1, particularly 4:1. In a specific embodiment, the composition of element c) of the immunogenic combination comprises recombinant HBc and recombinant HBs in a ratio of 4:1 or greater. In a specific embodiment, the composition comprises full-length recombinant hepatitis B surface antigen (HBs) (e.g., SEQ ID NO: 1), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant comprising MPL and QS-21. In a specific embodiment, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., amino acids 1-149 of HBc (e.g., SEQ ID NO: 2). For example, the composition of element c) of the immunogenic combination includes full-length recombinant HBs (SEQ ID NO: 1), amino acids 1 to 149 of HBc (SEQ ID NO: 2), and an adjuvant including MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in, for example, phosphate-buffered saline.

[0036] Thus, in one particular embodiment, the present invention provides a method for treating a pulmonary arthritis, comprising: a) a composition comprising a ChAd155 vector containing a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs; b) a composition comprising an MVA vector containing a polynucleotide vector insert encoding HBc, 2A, and HBs; and c) Compositions comprising recombinant HBs protein, recombinant truncated HBc protein, and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. The present invention provides an immunogenic combination comprising:

[0037] In one such embodiment, the immunogenic combination comprises: a) a composition comprising a ChAd155 vector encoding the amino acid sequence of SEQ ID NO:9 or comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:15; b) a composition comprising an MVA vector containing a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:5; and c) A composition comprising a recombinant HBs protein of SEQ ID NO: 1, a recombinant truncated HBc protein of SEQ ID NO: 2, and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. Includes.

[0038] In another embodiment, the immunogenic combination is: a) a composition comprising a ChAd155 vector containing a polynucleotide vector insert of SEQ ID NO: 10 or a polynucleotide vector insert of SEQ ID NO: 14; b) a composition comprising an MVA vector containing a polynucleotide vector insert of SEQ ID NO: 6; and c) A composition comprising a recombinant HBs protein of SEQ ID NO: 1, a recombinant truncated HBc protein of SEQ ID NO: 2, and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. Includes.

[0039] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant. together with instructions for administering the components sequentially or simultaneously for the treatment of CHB.

[0040] In one embodiment, the composition of component a) of the kit comprises a ChAd vector selected from the group consisting of ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also referred to as C7), and Pan 9, particularly ChAd63 or ChAd155, comprising vector inserts encoding HBc and HBs. In one embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). In a specific embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding a spacer incorporating the 2A cleavage region of FMDV. In a specific embodiment, HBc is fused to hIi. In a specific embodiment, the composition of component a) of the kit comprises a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs, e.g., a ChAd155 vector comprising an insert encoding a construct having the structure shown in FIG. 22. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto) separated by a sequence encoding a spacer (e.g., SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto) incorporating the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) is fused to an hIi (e.g., SEQ ID NO: 7 or an amino acid sequence at least 98% homologous thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 12). In one specific embodiment, the vector is a ChAd155 vector. In one embodiment, the composition of component a) of the kit comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:9.In one embodiment, the composition of component a) of the kit comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 10. In one embodiment, the composition of component a) of the kit comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 15. In one embodiment, the composition of component a) of the kit comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO: 14.

[0041] In one embodiment, the composition of component b) of the kit comprises an MVA vector comprising vector inserts encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). In a specific embodiment, the polynucleotides encoding HBs and HBc are separated by a sequence encoding a spacer incorporating the 2A cleavage region of FMDV. In a specific embodiment, the composition of component b) of the kit comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A, and HBs, e.g., an insert encoding a construct having the structure shown in FIG. 21. In a specific embodiment, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto), separated by a sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus (FMDV). In one embodiment, the composition of component b) of the kit comprises an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition of component b) of the kit comprises an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence set forth in SEQ ID NO:6.

[0042] In one embodiment, the composition of component c) of the kit comprises full-length recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant comprising MPL and QS-21. In a specific embodiment, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of the wild-type hepatitis B core antigen protein. In one embodiment, the composition of component c) of the kit comprises full-length recombinant HBs, amino acids 1-149 of HBc, and an adjuvant comprising MPL and QS-21. In a specific embodiment, the recombinant protein HBs and HBc antigen are in the form of virus-like particles. In one embodiment, the composition comprises recombinant HBc and recombinant HBs in a 1:1 ratio. In another embodiment, the ratio of HBc to HBs in the composition is greater than 1, e.g., the ratio of HBc to HBs may be 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1 or greater, particularly 3:1, 4:1, or 5:1, and particularly 4:1. In a specific embodiment, the composition of component c) of the kit comprises recombinant HBc and recombinant HBs in a ratio of 4:1 or greater. In a specific embodiment, the composition comprises full-length recombinant hepatitis B surface antigen (HBs) (e.g., SEQ ID NO: 1), C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant comprising MPL and QS-21. In a specific embodiment, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., amino acids 1-149 of HBc (e.g., SEQ ID NO: 2). For example, the composition of component c) of the kit includes full-length recombinant HBs (SEQ ID NO: 1), amino acids 1 to 149 of HBc (SEQ ID NO: 2), and an adjuvant including MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in, for example, phosphate-buffered saline.

[0043] In one embodiment, the kit comprises the following components: a) a composition comprising a ChAd155 vector containing a polynucleotide vector insert of SEQ ID NO: 10 or a polynucleotide vector insert of SEQ ID NO: 14; b) a composition comprising an MVA vector containing a polynucleotide vector insert of SEQ ID NO: 6; and c) A composition comprising a recombinant HBs protein of SEQ ID NO: 1, a recombinant truncated HBc protein of SEQ ID NO: 2, and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. together with instructions for administering the components sequentially or simultaneously for the treatment of CHB.

[0044] Regimen The present disclosure provides a powerful CD8 + A vaccine regimen providing a heterologous prime-boost schedule using two viral vectors encoding hepatitis B core (HBc) and hepatitis B surface (HBs) antigens to induce T cell responses, which results in a potent antigen-specific CD4 + Described are vaccine regimens involving the sequential or simultaneous administration of adjuvanted recombinant HBc and HBs proteins to induce T cell and antibody responses. The present disclosure further describes immunogenic compositions and combinations that may find use as vaccine compositions. Such compositions and combinations may be used in the disclosed regimens. The disclosed vaccine compositions and regimens have been shown to induce HBs- and HBc-specific antibodies and CD8 T cells in a mouse model that recapitulates the virological and immunological characteristics of human chronic HBV infection, without associated signs of liver alteration side effects. + T cell responses and HBs-specific CD4 + T cell responses were successfully restored.

[0045] The immunogenic compositions and immunogenic combinations described herein are useful in a method of treating chronic hepatitis B infection (CHB) in a human, comprising: a) administering to a human a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) administering to the human a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs) and a nucleic acid encoding a hepatitis B virus core antigen (HBc); and c) administering to the human a composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant. The present invention may find use in methods including:

[0046] In one embodiment, the steps of the method are performed sequentially, with step a) preceding step b), which in turn precedes step c). Optionally, step c) may be repeated. In certain embodiments, the period between steps of the method is 2 to 12 weeks, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In one embodiment, the period between steps of the method is 4 to 8 weeks. In one embodiment, the period between sequential administrations of compositions according to the method is 4 weeks. In another embodiment, step c) is performed simultaneously with step a) and / or step b). In certain embodiments, simultaneous steps b) and c) may be repeated. In a further embodiment, step c is repeated, and the steps of the method are performed in the following order: step a), step b), step c), step c). In certain embodiments, the period between steps of the method is 2 to 12 weeks, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In one embodiment, the period between steps of the method is 4 to 8 weeks. In one embodiment, the period between sequential administrations of compositions according to the method is 4 weeks.

[0047] In another aspect, the immunogenic compositions and immunogenic combinations are used in a method of treating chronic hepatitis B infection (CHB) in a human, comprising: a) administering to a human i) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B core antigen (HBc), and simultaneously ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B core antigen (HBc), and an adjuvant; and b) administering to the human: i) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B core antigen (HBc), and simultaneously a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B core antigen (HBc), and an adjuvant. The present invention may find use in methods including:

[0048] In one embodiment, the method steps are performed sequentially, with step a) preceding step b). Optionally, step b) may be repeated. Optionally, step a) may be repeated. In one embodiment, the method steps are performed in the order of step a), followed by step a), followed by step b). Alternatively, the method steps may be performed in the order of step a), followed by step b), followed by step a). Optionally, step b) may be repeated. In one embodiment, the method steps are performed in the order of step a), followed by step b), followed by step b). In an alternative embodiment, the method steps are performed in the order of step b), followed by step a), followed by step b). Optionally, step b) may be repeated more than once. Optionally, both step a) and step b) may be repeated. In one embodiment, the method steps are performed in the order of step a), followed by step b), followed by step b), followed by step b). In further embodiments, the steps of the method are performed in the following order: step a), followed by step a), followed by step b), followed by step b), optionally followed by step b). In certain embodiments, the period between steps of the method is 2 to 12 weeks, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In one embodiment, the period between steps of the method is 4 to 8 weeks. In one embodiment, the period between sequential administrations of compositions according to the method is 4 weeks.

[0049] antigen At least nine genotypes of HBV (A to I) have been identified, with genomes differing by more than 8%. Within a given HBV genotype, multiple genosubtypes have been identified, differing by 4-8%. Antigens for use in the disclosed methods are appropriately selected to provide immunological coverage across multiple, preferably all, HBV genotypes. The hepatitis B core protein antigen (HBc) is highly conserved across genotypes and genosubtypes, and the hepatitis B surface protein antigen (HBs) sequence is appropriately selected to contain important cross-genotype-preserved B-cell epitopes that enable the induction of a broad neutralizing response. Suitably, HBc and HBs sequences for use in the disclosed methods and compositions are based on those from genotype / subtype A2.

[0050] Suitably, HBs antigen for use in the disclosed methods and compositions is derived from the small, medium, or large surface antigen protein. In particular, a suitable HBs antigen includes the small (S) protein of HBV adw2 strain, genotype A. For example, a suitable HBs antigen has the 226 amino acid sequence of SEQ ID NO: 1. When used as a recombinant protein, the HBs antigen is preferably associated with virus-like particles. This antigen is included in well-studied, commercially available hepatitis B vaccines (Engerix B, Fendrix, Twinrix, and others) and has been demonstrated to protect against hepatitis B across genotypes. Preferably, recombinant HBs protein antigen is expressed from yeast and purified for use in the vaccine compositions and methods of the invention. Suitable methods for expression and purification are known, for example, from EP 1307473 B1.

[0051] Hepatitis B core protein (HBc) is a major component of the nucleocapsid shell that packages the viral genome. This protein (183-185 amino acids long) is expressed in the cytoplasm of infected cells and remains unglycosylated. HBc contains a 149-residue assembly domain and a 34-36-residue RNA-binding domain (at the C-terminus). HBc antigens for use in the disclosed methods and compositions can be full-length or C-terminally truncated proteins (lacking the RNA-binding C-terminus), e.g., containing 145-149 amino acids of the assembly domain of wild-type core antigen protein, e.g., amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of wild-type hepatitis B core antigen protein. Truncated proteins retain the ability to assemble into nucleocapsid particles. HBc antigens suitable for use in the disclosed methods and compositions have an amino acid sequence derived from HBV adw2 strain, genotype A. When used as a recombinant protein, the HBc antigen is suitably truncated from the wild-type at the C-terminus; in particular, the antigen may be amino acids 1-149 of HBc, e.g., having the amino acid sequence of SEQ ID NO: 2. Preferably, the recombinant HBc protein antigen is expressed from E. coli and purified for use in the vaccine compositions and methods of the invention. Methods for recombinant expression of viral proteins in E. coli are well known in the art.

[0052] When used as a recombinant protein, the HBc antigen is preferably associated with a virus-like particle. When expressed from a viral vector, the HBc antigen may be full-length or truncated, for example, suitably full-length HBc antigen (e.g., SEQ ID NO: 11). Suitable doses of recombinant HBs protein antigen for use in the methods disclosed herein are from 10 μg per administration to 100 μg per administration, e.g., 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg per administration. Suitable doses of recombinant HBc antigen protein for use in the methods disclosed herein are from 10 μg per dose to 100 μg per dose, e.g., 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, or 100 μg per dose.

[0053] Antigens are substances that induce immune responses, particularly antibody production, in the body. Antigens can be foreign, i.e., of pathogenic origin, or derived from the organism itself; the latter are called self antigens or auto antigens. Antigens can be presented on the surface of antigen-presenting cells by MHC molecules. There are two classes of MHC molecules: MHC class I (MHC-I) and MHC class II (MHC-II). MHC-II molecules are membrane-bound receptors that are synthesized in the endoplasmic reticulum and leave the endoplasmic reticulum in the MHC class II compartment. To prevent endogenous peptides, i.e., self antigens, from binding to and presenting the MHC-II molecules to generate an immune response, nascent MHC-II molecules bind to another protein, the invariant chain, which blocks the peptide-binding groove of the MHC-II molecules. The human invariant chain (hIi, also known as CD74 when expressed on the plasma membrane) is an evolutionarily conserved type II membrane protein with several roles within cells and throughout the immune system [Borghese, 2011]. When MHC class II compartments fuse with late endosomes containing phagocytosed and degraded foreign proteins, the invariant chain is cleaved, leaving only the CLIP domain bound to MHC-II molecules. In a second step, CLIP is removed by HLA-DM molecules, freeing MHC-II molecules to bind fragments of the foreign protein. Once the MHC class II compartment fuses with the plasma membrane, the fragments are presented on the surface of antigen-presenting cells, thus presenting foreign antigens to other cells, primarily T helper cells.

[0054] It is known that when an adenoviral expression system encoding a fusion of an invariant chain and an antigen is used for vaccination, the immune response to the antigen is increased (see WO2007 / 062656 (also published as US2011 / 0293704, incorporated by reference for the purpose of disclosing the invariant chain sequence)), i.e., the invariant chain enhances the immunogenicity of the antigen, and invariant chains such as hIi are sometimes referred to as "genetic adjuvants" in recognition of this effect. Furthermore, the adenoviral constructs have been found to be useful for priming immune responses in the context of prime-boost vaccination regimens (see WO2014 / 141176 (also published as US2016 / 0000904); and WO2010 / 057501 (also published as US2010 / 0278904, which is incorporated by reference for the purposes of disclosing invariant chain sequences and adenoviral vectors encoding invariant chain sequences)). In particular, the hIi sequence and hIi are intended to be useful in priming immune responses in the context of CD8 + It has the potential to increase T cell responses [Spencer, 2014; Capone, 2014]. In certain embodiments, nucleotide sequences included within vectors for use in the methods, uses, and compositions disclosed herein may include a nucleotide sequence encoding an hIi. The amino acid sequence for an hIi that may be included in the disclosed adenoviral vector ChAd155-hIi-HBV is set forth in SEQ ID NO:7, and an alternative sequence is set forth in SEQ ID NO:12. Nucleotide sequences encoding these amino acid sequences are set forth in SEQ ID NO:8 and SEQ ID NO:13. Suitably, the nucleotide sequence encoding the hIi is fused to a nucleotide sequence encoding an HBc antigen to generate a fusion protein in which the hIi polypeptide is fused N-terminally to the HBc antigen.

[0055] vector In addition to a polynucleotide encoding an antigen protein (also referred to herein as an "insert"), vectors for use in the methods and compositions disclosed herein may also contain conventional control elements operably linked to the encoding polynucleotide to permit transcription, translation, and / or expression in cells transfected with the vector. Thus, the vector insert polynucleotide encoding the protein antigen is incorporated into an expression cassette with appropriate control elements.

[0056] Expression control elements include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals, e.g., rabbit β-globin polyA; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product.

[0057] A promoter is a nucleotide sequence that allows RNA polymerase to bind and direct the transcription of a gene. Typically, promoters are located in the 5' non-coding region of a gene, proximal to the transcription start site of the gene. Sequence elements within a promoter that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters derived from bacteria, yeast, plants, viruses, and mammals (including humans). Numerous expression control sequences, e.g., endogenous, native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be utilized.

[0058] Examples of constitutive promoters include the TBG promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer; see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the CASI promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter (Invitrogen). Suitably, the promoter is a CMV promoter or a variant thereof, more suitably a human CMV (HCMV) promoter or a variant thereof.

[0059] Adenovirus vectors Adenoviruses are widely used for gene transfer applications due to their ability to achieve highly efficient gene transfer in various target tissues and their large transgene capacity. Typically, the E1 gene of adenovirus is deleted and replaced with a transgene cassette consisting of a selected promoter, a cDNA sequence of the gene of interest, and a poly(A) signal, resulting in a replication-deficient recombinant virus. Human adenovirus vectors have been shown to induce CD8 expression of transgenes in animal models and humans. +Adenoviruses have been shown to be potent vectors for inducing T cell responses. Adenoviruses have a broad tropism and are capable of infecting replicating and non-replicating cells. The main limitation of the clinical application of vectors based on human adenoviruses is the high prevalence of neutralizing antibodies in the general population. Adenoviruses isolated from alternative species are considered potential vaccine vectors to circumvent the problem of pre-existing anti-adenovirus immunity in humans. Among them, simian adenoviruses derived from chimpanzees, gorillas, or bonobos may be suitable for use in delivering antigens and eliciting targeted T cell and / or humoral responses against those antigens in humans. Simian adenoviruses, such as those derived from chimpanzees, are being tested in clinical studies. Chimpanzee adenoviral vectors have low / no seroprevalence in the human population, are not known to cause pathological disease in humans, and some ChAd vectors can be grown to high titers in cell lines previously used to generate clinical-grade material, e.g., human embryonic kidney 293 (HEK293).

[0060] Replication-incompetent or replication-deficient adenoviruses are adenoviruses that are unable to replicate because they have been engineered to contain at least a functional deletion (or "loss-of-function" mutation), i.e., a deletion or mutation that impairs the function of a gene without completely removing the gene, such as the introduction of an artificial stop codon, a deletion or mutation of an active site or interaction domain, a mutation or deletion of a regulatory sequence of a gene, or the complete removal of a gene encoding a gene product that is essential for viral replication (e.g., one or more of the adenoviral genes selected from E1A, E1B, E2A, E2B, E3, and E4 (e.g., E3 ORF1, E3 ORF2, E3 ORF3, E3 ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3 ORF9, E4 ORF7, E4 ORF6, E4 ORF5, E4 ORF4, E4 ORF3, E4 ORF2, and / or E4 ORF1)). Suitably, the E1 and E3 genes are deleted, more suitably, the E1, E3 and E4 genes are deleted.

[0061] Suitable vectors for use in the methods and compositions disclosed herein are replication-deficient chimpanzee adenovirus vectors, such as ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also referred to as C7), or Pan 9. Examples of such strains are described in WO03 / 000283, WO2005 / 071093, WO2010 / 086189, and WO2016 / 198621. The ChAd155 vector (see WO2016 / 198621, which is incorporated by reference for purposes of disclosing the ChAd155 vector sequence and methods) belongs to the same phylogenetic adenovirus group (group C) as the ChAd3 vector. In one embodiment, a vector for use in the methods and compositions disclosed herein is a ChAd vector of phylogenetic group C, such as ChAd3 or ChAd155. In one specific embodiment, the method of treating chronic hepatitis B disclosed herein comprises administering to a human a composition comprising a ChAd155 vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc). A suitable dose of ChAd vector for use in the methods disclosed herein is 1 x 10 per administration. 8 ~1×10 11 Viral particles (vp), e.g., about 1 x 10 per dose 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 or 1 x 10 11 virus particles (vp).

[0062] More specifically, in one embodiment, a vector for use in the methods and compositions disclosed herein is a replication-deficient chimpanzee adenoviral vector, ChAd155, encoding a fusion of sequences derived from two HBV proteins: HBc (core, nucleocapsid protein) and HBs (minor surface antigen). In a specific embodiment, the vector is ChAd155, encoding HBc and HBs separated by a spacer, SEQ ID NO:3, which incorporates sequences encoding the 2A cleavage region of foot-and-mouth disease virus for processing HBc and HBs into separate proteins (resulting in a 23-amino acid tail at the C-terminus of the upstream protein and a single proline at the N-terminus of the downstream protein). Cleavage of core from the surface antigen allows proper folding of HBs and the generation of an antibody response against the surface antigen. Alternatively, the adenoviral vector may be a dual-promoter (bicistronic) vector, allowing independent expression of HBs and HBc antigens.

[0063] In certain embodiments, the N-terminal portion of the gene encoding the HBc protein may be fused to a gene encoding the human major histocompatibility complex (MHC) class II-binding invariant chain p35 isoform (i.e., hIi or CD74). Accordingly, certain ChAd155 vectors for use in the methods and compositions disclosed herein contain a polynucleotide vector insert encoding a construct having the structure shown in FIG. 22, which includes hIi, HBc, 2A, and HBs. The amino acid sequence of such a construct is set forth in SEQ ID NO:9, and the nucleotide sequence encoding the amino acid sequence of the construct is set forth in SEQ ID NO:10. The amino acid sequence of an alternative such construct is set forth in SEQ ID NO:15, and the nucleotide sequence encoding the amino acid sequence of the construct is set forth in SEQ ID NO:14.

[0064] Modified vaccinia virus Ankara (MVA) vector Modified vaccinia virus Ankara (MVA) is replication-deficient in humans and other mammals and is derived from vaccinia virus. It belongs to the poxvirus family and was initially developed to improve the safety of smallpox vaccination by passage of vaccinia virus in chicken embryo fibroblasts (CEF) over 570 times, resulting in multiple deletions, after which the virus became highly attenuated and replication-deficient in humans and other mammals. The replication defect occurs late in virion assembly so that viral and recombinant gene expression are intact, making MVA an efficient single-round expression vector unable to cause infection in mammals. MVA has since been widely used as a viral vector to induce antigen-specific immunity against transgenes in both animal models and humans. A description of MVA can be found in Mayr A, et al. (1978) and Mayr, A. et al. (1975).

[0065] In one embodiment, the MVA is derived from virus seed batch 460 MG obtained from the 571st passage of vaccinia virus on CEF cells. In another embodiment, the MVA is derived from virus seed batch MVA 476 MG / 14 / 78. In a further embodiment, the MVA was derived or produced before December 31, 1978 and is free of prion contamination. A suitable dose of MVA vector for use in the methods disclosed herein is 1 x 10 per administration. 6 ~1×10 9 Plaque-forming units (pfu), e.g., about 1 x 10 per dose 6 , 2 × 10 6 , 5×10 6 , 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 or 1 x 10 9 It is pfu.

[0066] In one specific embodiment, a vector for use in the methods and compositions disclosed herein is an MVA encoding a fusion of sequences derived from two HBV proteins: HBc (core nucleocapsid protein) and HBs (minor surface antigen). In a particular embodiment, a vector for use in the methods and compositions disclosed herein is an MVA encoding HBc and HBs separated by a nucleotide sequence encoding SEQ ID NO:3, a spacer incorporating sequences encoding the 2A cleavage region of foot-and-mouth disease virus for processing HBc and HBs into separate proteins (resulting in a 23-amino acid tail at the C-terminus of the upstream protein and a single proline at the N-terminus of the downstream protein). Accordingly, a particular MVA vector for use in the methods and compositions disclosed herein comprises a polynucleotide vector insert encoding a construct having the structure shown in FIG. 21, comprising HBc, 2A, and HBs. The amino acid sequence of such a construct is set forth in SEQ ID NO:5, and the nucleotide sequence encoding the amino acid insert construct is set forth in SEQ ID NO:6.

[0067] Pharmaceutical Composition The immunogenic compositions disclosed herein find use in the disclosed methods and are suitably pharmaceutically acceptable compositions. Suitably, pharmaceutical compositions include a pharmaceutically acceptable carrier.

[0068] Immunogenic compositions containing ChAd or MVA vectors may be prepared for administration by a suspension of viral vector particles in a pharmaceutically or physiologically acceptable carrier, such as isotonic saline or other isotonic salt solution. Suitable carriers will be apparent to those skilled in the art and will depend largely on the route of administration.

[0069] Compositions containing recombinant protein antigens may be prepared by isolating and purifying the proteins from cell cultures in which they are expressed, suspending them in a formulation buffer containing one or more salts, surfactants, and / or cryoprotectants, and then lyophilizing them. For example, a suitable formulation buffer may contain a sugar or a mixture of sugars, such as sucrose, trehalose, or sucralose, as a cryoprotectant and a non-ionic copolymer, such as poloxamer, as a surfactant. For administration, the lyophilized recombinant protein formulation is reconstituted in a pharmaceutically or physiologically acceptable carrier, such as isotonic saline or other isotonic salt solutions for injection or inhalation. Suitable carriers will be apparent to those skilled in the art and will largely depend on the route of administration. The reconstituted composition may also contain an adjuvant or mixture of adjuvants. In one embodiment, the lyophilized recombinant protein is reconstituted in a liquid adjuvant-based formulation.

[0070] The term "carrier," as used herein, refers to a pharmacologically inert substance, such as, but not limited to, a diluent, excipient, or vehicle with which a therapeutically active ingredient is administered. Liquid carriers include, but are not limited to, sterile liquids such as saline solutions in water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0071] Compositions for use in the methods disclosed herein may include an adjuvant system in addition to the vector or recombinant protein of the composition. The term "adjuvant" refers to an agent that augments, stimulates, activates, strengthens, or modulates the immune response to the antigen of the composition, either at the cellular or humoral level; for example, an immunological adjuvant stimulates the immune system's response to an antigen but does not have an immunological effect by itself. The immunogenic compositions disclosed herein may include an adjuvant as a separate component in the formulation, regardless of whether the vector included in the composition also encodes a "genetic adjuvant," such as a hIi.

[0072] Appropriate adjuvants are those that can enhance immune responses in subjects with chronic conditions and disrupted immune capabilities. CHB patients are characterized by an inability to mount efficient innate and adaptive immune responses against viruses, making the development of an effective vaccine challenging. In these patients, one important function of adjuvanted vaccine formulations should be to target the cellular immune response toward a T helper 1 (Th1) profile, which is recognized as important for the elimination of intracellular pathogens.

[0073] Examples of suitable adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts, such as aluminum phosphate or aluminum hydroxide), organic non-peptide adjuvants (e.g., saponins, such as QS21, or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immune stimulating complex (IFC) ( Examples of adjuvants include ISCOMS, liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A (MPL), e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL), or muramyl peptides), synthetic adjuvants (e.g., non-ionic block copolymers, muramyl peptide analogs, or synthetic lipid A), synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine), and immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides ("CpG"). In particular, the adjuvant may be an organic non-peptide adjuvant (e.g., a saponin, e.g., QS21, or squalene) and / or a bacterial adjuvant (e.g., monophosphoryl lipid A (MPL), e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL)).

[0074] One suitable adjuvant is monophosphoryl lipid A (MPL), particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Chemically, it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with 4, 5, or 6 acylated chains. It can be purified and prepared by the methods taught in GB 2122204B, which also discloses the preparation of diphosphoryl lipid A and its 3-O-deacylated variant. Other purified synthetic lipopolysaccharides have been described [U.S. Patent No. 6,005,099 and EP 0 729 473 B1; Hilgers, 1986; Hilgers, 1987; and EP 0 549 074 B1].

[0075] Saponins are also suitable adjuvants (Lacaille-Dubois, 1996). For example, the saponin Quil A (derived from the bark of the South American tree Quillaja saponaria Molina) and its fractions are described in U.S. Patent No. 5,057,540 and Kensil, 1996; and EP 0 362 279 B1. Purified fractions of Quil A, such as QS21 and QS17, are also known as immunostimulants; methods for their production are disclosed in U.S. Patent No. 5,057,540 and EP 0 362 279 B1. The use of QS21 is further described in Kensil, 1991. Combinations of QS21 with polysorbates or cyclodextrins are also known (WO 99 / 10008). Particulate adjuvant systems comprising fractions of Quil A, such as QS21 and QS7, are described in WO 96 / 33739 and WO 96 / 11711.

[0076] Adjuvants such as those described above may be formulated with carriers such as liposomes, oil-in-water emulsions, and / or metal salts (including aluminium salts such as aluminium hydroxide). For example, 3D-MPL may be formulated with aluminium hydroxide (EP 0 689 454) or an oil-in-water emulsion (WO 95 / 17210); QS21 may be formulated with cholesterol-containing liposomes (WO 96 / 33739), an oil-in-water emulsion (WO 95 / 17210) or alum (WO 98 / 15287).

[0077] Combinations of adjuvants may also be utilized in the disclosed compositions, particularly combinations of monophosphoryl lipid A and saponin derivatives (see, e.g., WO 94 / 00153; WO 95 / 17210; WO 96 / 33739; WO 98 / 56414; WO 99 / 12565; WO 99 / 11241), more particularly the combination of QS21 and 3D-MPL as disclosed in WO 94 / 00153, or a composition in which QS21 is quenched in cholesterol-containing liposomes (DQ), as disclosed in WO 96 / 33739. A potent adjuvant formulation comprising QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion is described in WO 95 / 17210 and is another formulation that may find use in the disclosed compositions. Thus, suitable adjuvant systems include, for example, monophosphoryl lipid A, preferably 3D-MPL, in combination with an aluminum salt (e.g., as described in WO 00 / 23105). Further exemplary adjuvants include QS21 and / or MPL and / or CpG. As disclosed in WO 96 / 33739, QS21 may be quenched in cholesterol-containing liposomes.

[0078] Thus, a suitable adjuvant for use in the disclosed immunogenic compositions is AS01, a liposome-based adjuvant containing MPL and QS-21. Liposomes are vehicles for the MPL and QS-21 immune enhancers and are composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in phosphate-buffered saline. B-4 is a particularly preferred variant of the AS01 adjuvant, consisting of the immune enhancer QS-21 (a triterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A) in a PBS solution, along with DOPC / cholesterol liposomes and sorbitol as a vehicle for the immune enhancer. B-4 A single human dose (0.5 mL) of AS01 contains 50 μg of QS-21 and 50 μg of MPL.E-4 is AS01 B-4 This corresponds to a two-fold dilution of QS-21 and MPL, i.e., 25 μg of QS-21 and 25 μg of MPL per human dose.

[0079] In one embodiment, an immunogenic composition is provided comprising a recombinant hepatitis B surface antigen (HBs), a truncated recombinant hepatitis B core antigen (HBc), and an adjuvant comprising MPL and QS-21. In one embodiment, the immunogenic composition comprises recombinant HBs, truncated recombinant HBc, and an AS01 adjuvant. In a specific embodiment, the immunogenic combination comprises a ratio of truncated recombinant HBc and recombinant HBs of 4:1 or greater, and an AS01 adjuvant, e.g., AS01 B-4 or AS01 E-4 The composition includes:

[0080] In one embodiment, the following: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. An immunogenic combination comprising:

[0081] In another embodiment, the following: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a truncated recombinant HBc and full-length recombinant HBs in a ratio of 4:1 or greater, and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. An immunogenic combination comprising:

[0082] In another embodiment, the following: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising truncated recombinant HBc and full-length recombinant HBs in a ratio of 4:1 or greater, and an AS01 adjuvant. An immunogenic combination comprising:

[0083] In another embodiment, the following: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) a 4:1 ratio of truncated recombinant HBc consisting of amino acids 1 to 149 of HBc (e.g., SEQ ID NO: 2) and full-length recombinant HBs (e.g., SEQ ID NO: 1), and AS01 B-4 A composition comprising An immunogenic combination comprising:

[0084] In one embodiment, the following: a) a composition comprising a replication-deficient ChAd155 vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. An immunogenic combination comprising:

[0085] In another embodiment, the following: a) a composition comprising a replication-deficient ChAd155 vector comprising a polynucleotide encoding HBs and a polynucleotide encoding HBc, separated by a polynucleotide encoding a linker incorporating the 2A cleavage region of foot-and-mouth disease virus; b) a composition comprising an MVA vector comprising a polynucleotide encoding HBs and a polynucleotide encoding HBc, separated by a polynucleotide encoding a linker incorporating the 2A cleavage region of foot-and-mouth disease virus; and c) A composition comprising a truncated recombinant HBc and full-length recombinant HBs in a ratio of 4:1 or greater, and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol. An immunogenic combination comprising:

[0086] In another embodiment, the following: a) a composition comprising a replication-deficient ChAd155 vector comprising a polynucleotide encoding HBs and a polynucleotide encoding the human invariant chain (hIi) fused to the N-terminus of HBc; b) a composition comprising an MVA vector comprising a polynucleotide encoding HBs and a polynucleotide encoding HBc, separated by a polynucleotide encoding a linker incorporating the 2A cleavage region of foot-and-mouth disease virus; and c) A composition comprising truncated recombinant HBc and full-length recombinant HBs in a ratio of 4:1 or greater, and an AS01 adjuvant. An immunogenic combination comprising:

[0087] In a specific embodiment, the vector insert of the ChAd vector of component a) of the immunogenic combination encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto), separated by a sequence encoding a spacer (e.g., SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto) incorporating the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, HBc (e.g., SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto) is fused to an hIi (e.g., SEQ ID NO: 7 or an amino acid sequence at least 98% homologous thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to an hIi (e.g., SEQ ID NO: 12).

[0088] In a specific embodiment, the vector insert of the MVA vector of component b) of the immunogenic combination encodes HBc (e.g., SEQ ID NO:11 or an amino acid sequence at least 98% homologous thereto) and HBs (e.g., SEQ ID NO:1 or an amino acid sequence at least 98% homologous thereto), separated by a sequence encoding a spacer (e.g., SEQ ID NO:3 or an amino acid sequence at least 98% homologous thereto) incorporating the 2A cleavage region of foot-and-mouth disease virus. For example, the vector insert encodes HBc (e.g., SEQ ID NO:11) and HBs (e.g., SEQ ID NO:1), separated by a sequence encoding a spacer (e.g., SEQ ID NO:3) incorporating the 2A cleavage region of foot-and-mouth disease virus.

[0089] In another embodiment, the following: a) a composition comprising a replication-deficient ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:9 or a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:15; b) a composition comprising an MVA vector containing a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO:5; and c) a 4:1 ratio of truncated recombinant HBc consisting of amino acids 1 to 149 of HBc (e.g., SEQ ID NO: 2) and full-length recombinant HBs (e.g., SEQ ID NO: 1), and AS01 B-4 A composition comprising An immunogenic combination comprising:

[0090] In another embodiment, the following: a) a composition comprising a replication-deficient ChAd155 vector comprising a polynucleotide vector insert comprising SEQ ID NO: 10, or a polynucleotide vector insert comprising SEQ ID NO: 14; b) a composition comprising an MVA vector comprising a polynucleotide vector insert comprising SEQ ID NO:6; and c) a 4:1 ratio of truncated recombinant HBc consisting of amino acids 1 to 149 of HBc (e.g., SEQ ID NO: 2) and full-length recombinant HBs (e.g., SEQ ID NO: 1), and AS01 B-4 A composition comprising An immunogenic combination comprising:

[0091] Administration In certain embodiments, the disclosed immunogenic compositions and immunogenic combinations are administered via intranasal, intramuscular, subcutaneous, intradermal, or topical routes. Preferably, administration is via the intramuscular route.

[0092] Intranasal administration refers to the administration of an immunogenic composition to the mucosa of the entire respiratory tract, including the lungs. More specifically, the composition is administered to the nasal mucosa. In one embodiment, intranasal administration is achieved by spray or aerosol. Intramuscular administration refers to the injection of the composition into any muscle of an individual. Exemplary intramuscular injections are into the deltoid, vastus lateralis, or ventral gluteal and dorsoventral gluteal regions. Preferably, administration is into the deltoid muscle. Subcutaneous administration refers to the injection of the composition into subcutaneous tissue. Intradermal administration refers to the injection of the composition into the dermis between the layers of skin. Topical administration refers to the administration of the composition to any part of the skin or mucosa without penetrating the skin with a needle or equivalent device. The composition may also be administered topically to the mucosa of the mouth, nose, genital area, and / or rectum. Topical administration includes administration means such as sublingual and / or buccal administration. Sublingual administration refers to the administration of the composition under the tongue (e.g., using an oral thin film (OTF)). Buccal administration is the administration of the vector through the buccal mucosa of the cheek.

[0093] The immunogenic compositions and combinations disclosed herein may find use in prime-boost immunization regimens. Accordingly, compositions for use in methods for treating CHB, which are prime-boost immunization methods, are disclosed herein. Often, a single administration of an immunogenic composition is not sufficient to generate the number of long-term immune cells required for effective protection or therapeutic treatment of a disease. As a result, repeated administration of a pathogen- or disease-specific biological preparation may be necessary to establish sustained and protective immunity against a particular pathogen or disease, or to treat or functionally cure a given disease. An administration regimen involving repeated administration of an immunogenic composition or vaccine against the same pathogen or disease is referred to as a "prime-boost regimen." In one embodiment, a prime-boost regimen includes at least two administrations of an immunogenic composition against hepatitis B. The first administration of an immunogenic composition is referred to as "priming," and any subsequent administrations of the same immunogenic composition or an immunogenic composition against the same pathogen are referred to as "boosting." It should be understood that two, three, four, or even five administrations to boost the immune response are also contemplated. The period between prime and boost is optionally 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks. More specifically, it is 4 weeks or 8 weeks. When two or more boosts are administered, the subsequent boost is administered 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 12 weeks, 6 months, or 12 months after the preceding boost. For example, the interval between any two boosts may be 4 weeks or 8 weeks.

[0094] The immunogenic compositions disclosed herein can be administered in a therapeutic regimen that includes the administration of additional immunogenic components, each formulated in a different composition (e.g., an immunogenic combination). The compositions are conveniently administered co-locationally, at or near the same site. For example, the components can be administered intramuscularly to the same side or limb ("co-lateral" administration) or to the opposite side or limb ("contra-lateral" administration). For example, in contralateral administration, a first composition can be administered to the left deltoid muscle and a second composition can be administered, either sequentially or simultaneously, to the right deltoid muscle. Alternatively, in ipsilateral administration, a first composition can be administered to the left deltoid muscle and a second composition can be administered, either sequentially or simultaneously, to the left deltoid muscle.

[0095] General Manufacturing Process ●ChAd155-hIi-HBV: The DNA fragment inserted as a transgene into the recombinant replication-deficient simian (chimpanzee-derived) adenovirus group C vector ChAd155 is derived from two HBV protein antigens, the core nucleocapsid protein antigen HBc and the small surface antigen HBs, separated by the self-cleaving 2A region of foot-and-mouth disease virus (FMDV) [Donnelly et al. 2001]. The 2A region of FMDV allows processing of the HBc-HBs fusion into separate protein antigens. Furthermore, the N-terminal portion of the gene encoding the HBc protein is fused to a gene encoding the human major histocompatibility complex (MHC) class II-binding invariant chain p35 isoform (hIi). A schematic diagram of the hIi-HBV transgene sequence is provided in (Figure 22).

[0096] The 2A region (18 amino acids) is supplemented with a 6-amino acid spacer at its N-terminus; this type of spacer has been reported to increase the efficiency of 2A-mediated cleavage. Protease cleavage via region 2A occurs at the C-terminus of 2A, just before the last proline in the 2A amino acid sequence. The proline remains at the N-terminus of the HBs protein, while the 23 amino acids preceding the proline cleavage site remain with the hIi-HBc-2A polypeptide.

[0097] Expression of the transgene thereby results in the production of two distinct polypeptides, hIi-HBc-spacer-2A and HBs, following protease processing. For brevity, the hIi-HBc-spacer-2A polypeptide is referred to as the hIi-HBc protein. When expressed in cell culture, the hIi-HBc antigen is detected in the cell culture supernatant, while the HBs protein is detected in the intracellular fraction.

[0098] An expression cassette encoding the antigen protein (operably linked to regulatory components to permit expression in the host cell) is assembled into a ChAd155 vector plasmid construct, as previously described (see WO 2016 / 198621, incorporated by reference for the purposes of disclosing the ChAd155 vector sequence and methods), to yield ChAd155-hIi-HBV. The hIi-HBV transgene is under the transcriptional control of the human cytomegalovirus (hCMV) promoter and bovine growth hormone polyadenylation signal (BGH pA). The expression cassette encodes HBs, HBc, and hIi amino acid sequences, in which the hIi sequence is fused to the HBc N-terminus of HBc, and the HBs and HBc sequences are separated by a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus for processing HBc and HBs into separate proteins.

[0099] To generate a replication-deficient recombinant ChAd155 adenovirus, the functions of the deleted gene regions required for adenoviral replication and infectivity must be supplied to the recombinant virus by a helper virus or cell line, i.e., a complementing or packaging cell line. A particularly suitable complementing cell line is the Procell92 cell line. The Procell92 cell line is based on HEK 293 cells expressing the adenovirus E1 gene transfected with the Tet repressor under the control of the human phosphoglycerate kinase-1 (PGK) promoter and a G418 resistance gene (Vitelli et al. PLOS One (2013) 8(e55435):1-9). Procell92.S is adapted for growth in suspension and is useful for generating adenoviral vectors expressing toxic proteins.

[0100] Production of ChAd155-hIi-HBV Drug Substance: The production of ChAd155-hIi-HBV viral particles (drug substance) was 5 × 10 5 The method involves culturing Procell-92.S cells at a cell density of 100 cells / ml. The cells are then infected with ChAd155-hIi-HBV master virus seed (MVS) using a multiplicity of infection of 200 vp / cell. The ChAd155-hIi-HBV virus harvest is purified by a multi-step process including cell lysis, lysate clarification, and concentration (filtration step), followed by anion exchange chromatography.

[0101] Vaccine Formulation and Filling The purified ChAd155-hIi-HBV bulk drug substance is then processed as follows: - Dilution of purified ChAd155-hIi-HBV drug substance in formulation buffer. - Sterile filtration. - Filling of final containers.

[0102] The ChAd155-hIi-HBV vaccine is a liquid formulation packaged in a vial. The formulation buffer contains Tris (10 mM), L-histidine (10 mM), NaCl (75 mM), MgCl (1 mM), and EDTA (0.1 mM), along with sucrose (5% w / v), polysorbate-80 (0.02% w / v), and ethanol (0.5% w / v), adjusted to pH 7.4 with HCl (water for injection to final volume).

[0103] ●MVA-HBV: MVA-HBV is a recombinant modified vaccinia virus Ankara (MVA) carrying two distinct proteins of HBV: the core and S proteins (separated by the 2A peptide). The MVA-HBV construct was generated from the MVA-Red vector system [Di Lullo et al. 2010], which was derived from an MVA virus seed batch from the attenuated passage 571 (referred to as MVA-571) described by Professor Anton Mayr [Mayr, A. et al. 1978].

[0104] The MVA-HBV transgene encodes the HBV core nucleocapsid protein, HBc, and the minor surface antigen, HBs. The HBc-HBs sequences are separated by the self-cleaving 2A region of foot-and-mouth disease virus, which allows for processing of the fusion protein into separate HBc and HBs antigens, as described above for adenoviral vectors. A schematic diagram of the transgene is provided in Figure 21.

[0105] Expression of the transgene results in the production of two distinct polypeptides after protease processing: HBc-spacer-2A and HBs. For simplicity, the HBc-spacer-2A polypeptide will be referred to as the HBc protein.

[0106] The expression cassette was subcloned into the MVA shuttle vector p94-elisaRen to generate the transfer vector p94-HBV, which contains the antigen expression cassette under the control of the vaccinia P7.5 early / late promoter, flanked by the FlankIII-2 and FlankIII-1 regions, allowing insertion into the delIII region of MVA by homologous recombination.

[0107] The generation of recombinant virus was based on two events of in vivo recombination in CEF cells.

[0108] Briefly, primary chicken embryo fibroblasts (CEFs) were infected with MVA-Red and then transfected with p94-HBV carrying an antigen transgene (and an EGFP marker gene under the control of the synthetic promoter sP). An initial recombination event occurred between homologous sequences (FlankIII-1 and -2 regions) present in both the MVA-Red genome and the transfer vector p94-HBV, resulting in replacement of the Hcred protein gene with the transgene / eGFP cassette. Infected cells containing the MVA-Green intermediate were isolated by FACS sorting and used to infect fresh CEFs. The intermediate recombinant MVA resulting from the initial recombination carried both the transgene and eGFP cassette but was unstable due to the presence of the repeated Z region.

[0109] Thus, a spontaneous second recombination event involving the Z region occurs, removing the eGFP cassette. The resulting recombinant MVA is colorless and carries the transgene cassette.

[0110] Finally, markerless recombinant virus (MVA-HBV) infected cells were sorted by FACS, and MVA-HBV was cloned by terminal dilution and propagated in CEF by conventional methods.

[0111] MVA-HBV drug substance production MVA-HBV viral particles (drug substance) were 1 × 10 6 ~2×10 6 Primary cell cultures of chicken embryo fibroblasts (CEFs) are grown to a cell density of 100 cells / ml and then infected with MVA-HBV master virus seeds (MVS) at a multiplicity of infection of 0.01-0.5 PFU / cell. MVA-HBV virus harvests are purified by a multistep process based on centrifugation pelleting, resuspension, and differential gradient centrifugation steps.

[0112] Vaccine Formulation and Filling The purified MVA-HBV bulk drug substance is then processed as follows: - Dilution of purified MVA-HBV DS in formulation buffer. - Filling of final containers.

[0113] The MVA-HBV vaccine is a liquid formulation packaged in a vial. The formulation buffer contains tris(hydroxymethyl)aminomethane pH 7.7 (10 mM), NaCl (140 mM), and water for injection to the final volume.

[0114] HBs-HBc recombinant protein mixture: HBc drug substance production The HBc recombinant protein (drug substance) manufacturing process consists of inoculating pre-culture flasks with recombinant Escherichia coli (E. coli) working seed, followed by a fermentation process and a multi-step purification process that includes harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0115] HBs drug substance production The HBs recombinant protein (drug substance) manufacturing process consists of inoculating pre-culture flasks with recombinant S. cerevisiae working seeds, followed by a fermentation process and a multi-step purification process including harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0116] Vaccine Formulation and Filling The purified HBs drug substance and HBc drug substance are diluted in a formulation buffer containing sucrose as a cryoprotectant and poloxamer as a surfactant, filled into 4 mL clear glass vials, and lyophilized. [Example]

[0117] Non-clinical development objectives: Strong and functional CD8, especially against HBcAg + and CD4 + T cell responses are associated with HBV clearance and resolution of infection [Boni, 2012; Li, 2011; Liang, 2011; Lau, 2002; Bertoletti, 2012]. Furthermore, anti-S antibodies may be key to preventing the spread of HBV to uninfected hepatocytes and controlling post-treatment relapse of HBV replication [Rehermann 2005; Neumann 2010]. The proposed vaccination regimen involves the development of a potent CD8 + A heterologous prime-boost schedule using two viral vectored vaccines (ChAd155-hIi-HBV and MVA-HBV) encoding hepatitis B core (HBc) antigen and hepatitis B surface (HBs) antigen to induce T cell responses was shown to result in potent antigen-specific CD4 T cell responses in CHB patients. + AS01 for inducing T cell and antibody responses B-4 This vaccine-induced immune response, including that with sequential or simultaneous administration of adjuvanted HBc-HBs proteins, should ultimately lead to a substantial reduction in HBsAg concentrations or HBsAg loss (i.e., HBsAg concentrations below detectable levels), which is considered a marker of complete and durable control of HBV infection.

[0118] The primary objectives of non-clinical development were to: Investigational vaccine components, e.g., ChAd155-hIi-HBV, MVA-HBV, and HBc-HBs / AS01, to guide the selection of vector constructs, the inclusion of hIi, the composition of protein formulations including adjuvant system selection, and the immunization schedule. B-4To demonstrate the immunogenicity of in naive and HBV-tolerant mice. • To demonstrate the safety of the complete vaccine regimen in HBV-tolerant mice (non-GLP study) and in a repeat-dose GLP toxicity study conducted in NZW rabbits. • To document the biodistribution of vector in vaccinated animals (GLP study).

[0119] Preclinical strategies and rationale for the selection of animal models Immunogenic packages were first generated in healthy mice to guide the selection of vector constructs, protein formulations including adjuvant system selection, and immunization schedules.

[0120] To support the selection of vector constructs, protein formulations including adjuvant system selection, and immunization schedules, most preclinical experiments were performed in inbred CB6F1 (a hybrid of C57Bl / 6 and BALB / c mice) mice, a model previously used to evaluate T cell responses elicited by AS01-adjuvanted candidate vaccines and adenoviral vectors [Lorin, 2015].

[0121] Using HLA.A2 / DR1 mice (transgenic for human HLA-A2 and HLA-DR1 molecules), candidate vaccines were tested against HBc-specific CD8 + The ability to induce T cell responses was assessed (since no such responses were detected against this antigen in inbred CB6F1 mice), likely due to the presence of H2-K in the HBc sequence of the investigational vaccine. b This is due to the absence of the MHC-I-restricted immunodominant epitope (MGLKFRQL). The HBc sequence of the investigational vaccine is based on the sequence of HBV genotype A / subtype adw, which differs by one amino acid, with an isoleucine (I) replacing a phenylalanine (F) in the epitope (MGLKIRQL), as reported by Riedl et al. [Riedl, 2014]. HBV-specific CD4 + T cells and antibodies were evaluated in the same HLA.A2 / DR1 mice.

[0122] Because HBV naturally infects only chimpanzees and humans, there are limited animal models available for evaluating the efficacy of therapeutic vaccines. Mouse models have been developed in which the entire HBV genome is expressed by integration of the viral genome into the host genome (HBV transgenic mice), by infection with replicating HBV DNA, or by a vector expressing the HBV genome. Although these do not recapitulate chronic HBV pathogenesis, viral replication intermediates and proteins can be detected in the liver, and immune tolerance is observed.

[0123] The AAV2 / 8-HBV-transduced HLA.A2 / DR1 mouse model recapitulates the virological and immunological characteristics of chronic HBV infection. To demonstrate that the vaccine regimen can overcome tolerance to HBs and HBc antigens To assess the effect of liver-infiltrating HBc-specific CD8+ T cells, potentially targeting HBcAg-expressing hepatocytes, on liver histology (hematoxylin-eosin [H&E] staining) and ALT / AST levels. selected [Dion, 2013; Martin, 2015].

[0124] Finally, standard animal models for biodistribution (Sprague-Dawley rats) and toxicology studies (NZW rabbits) have been chosen to evaluate candidate vaccines because, although they are not models of infectious disease, they are capable of mounting pharmacologically relevant immune responses to vector-expressed and recombinant proteins and are well-accepted species for vaccine toxicity testing. These species also demonstrate the efficacy of AS01. B It has been previously used in toxicology testing programs for the adjuvants and their immune enhancers MPL and QS-21.

[0125] Nonclinical Pharmacology Investigational vaccine components, e.g., ChAd155-hIi-HBV, MVA-HBV, and HBc-HBs / AS01, following intramuscular administration B-4 Several preclinical studies were conducted to demonstrate the immunogenicity of the vaccine in naive and HBV-tolerant animals. Antigen-specific immunogenicity profiles were first assessed using viral vectors (ChAd155-hIi-HBV and MVA-HBV) and an investigational HBV recombinant protein-adjuvanted vaccine (HBc-HBs / AS01). B-4 The immunogenicity and safety profile of the complete vaccine regimen intended for first time in humans (FTiH) administration was evaluated in Phase 2.

[0126] material Doses of the AS01 adjuvant system used in nonclinical immunogenicity studies AS01 B-4 The adjuvant system is composed of the immune enhancers QS-21 (a triterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A), along with liposomes and sorbitol as vehicles for these immune enhancers. B-4 A single human dose (0.5 mL) contains 50 μg of QS-21 and 50 μg of MPL. 1 / 10 of the human dose, i.e., 50 μl, is the volume injected into mice (equivalent to 5 μg of QS-21 and MPL).

[0127] AS01 E-4 The adjuvant system is AS01 B-4 This corresponds to a 2-fold dilution of the dilution. 1 / 10 of the human dose, i.e., 50 μl, is the volume injected into mice (corresponding to 2.5 μg of QS-21 and MPL).

[0128] Cellular immune response - Intracellular cytokine staining (ICS) Fresh pools of peripheral blood leukocytes (PBLs), splenocytes, or liver-infiltrating lymphocytes collected at various time points were stimulated ex vivo for 6 hours with pools of 15-mers overlapping 11 amino acids covering the HBc or HBs sequence. HBc- and HBs-specific cellular responses were observed in CD4+ cells expressing IFN-γ and / or IL-2 and / or tumor necrosis factor (TNF)-α. + or CD8 + The results were assessed by ICS, which measures the amount of CD8 T cells. + T or CD4 + The minimum number of T cells included >3000 events. Alternatively, IFN-γ-ELISpot was performed after restimulation of splenocytes with the same peptides as for ICS.

[0129] Humoral immune response - enzyme-linked immunosorbent assay (ELISA) HBc- and HBs-specific antibody responses were measured by ELISA using sera from immunized mice at various time points. Briefly, 96-well plates were coated with HBc or HBs antigen. Individual serum samples were then added in serial dilutions and incubated for 2 hours. Biotinylated anti-mouse F(ab)'2 fragments were then added, and antigen-antibody complexes were revealed by incubation with streptavidin-horseradish peroxidase complex and the peroxidase substrate ortho-phenylenediamine dihydrochloride / H2O2. For each time point and each antigen (HBc, HBs), an analysis of variance (ANOVA) model was fitted to the log10 titers using a heteroscedastic model (identical variances between groups were not assumed), including group, study, and interaction as fixed effects. This model was used to estimate geometric means (and their 95% CI) and geometric mean ratios and their 95% CI. As no predefined criteria were set, the analysis was descriptive and the 95% CI of the ratios between groups was calculated without adjustment for multiplicity.

[0130] ALT / AST measurement ALT and AST levels in mouse serum were quantified using the following commercially available kits: Alanine Aminotransferase Activity Assay Kit Sigma Aldrich Catalog #MAK052 Aspartate Aminotransferase Activity Assay Kit Sigma Aldrich Catalog #MAK055

[0131] Quantification of serum HBs antigen Circulating HBs antigen in mouse serum was quantified using Monolisa Anti-HBs PLUS from BIO-RAD (catalog #72566) and an international standard (Abbott Diagnostics).

[0132] Histopathology analysis Livers (one lobe per liver) were harvested and preserved in 10% formaldehyde fixative. All samples for microscopic examination were trimmed according to RITA guidelines [Ruehl-Fehlert, 2003; Kittel 2004; Morawietz 2004], embedded in paraffin wax, sectioned to a thickness of approximately 4 microns, and stained with H&E. Histological activity (necroinflammatory lesions) and fibrosis were graded according to the METAVIR scoring system [Bedossa, 1996; Mohamadnejad, 2010; Rammeh, 2014]. Inflammatory cell foci were graded according to the Desmet score as described by Buchmann et al. [Buchmann, 2013].

[0133] The statistical analyses performed in each study are detailed in the sections relating to each individual study.

[0134] Example 1 - Evaluation of ChAd155-HBV (with and without hIi) prime and MVA-HBV boost in an HLA.A2 / DR1 transgenic mouse model the purpose The primary objective of this study was to demonstrate that priming with a single dose of ChAd155-HBV (with or without hIi) followed by a booster dose of MVA-HBV results in potent CD8 responses against HBc in HLA.A2 / DR1 mice transgenic for human MHC-I / II molecules. + The aim of this study was to determine whether ChAd155-HBV with and without hIi could induce HBc-specific CD8 T cell responses. Furthermore, a direct comparison was performed between ChAd155-HBV with and without hIi, and the results showed that HBc-specific CD8 T cell responses were significantly higher than those reported previously for other antigens [Spencer, 2014; Capone, 2014]. + The potential of the hIi sequence to further enhance T cell responses was investigated. + T cell responses and HBc and HBs-specific CD4 + T cell and antibody responses were also assessed.

[0135] research design HLA.A2 / DR1 mice (11 mice per group) were vaccinated with 10 8 Immunized with ChAd155-HBV (with or without hIi) in the vp and 10 days after immunization on day 28. 7 Mice were boosted with pfu of MVA-HBV (without hIi) (Table 1). Mice were sacrificed 14 days after the first immunization (prime) (14 dpI) or 7 days after the second immunization (boost) (7 dpII), and HBc- and HBs-specific humoral and cellular immune responses were determined in serum and spleen, respectively.

[0136] [Table 1]

[0137] statistical analysis The ANOVA model included the two groups (with or without hIi) and the experiment (results of three experiments after prime and two experiments after boost) as fixed parameters, using a heteroscedastic model to estimate the log CD8 +The model was fitted to T cell frequencies. The geometric means (and their 95% CI) and geometric mean ratios and their 95% CI were estimated using this model.

[0138] result HBc and HBs-specific T cell responses Both the ChAd155-HBV and ChAd155-hIi-HBV vectors express HBc-specific CD8 + The presence of hIi induced a higher CD8 T cell response to HBc antigen (Fig. 1A). + MVA-HBV boost in mice immunized with ChAd155-hIi-HBV tends to induce HBc-specific CD8 T cell responses. + It induced a three-fold increase in T cell responses, whereas no increase was observed for the ChAd155-HBV group.

[0139] Both vectors express HBs-specific CD8 + T cell responses were induced, and the MVA-HBV booster effect was more pronounced in mice primed with the ChAd155-HBV construct (Fig. 1B).

[0140] HBc- and HBs-specific CD4 + T cell responses were low (data not shown).

[0141] The results of this experiment were consistent with those of two other similar independent experiments (data not shown). Although each independent study was not statistically powered to compare groups due to limitations related to animal availability, a meta-analysis of the three studies was performed to compare the HBc-specific CD8+ / -induced by ChAd155-HBV versus ChAd155-hIi-HBV after priming and MVA-HBV boost. + T cell responses were compared.

[0142] Statistical analysis showed that HBc-specific CD8+ cells were induced by the prime-boost regimen using the ChAd155-hIi-HBV construct after the prime and MVA-HBV boost.+ The T cell responses were shown to be significantly higher than those elicited by a prime-boost regimen using ChAd155-HBV, supporting the use of a human invariant chain sequence fused to an HBc sequence. a. 14 days after administration I, the geometric mean ratio (hIi / hIi-free) was estimated to be 3.27 (95% CI: 1.57-6.79). b. Seven days after administration II, the geometric mean ratio (hIi / hIi-free) was estimated to be 6.25 (95% CI: 2.11-18.51).

[0143] HBc and HBs-specific antibody responses Immunization with ChAd155-HBV, but not with ChAd155-hIi-HBV, induced HBc-specific antibodies. After the MVA-HBV booster, anti-HBc antibody responses were comparable between groups primed with ChAd155-HBV or ChAd155-hIi-HBV (Figure 2). No HBs-specific antibody responses were detected (data not shown).

[0144] conclusion ChAd155-hIi-HBV had the highest CD8 cytotoxicity against HBc when compared with ChAd155-HBV. + It induced a T cell response that was further increased after an MVA-HBV boost.

[0145] Example 2 - HBc-HBs / AS01 in inbred mice (CB6F1) B-4 Immunogenicity research] the purpose The primary objective of this immunogenicity study was to determine whether HBc and HBs proteins are resistant to AS01 B-4 The objective of this study was to determine whether HBc- and HBs-specific HBV-specific antibodies could induce both humoral and T cell responses when co-formulated into a HBV-specific antibody.

[0146] research design Six to eight-week-old CB6F1 mice (30 mice per group) were treated with 50 μl of AS01 B-4Mice were immunized three times intramuscularly with HBc, HBs, or HBc-HBs formulated in HBV (listed in Table 2 below) on days 0, 14, and 28. HBc- and HBs-specific T cell responses were measured in fresh PBL 7 days after the second and third doses, and anti-HBs and anti-HBc antibody responses were measured 14 days after the second and third doses.

[0147] [Table 2]

[0148] statistical analysis Statistical analysis was performed using ANOVA on log10 values ​​with one factor (group) using a heteroscedastic model, i.e., identical variances were not assumed for different levels of the factor. This analysis included time points (after the second and third immunizations), T cell responses (CD4 + and CD8 + The data were collected by antigen specificity (HBs and HBc) and by T cell type. The estimates of geometric mean ratios between groups and their 95% confidence intervals (CI) were obtained using a back-transformation on the log values. Multiplicity adjustment was performed using Tukey's method. Multiplicity-adjusted 95% CIs were provided.

[0149] result HBc and HBs-specific T cell responses HBc / AS01 B-4 , HBs / AS01 B-4 or HBc-HBs / AS01 B-4 Immunization of mice with β-lactam elicited potent CD4 responses to both antigens. + induced a T cell response (Figure 3). + The magnitude of the T cell response was HBc-HBs / AS01 B-4 Regarding the preparation, HBc / AS01 B-4 The HBc-specific CD4 +T cell levels far exceeded those in the control group and appeared to remain robust. + No such interference was observed on the level of T cell responses.

[0150] HBs / AS01 B-4 or HBc-HBs / AS01 B-4 The formulation contains a potent HBs-specific CD8 + As reported by others [Riedl, 2014], the HBc sequence of our vaccine candidate contains the H2-K b None of the vaccine candidates elicited detectable HBc-specific CD8+ cells, as expected in this mouse model due to the absence of the MHC-I restricted immunodominant epitope -MGLKFRQL-. + It did not induce a T cell response (data not shown).

[0151] HBc and HBs-specific antibody responses High levels of anti-HBc and / or anti-HBs antibodies were induced by each of the three formulations (see Figure 5). B-4 In the preparation, HBc / AS01 B-4 A significantly lower level of anti-HBc antibody response was observed compared with HBc-HBs / AS01 (2.35-fold, P<0.0001). B-4 The presence of HBc in the combination did not negatively affect the anti-HBs antibody response (Fig. 5).

[0152] conclusion All preparations (HBs / AS01 B-4 , HBc / AS01 B-4 and HBc-HBs / AS01 B-4 ) was immunogenic and induced both cellular and humoral responses to both antigens (HBc-specific CD8 + HBc-HBs / AS01) except for T cell responses (as expected in this model). B-4 The anti-HBc response induced by the formulation was HBc / AS01 B-4This was lower than that induced by HBs in this mouse model, suggesting interference related to the presence of HBs in this mouse model. This interference was further evaluated; see Example 7, where a 4:1 ratio of HBc to HBs suppressed the anti-HBc immune response (antibodies and specific CD4 + This preparation, HBc-HBs 4-1 / AS01, was able to restore T cells. B-4 was selected for subsequent non-clinical immunogenicity studies with adjuvanted protein formulations.

[0153] Example 3 - Comparative adjuvant experiment in inbred mouse strain (CB6F1) the purpose The main purpose of this experiment was to compare the effects of various adjuvants (Alum, AS01 B-4 or AS01 E-4 ) or without adjuvant, a 4:1 ratio of HBcAg and HBsAg, which are potent CD4 antagonists against both antigens. + The purpose was to compare their ability to induce T cell and humoral responses.

[0154] research design Six to eight week old CB6F1 mice (35 mice for groups 1 to 4 and 25 mice for group 5) were cultured in a medium containing alum, AS01 B-4 or AS01 E-4 The mice were immunized three times intramuscularly on days 0, 14, and 28 with HBc-HBs antigen (4 μg-1 μg) formulated using the formula (listed in Table 3 below). E-4 The adjuvant system is AS01 B-4 It contains half the amount of immune enhancers QS-21 and MPL compared to the first 2 doses. HBc- and HBs-specific T cell responses were measured in fresh PBLs 7 days after the second and third doses after 6 hours of ex vivo restimulation with the peptide pool, and anti-HBs and anti-HBc antibody responses were measured by ELISA 14 days after the second and third doses.

[0155] [Table 3]

[0156] statistical analysis For statistical analysis, an ANOVA model was fitted to the log2 T cell frequencies and log10 antibody titers using a heteroscedastic model, including group as a fixed effect (identical variances between groups were not assumed, and the NaCl group was excluded from the analysis). This model was used to calculate the geometric means (and their 95% CI) and the geometric mean ratios (ASO1 relative to the three other groups). B ) and their 95% CIs were estimated. Dunnett's adjustment was performed for HBc- and HBs-specific CD4 + This was applied to T cell frequencies (primary endpoint) and anti-HBs antibody titers (secondary endpoint) measured 14 days after the third dose. For other responses / time points, the analysis was descriptive and no adjustment was applied.

[0157] result HBc and HBs-specific T cell responses The AS01-adjuvanted formulation produced significantly higher HBc-specific CD4 than the alum-adjuvanted and non-adjuvanted formulations. + induced a T cell response (Figure 6B). B-4 Formulation and AS01 E-4 No statistically significant differences were observed between the formulations. As previously observed in CB6F1 mice, HBc-specific CD8 + T cell responses were undetectable whatever the formulation tested (data not shown).

[0158] The AS01-adjuvanted formulation significantly increased HBs-specific CD4 expression compared with alum-adjuvanted and non-adjuvanted formulations. + (Figure 6A) and CD8 + (Fig. 6C) T cell responses were significantly higher. B-4 Formulation and AS01 E-4 No statistically significant differences were observed between the formulations.

[0159] HBc and HBs-specific antibody responses The AS01-adjuvanted formulation induced significantly higher anti-HBc and anti-HBs total IgG responses compared to the alum-adjuvanted and non-adjuvanted formulations (Figure 7). B-4 and AS01 E-4 The total IgG antibody responses elicited by the adjuvanted formulations were not statistically different.

[0160] conclusion Overall, the AS01 adjuvant system (AS01 E-4 or AS01 B-4 ) induced the highest humoral and cellular responses against HBc and HBs in CB6F1 mice compared with alum-based or non-adjuvanted formulations.

[0161] Example 4 - ChAd155-hIi-HBV / MVA-HBV / HBs-HBc / AS01 in HLA.A2 / DR1 transgenic mice B-4 Immunogenicity evaluation of vaccine regimens] the purpose The objective of this study was to evaluate the efficacy and safety of ChAd155-hIi-HBV / MVA-HBV viral vector prime / boost followed by or co-administered with two doses of HBc-HBs 4-1 / AS01. B-4 The objective of this study was to evaluate the immunogenicity of various protein-based vaccine regimens.

[0162] research design Group 1 mice (N=16) were immunized with ChAd155-hIi-HBV on day 0, followed by MVA-HBV 28 days later. Two doses of HBc-HBs 4-1 μg / AS01 B-4 were injected at 14-day intervals after this prime / boost viral vector regimen (Table 4). Mice in Group 2 (N=16) were injected with ChAd155-hIi-HBV and HBc-HBs 4-1 / AS01 on day 0. B-4 immunization with HBc-HBs 4-1 / AS01 28 days later. B MVA-HBV and HBc-HBs 4-1 / AS01 were co-administered with MVA-HBV.B Two subsequent co-immunizations of HBsAg were performed 14 days apart (Table 4). A third group of mice (N=8) was injected with NaCl as a negative control. Mice were sacrificed 7 days after the second and fourth immunizations (7 dpII and 7 dpIV, respectively) to determine HBc- and HBs-specific humoral (serum) and cellular immune responses (in splenocytes and liver-infiltrating lymphocytes).

[0163] This study was descriptive and no statistical sample size justification and analysis was performed.

[0164] [Table 4]

[0165] result HBc- and HBs-specific CD8 + T cell response (splenocytes) HBc-HBs 4-1 / AS01 B-4 Co-administration of ChAd155-hIi-HBV vector (as prime) and MVA-HBV vector (as boost) (Group 2) significantly increased HBc-specific CD8+ expression at 7 dpII when compared with injection of ChAd155-hIi-HBV / MVA-HBV alone (Group 1). + Induced a four-fold increase in T cell responses against HBs (Fig. 8). + T cell responses were induced in both groups (Figure 8).

[0166] At 7dpIV, HBc-specific CD8 + T cell responses are HBc-HBs / AS01 B-4 Although there was a clear boost after subsequent administration of HBs-specific CD8 + T cell responses were not boosted (Group 1). MVA-HBV / HBc-HBs 4-1 / AS01 B-4 When two additional doses of + No further increase in T cells was observed (group 2).

[0167] HBc- and HBs-specific CD4 + T cell response (splenocytes) Low levels of HBc- and HBs-specific CD4 + T cells were detected after prime-boost ChAd155-hIi-HBV / MVA-HBV immunization (median 0.17% and 0.11%, respectively) (group 1), whereas HBc-HBs 4-1 / AS01 B-4 When co-administered with prime-boost ChAd155-hIi-HBV / MVA-HBV, a strong response to both antigens was observed at 7 dpII (group 2) (FIG. 9).

[0168] ChAd155-hIi-HBV / MVA-HBV prime-boost HBc-HBs 4-1 / AS01 B-4 Subsequent administration of (Group 1) increased HBc- and HBs-specific CD4 + HBc-HBs / AS01 significantly enhanced T cell responses (median 1.64% and 2.32%, respectively). B-4 Mice already vaccinated with prime-boost ChAd155-hIi-HBV / MVA-HBV co-administered with two additional doses of MVA-HBV and HBc-HBs / AS01 B-4 When co-administered with HBs-specific CD4 + A robust increase in T cells was observed (Group 2). HBc-specific CD4 + T cells remained at the same level 7 days after II (7dpost II) in that same group.

[0169] HBc- and HBs-specific T-cell responses measured in liver-infiltrating lymphocytes. Seven days after the last immunization, the presence of vaccine-induced T cell responses in the liver was investigated by ICS. To ensure a sufficient number of liver-infiltrating lymphocytes for in vitro restimulation and ICS, a pool of cells collected after perfusion of three or four livers was constructed for each data point. Due to the small number of data points, no statistical analysis was performed, and the results are descriptive.

[0170] Both vaccine regimens resulted in detectable HBc- and HBs-specific CD4 in the livers of vaccinated mice. + T cells (Figure 10). + T cell responses were measured in the livers of animals vaccinated with both vaccine regimens, while much lower frequencies of HBs-specific CD8 + T cells were measured.

[0171] HBc and HBs-specific antibody responses ChAd155-hIi-HBV / MVA-HBV, HBc-HBs 4-1 / AS01 B-4 Co-administration of MVA-HBV+HBc-HBs / AS01 (Group 2) induced the highest level of anti-HBc antibodies at 7 days post-immunization (Figure 11). B-4 Subsequent injection of HBs-HBs / AS01 did not further increase the level of anti-HBc antibody responses (7 days after IV). B-4 A clear increase in anti-HBc-specific antibody responses was observed 7 days after injection of ChAd155-hIi-HBV / MVA-HBV (Group 1). Since no anti-HBs antibody responses were detected in animals after immunization with ChAd155-hIi-HBV / MVA-HBV, the HBc-HBs / AS01 B-4 The presence of the components appeared to be important in the schedule for eliciting strong anti-HBs antibodies (Figure 11). The greatest magnitude of the response was observed in the co-administration group (Group 2) after the last immunization.

[0172] conclusion In HLA.A2 / DR1 transgenic mice, ChAd155-hIi-HBV / MVA-HBV induced low but detectable HBc-specific CD4 + It induces a T cell response, which is a HBc-HBs 4-1 / AS01 B-4 The initial prime-boost immunization with ChAd155-hIi-HBV / MVA-HBV resulted in potent HBc- and HBs-specific CD8 +T cell responses were induced, and HBc-specific responses were observed with sequentially given HBc-HBs / AS01. B-4 It increased even more after the boost.

[0173] Interestingly, ChAd155-hIi-HBV / MVA-HBV inhibited HBc-HBs 4-1 / AS01 B-4 When coadministered with 、 High levels of HBc- and HBs-specific CD4 after only two immunizations + and CD8 + T cells and antibodies were induced. MVA-HBV+HBc-HBs / AS01 B-4 Further immunization with did not further increase the level of these responses.

[0174] Furthermore, vaccine-induced HBc- and HBs-specific CD4 + and CD8 + T cells were also detected in the livers of animals vaccinated with both vaccine regimens.

[0175] Example 5 - Evaluation of T and B cell tolerance to the "invariant chain" sequence Ii encoded by the ChAd155 vector: Use of a ChAd155 construct encoding the murine Ii sequence (mIi) in CB6F1 mice. the purpose Immunogenicity studies were performed in CB6F1 mice to investigate T and B cell tolerance to the "invariant chain" sequence Ii in an allogeneic model using a ChAd155 construct encoding the murine Ii sequence (mIi): ChAd155-mIi-HBV.

[0176] research design The induction of autologous mIi-specific immune responses was investigated using high doses (10 9 vp) with the ChAd155-mIi-HBV vector, were assessed by IFN-γ ELISpot (in splenocytes) and ELISA (in serum) (Table 5).

[0177] [Table 5]

[0178] method For T cell responses, pooled 15-mer peptides encompassing the mouse Ii sequence and overlapping by 11 amino acids were used as antigens in IFN-γ-ELISpot assays. For antibody responses, commercially available mouse Ii recombinant protein and a monoclonal antibody specific for mouse Ii were used to coat ELISA plates and as positive controls, respectively. As a positive control for "vaccination," HBc- and HBs-specific T cell responses were monitored in IFN-γ-ELISpot assays.

[0179] result Strong HBc-specific T cell responses and low but detectable HBs-specific T cell responses were measured after the first and second immunizations with ChAd155-mIi-HBV. Notably, the HBc Kb-restricted dominant class I epitope (MGLKFRQL) was added to this construct, resulting in the induction of HBc-specific CD8 T cells in this mouse strain. + To monitor T cell responses, splenocytes were restimulated with this specific sequence in an ELISpot assay. Two weeks after the first or second immunization, no anti-mIi antibodies (Figure 13) or mIi-specific T cells (Figure 12) were detected in any of the animals, suggesting that immune tolerance to the mIi sequence was maintained.

[0180] Example 6 - ChAd155-hIi-HBV / MVA-HBV / HBc-HBs / AS01 in AAV2 / 8-HBV-transduced HLA.A2 / DR1 mice B-4 Immunogenicity and safety evaluation of vaccine regimens] the purpose The AAV2 / 8-HBV-transduced HLA.A2 / DR1 mouse model recapitulates the virological and immunological characteristics of chronic HBV infection. In this model, the liver of mice is transduced with an adeno-associated virus serotype 2 / 8 (AAV2 / 8) vector carrying a replication-competent HBV DNA genome.

[0181] 5 × 10 of AAV2 / 8-HBV vector 10 A single tail vein injection of vg (viral genome) results in HBV replication and gene expression in the liver of AAV2 / 8-HBV-transduced mice [Dion; 2013]. HBV DNA replication intermediates, HBV RNA transcripts, and HBcAg are detectable in the liver for up to one year after injection without associated significant liver inflammation. HBs and HBeAg, as well as HBV DNA, can be detected in serum for up to one year. Furthermore, the establishment of immune tolerance to HBV antigens is observed in this surrogate model of chronic HBV infection.

[0182] The objectives of this study, conducted in AAV2 / 8-HBV transduced HLA.A2 / DR1 mice, were to: Demonstrate that the vaccine regimen can overcome tolerance to HBs and HBc antigens Liver-infiltrating HBc-specific CD8, potentially targeting HBcAg-expressing hepatocytes, on liver histology (H&E staining) and AST and ALT levels (as surrogate parameters for liver function) + To assess the influence of T cells It was.

[0183] research design Alone or HBc-HBs 4-1 / AS01 B-4 ChAd155-hIi-HBV and MVA-HBV (both encoding HBV core [HBc] and surface [HBs] antigens) in combination with HBc-HBs 4-1 / AS01, followed by two booster doses of HBc-HBs 4-1 / AS01. B-4 Two different vaccine regimens based on sequential immunization with HBV (alone or in combination with MVA-HBV) were tested (Table 6).

[0184] HLA.A2 / DR1 mice in groups 1, 2, and 3 were treated with 5 × 10 10 vg of AAV2 / 8-HBV vector (administered intravenously), while group 4 served as a positive control for immunogenicity (no establishment of tolerance before vaccination).

[0185] Group 1 animals (N=21) were immunized with ChAd155-hIi-HBV on day 31 followed by MVA-HBV on day 58. Two doses of HBc-HBs 4-1 μg / AS01 B-4 were injected on days 72 and 86 after this prime / boost viral vector regimen (Table 6).

[0186] Group 2 animals (N=21) were immunized with ChAd155-hIi-HBV on day 31 and HBc-HBs 4-1 / AS01 B-4 followed by HBc-HBs 4-1 / AS01 on day 58. B MVA-HBV and HBc-HBs 4-1 / AS01 were co-administered with MVA-HBV. B Two subsequent co-immunizations were performed on days 72 and 86 (Table 6).

[0187] Group 3 animals (N=21) were injected with NaCl on days 31, 58, 72 and 86 as a negative control.

[0188] Group 4 animals (N=8) received the same vaccine regimen as Group 2 (except they were not transduced with AAV2 / 8-HBV).

[0189] All vaccines were administered intramuscularly.

[0190] Circulating HBs antigen levels were measured in serum on days 23, 65 and 93 (groups 1, 2 and 3).

[0191] HBs- and HBc-specific antibody responses were measured by ELISA in the serum of all animals on days 23 (after AAV2 / 8-HBV transduction), 65 (7 days after the second immunization), and 93 (7 days after the fourth immunization). HBs- and HBc-specific CD4 + and CD8 + T cell responses were assessed in splenocytes and liver-infiltrating lymphocytes after ex vivo restimulation and ICS on days 65 (9 animals / group) and 93 (12 animals / group) (Groups 1, 2, and 3). These immunogenicity readouts were performed only on day 93 for Group 4 animals (8 animals).

[0192] Regarding liver-related safety parameters, AST and ALT levels were measured in serum on days 38, 65, and 93, and microscopic examination of H&E-stained liver sections was performed on days 65 and 93 to detect potential vaccine-related histopathological changes or inflammation (Groups 1, 2, and 3).

[0193] [Table 6]

[0194] statistical analysis AST and ALT levels An ANOVA model for repeated measures, including sex, day, group, and three 2 × 2 interactions, was fitted to the log10-transformed enzyme activity values ​​using an unstructured covariance structure. Model assumptions were tested. Interactions that were not significant at the 5% level were removed from the model. For both enzymes, the final model included sex, day, group, and the interaction between group and day. The geometric mean of enzyme activity for each group at each time point was derived from this model. Group comparisons of interest are reported by geometric mean ratios (GMRs), also derived from this model. All these statistics are presented with two-sided 95% confidence intervals. Multiplicity was not considered when calculating these GMRs.

[0195] All analyses were performed using SAS 9.2.

[0196] Humoral response Descriptive statistics were performed to calculate the number of responders. Cutoffs for responsiveness of anti-HBc or anti-HBs antibody responses were defined based on the geometric mean titers calculated in group 3 (AAV2 / 8-HBV transduced but unvaccinated).

[0197] Cellular response HBc, HBs-specific CD4 + or CD8 + Descriptive analysis was performed to define the number of responders for any of the T cells. The cutoff for responsiveness was defined as the 95th percentile of measurements performed in group 3 (AAV2 / 8-HBV transduced but unvaccinated).

[0198] result HBc-specific CD8 + and CD4 + T cells In AAV2 / 8-HBV-transduced HLA-A2 / DR1 mice, HBc-specific CD8 + or CD4 + Background levels of T cells were very low to undetectable without immunization at all time points tested (Group 3).

[0199] alone (group 1) or HBc-HBs 4-1 / AS01 B-4 Immunization with ChAd155-hIi-HBV and MVA-HBV vectors in combination with (Group 2) increased HBc-specific CD8 + T cells were induced (6 / 7 and 9 / 9 responders, respectively, 7 days after II), demonstrating the escape of tolerance to HBc antigen (Figure 14A). B-4 Two additional doses of + It only modestly increased T cell responses, reaching a median frequency of 1% in group 1 and 1.45% in group 2. HBc-specific CD8 T cells induced by the same vaccine regimen as in group 2. +The frequency of HBc-specific CD8 T cells was higher in non-transduced HLA.A2 / DR1 mice in group 4 (8 / 8 responders, frequency approximately 4-fold higher 7 days after IV), as expected due to immune tolerance to HBc antigen. + T cells were also detected in the livers of vaccinated mice with the same profile as in the spleen (FIG. 14B).

[0200] Both vaccine regimens induced very low to undetectable HBc-specific CD4 responses in AAV2 / 8-HBV-transduced HLA-A2 / DR1 mice. + T cells against HBc antigen (groups 1 and 2), whereas a robust response was measured in non-transduced mice (group 4), indicating that the vaccine regimen was able to induce CD4 T cells against HBc antigen under these experimental conditions. + This suggests that T cell tolerance was not overcome (Fig. 15A, B).

[0201] HBs-specific CD8 + and CD4 + T cells alone (group 1) or HBc-HBs 4-1 / AS01 B-4 Immunization with ChAd155-hIi-HBV and MVA-HBV vectors in combination with (Group 2) increased HBs-specific CD8 expression in AAV2 / 8-HBV-transduced mice. + T cells were induced and two additional doses of HBc-HBs 4-1 / AS01 alone or in combination with MVA-HBV were administered. B-4 There was no further increase in intensity afterwards (Fig. 16A). At the end of the vaccination schedule (7 days after the fourth dose), HBs-specific CD8 + The frequency of T cells was close to that detected in group 4 (non-transduced HLA.A2 / DR1 mice, median 7 days post-IV = 0.62%, 5 / 8 responders), suggesting overcoming T cell tolerance to HBsAg. + T cells were detected in the livers of animals in groups 1, 2 and 4 in most of the vaccinated animals (FIG. 16B).

[0202] HBs-specific CD4 + T cells were administered starting 7 days after the second vaccination in group 2 and 7 days after the fourth vaccination in group 1 with HBc-HBs 4-1 / AS01 alone or in combination with vector. B-4 The vaccine schedule used in animals in group 2 significantly increased HBs-specific CD4 responses compared to the vaccine schedule used in animals in group 1 (median 7 days post-IV = 1.34%, 11 / 12 responders). + Approximately three times higher frequencies of T cells were induced (median 7 days post-IV = 3.7%, 11 / 11 responders), reaching levels similar to those in Group 4 (non-transduced HLA.A2 / DR1 mice, median 7 days post-IV = 3%, 8 / 8 responders), suggesting complete overcoming of T cell tolerance to HBsAg. + Similar to the T cell response, HBs-specific CD4 + T cells were detected in the livers of animals in groups 1, 2 and 4 in all vaccinated animals (FIG. 17B).

[0203] HBs- and HBc-specific antibody responses Twenty-three days after injection of the AAV2 / 8-HBV vector, no anti-HBs antibody response was detected in HLA.A2 / DR1 mice, suggesting strong humoral tolerance to HBs antigen. Immunization with ChAd155-hIi-HBV and MVA-HBV vectors alone (Group 1) did not disrupt this tolerance, whereas HBc-HBs 4-1 / AS01 B-4 Immunization with the vector in combination with two doses of HBc-HBs 4-1 / AS01 in Group 1 resulted in the induction of anti-HBs antibody responses in 15 of 21 animals at day 65 (Group 2) (Figure 18A). B-4 Further administration of HBs 4-1 / AS01 induced detectable anti-HBs antibodies (geometric mean titer (GMT) of 116.8 at day 93 and 8 / 12 responders), and HBc-HBs 4-1 / AS01 in Group 2 B-4Two additional doses of MVA-HBV in combination with HBV further increased the strength of the anti-HBs antibody response in 11 / 11 responders to a GMT of 775 (although it remained approximately 5-fold lower than in non-AAV2 / 8-HBV-transduced animals in Group 4 (GMT = 3933) at day 93).

[0204] Similarly, HBc-HBs 4-1 / AS01 B-4 Only when the components were present in the vaccine regimen was an anti-HBc antibody response induced, with 3-fold higher levels measured at day 93 in animals from group 2 (GMT=1335.5; 11 / 11 responders) compared with group 1 (GMT=442.8; 12 / 12 responders) (FIG. 18B). Anti-HBc antibody titers induced in non-transduced mice (group 4) with the same vaccine regimen as in group 2 were higher (approximately 27-fold, GMT=35782).

[0205] These results indicate that the presence of adjuvanted protein components in the vaccine regimen is important for breaking humoral tolerance to both HBcAg and HBsAg. B-4 The vaccine regimen used in Group 2, which included four doses of HBV, induced the highest anti-HBc and anti-HBs antibody responses (although they remained lower than in non-AAV2 / 8-HBV-transduced mice (Group 4)).

[0206] AST / ALT levels As liver-related inflammatory parameters, serum activity of AST and ALT was measured (all groups) on day 38 (7 days after the first vaccination), day 65 (7 days after the second vaccination), and / or day 93 (7 days after the fourth immunization). Overall, AST and ALT levels remained stable throughout the course of the vaccine regimen in AAV2 / 8-HBV-transduced HLA.A2 / DR1 mice (groups 1 and 2) and were similar to those measured in unvaccinated mice (group 3) (Figure 19). AST levels were found to be statistically significantly higher in animals in the vaccine groups (groups 1 and 2) compared with control group 3 on day 65. However, AST levels were surprisingly low in group 3 animals on day 65 compared with the remaining kinetics, suggesting that these differences were due to the particularly unexpectedly low values ​​obtained in control group 3 at this time point, rather than increased AST levels in the vaccine groups (groups 1 and 2) (Figure 19A).

[0207] Although slightly lower ALT levels were measured at day 38 in Group 1 animals compared with Group 3 control animals, this difference was not considered clinically relevant (FIG. 19B).

[0208] Liver microscopy Microscopic examination of H&E stained liver sections was performed on days 65 and 93 to detect potential vaccine-related histopathological changes or inflammation (Groups 1, 2, and 3) (Table 7).

[0209] In AAV2 / 8-HBV transduced HLA-A2 / DR mice, day 65 (HBc-HBs 4-1 / AS01 B-4 There were no microscopic findings related to the laboratory parameters on either day 93 (7 days after the second injection of viral vectored vaccine MVA-HBV with or without the vaccine components ChAd155-hIi-HBV, MVA-HBV, and HBc-HBs 4-1 / AS01) or day 93 (7 days after the last injection). B-4 There were no histopathological changes that could be related to the use of

[0210] Furthermore, with the exception of control animal 3.13 (which showed focal grade 1 piecemeal necrosis), none of the animals showed morphological signs of chronic hepatitis.

[0211] Other microscopic findings observed in treated animals were considered incidental because they also occurred in controls, were of low incidence / magnitude, and / or were common background findings in mice of similar age [McInnes, 2012].

[0212] [Table 7]

[0213] HBs antigen levels in the serum of AAV2 / 8-HBV-injected mice As previously reported by Dion et al. [Dion, 2013], 23 days after injection with the AAV2 / 8-HBV vector, HBsAg levels were higher in males compared to females. These levels remained stable in all groups, without detectable effects of the vaccination regimen (Figure 20). However, AAV2 / 8-HBV-injected mice are not an animal model for studying vaccine efficacy against HBsAg.

[0214] conclusion In a surrogate model of chronic HBV infection in which immune tolerance to HBc and HBs antigens is established, i.e., AAV2 / 8-HBV-transduced HLA-A2 / DR1 mice, both tested vaccine regimens elicited HBc- and HBs-specific IgG and CD8 responses, albeit at lower levels than in non-transduced mice, as expected due to strong immune tolerance. + T cell responses and HBs-specific CD4 + The ChAd155-hIi-HBV / MVA-HBV vector circumvented tolerance by inducing T cell responses. B-4 When coadministered with 、The strength of vaccine-induced antibody and T cell responses was higher than that observed with a vaccine regimen in which the vector and adjuvanted protein were administered sequentially. Furthermore, vaccine-associated liver inflammation was assessed by measuring serum activity of AST and ALT and performing liver histopathological evaluation. No increases in liver enzymes were detected in the vaccine group compared with non-vaccinated groups, and none of the microscopic findings could be related to vaccine treatment. Collectively, these results demonstrate that the tested vaccine candidate induced HBs- and HBc-specific antibodies and CD8+ antibodies under these experimental conditions without the detection of relevant signs of liver changes. + T cell responses and HBs-specific CD4 + 1 shows that T cell responses were successfully restored.

[0215] Summary of Nonclinical Immunology Data for Examples 1-6 AS01 B-4 Studies in CB6F1 mice vaccinated with vaccine protein (HBc-HBs) formulated in an adjuvant system demonstrated that HBc-induced antibodies and CD4 + This suggests a negative interference of HBsAg on T cell responses. Nevertheless, HBc-HBs / AS01 B-4 The combination vaccine showed robust specific CD4 responses to both vaccine antigens. + It was possible to mount a T cell and antibody response.

[0216] - When compared with alum-based or non-adjuvanted formulations, the AS01 family-based formulation demonstrated significantly higher CD4 responses to both HBcAg and HBsAg. + Induced T cell and antibody responses.

[0217] Administration of ChAd155-hIi-HBV in HLA.A2 / DR1 transgenic mice resulted in potent CD8 expression against HBc antigen. + Induce T cell responses and, to a lesser extent, CD8 against HBsAg. +The MVA-HBV administration induced a CD8 T cell response against HBcAg. The response against HBcAg was clearly enhanced by the presence of the hIi in the construct. Subsequent administration of MVA-HBV resulted in a CD8 T cell response against HBcAg. + Further increased T cell responses: after MVA boost, HBc-specific CD8 T cells were detected in ChAd155-hIi-HBV-primed mice versus ChAd155-HBV-primed mice. + Higher frequencies of T cells were observed, while HBs-specific CD8 + The T cell response was not further enhanced.

[0218] When administered to HLA.A2 / DR1 transgenic mice, the complete vaccination regimen (i.e., sequential or simultaneous administration of viral vector and adjuvanted protein) resulted in robust CD4 expression against both vaccine antigens. + T cells, CD8 + In addition, vaccine-induced HBs- and HBc-specific CD4 + and CD8 + T cells were detected in the livers of animals vaccinated with both vaccine regimens.

[0219] Immunogenicity studies were performed in CB6F1 to investigate T and B cell tolerance to the "invariant chain" sequence (Ii) in an allogeneic model using the ChAd155 construct: ChAd155-mIi-HBV, encoding the murine Ii sequence (mIi). The induction of autologous mIi-specific immune responses was evaluated using a high dose (10 9 The HBV-specific immune response was assessed after two immunizations (days 0 and 14) with the ChAd155-mIi-HBV vector (vp). Two weeks after the first or second immunization, no anti-mIi antibodies or mIi-specific T cells were detected in any of the animals, suggesting that immune tolerance to the mIi sequence was maintained.

[0220] In a preclinical HBV persistent mouse model (AAV2 / 8-HBV transduced HLA.A2 / DR1 mice) in which immune tolerance to HBV antigens is observed, a vaccine regimen can break tolerance and induce HBc- and HBs-specific CD8 + T cells, HBs-specific CD4 + T cell and antibody responses against both HBsAg and HBcAg were induced (HBc-specific CD4 + (Though no T cell responses were observed). However (and as expected), the level of vaccine-induced responses in AAV-transduced mice was lower than that detected in naive HLA.A2 / DR1 mice. Furthermore, vaccine-associated liver inflammation was assessed by measuring serum activity of aspartate aminotransferase (AST) and ALT and performing liver histopathological evaluation; however, no increase in liver enzymes was detected in the vaccine group compared with the non-vaccinated group, and none of the microscopic findings could be related to vaccine treatment. Taken together, these results demonstrate that the tested vaccine candidate induced HBs- and HBc-specific antibodies and CD8+ expression levels without the detection of relevant signs of liver changes under these experimental conditions. + T cell responses and HBs-specific CD4 + 1 shows that T cell responses were successfully restored.

[0221] Example 7 - Immunogenicity evaluation of various adjuvanted recombinant protein HBc / HBs ratios the purpose The purpose of the experiment was to measure HBc-induced CD4 as seen in Example 2 (HBsAg and HBcAg were mixed in a 1:1 ratio) 7 days after the third immunization. + The objective of this study was to confirm the negative interference of HBsAg on T cell responses. A further objective was to limit this interference and to prevent the development of at least potent HBc-specific CD4 + To ensure a T cell response (while simultaneously generating robust HBc- and HBs-specific antibody responses), various ratios of HBs / HBc were evaluated.

[0222] research design Six to eight-week-old CB6F1 mice (30 mice per group) were treated with 50 μl of AS01 B-3 or AS01 B-4 Mice were immunized three times intramuscularly (gastrocnemius) on days 0, 14, and 28 with various formulations (listed in Table 8) containing HBc and HBs antigens in the immunized mice. CB6F1 mice were randomly assigned to one of the study groups. Evaluation of HBc- and HBs-specific T cell responses by intracellular cytokine staining (ICS) was performed using leukocytes collected 7 days after the second and third immunizations from six pools of five mice per group. Serum was collected from individual mice 14 days after the second and third immunizations, and as a result of statistical sample size analysis, only sera from 20 randomized mice were tested for evaluation of HBc- and HBs-specific antibody total Ig responses.

[0223] [Table 8]

[0224] statistical analysis Non-inferiority of the HBc-HBs group compared with the corresponding HBc group was evaluated. HBc-specific CD4+ cells expressing at least one cytokine (IL-2 and / or IFN-γ and / or TNF-α) for the HBc group versus the corresponding HBc-HBs group were evaluated. + This non-inferiority is reached if the UL with 95% CI of the geometric mean ratio of T cell frequencies (%) is less than 2 7 days after Dose III. As this was an initial assessment and the criteria were not predefined, no adjustment for multiplicity was applied.

[0225] Two primary objectives were answered using an ANOVA (analysis of variance) model. This model included group (4–12) as a fixed effect, interactions, and a heteroscedastic model (identical variances between groups were not assumed). The log10 CD4 + Frequencies were fitted. Using this model, geometric means (and their 95% CI) and geometric mean ratios and their 95% CI were estimated using inverse log10 transformations of the means and differences.

[0226] result Antigen-specific CD4+ T cell response All formulations showed potent anti-HBc and HBs-specific CD4 responses after the second immunization. + Induced T cell responses (±1%). B-4 HBc-specific CD4 induced by a preparation containing equal amounts of HBc and HBs + The magnitude of the T cell response was B-4 There was a tendency for the levels to be lower when compared to the single group (Figure 23), but these differences were not statistically significant as the ratio was ±1.4 (p=0.143) (Figure 23). B-4 HBc-specific CD4 induced by a preparation containing equal amounts of HBc and HBs + The magnitude of the T cell response was B-4 When compared with the HBc-specific CD4 monotherapy group, the GMR was statistically lower (GMR 0.391 and 95% CI [0.184-0.828]) (Figure 24). + The observed interference of HBs on T cell responses was confirmed. When the antigens were formulated at a 4:1 ratio of HBc and HBs antigens, the interference was less pronounced. HBs-specific CD4 + No such negative effects were observed when looking at T cell responses (Figures 23 and 24).

[0227] By further increasing the HBc-to-HBs ratio, such HBc-specific CD4 + There was a trend (although not statistically significant) towards a more restored T cell response, with total HBc-specific CD4 + The median T cell count was up to 1.7%.

[0228] HBs-specific CD8 + T cell responses were evaluated after the second and third immunizations (Figure 25). HBs-specific dose-ranging responses were observed after the second and third immunizations. After the third immunization, HBs-specific CD8 +T cell responses tended to be reduced when co-formulated with an equal amount of HBcAg (GMR 0.66 and 95% CI [0.337-1.295]), but this ratio was not statistically significant (p=0.1717). + T cell responses were low to undetectable (less than 0.1%, data not shown).

[0229] antigen-specific antibody response All formulations were AS01 after the second immunization. B-4 Co-formulation of HBs and HBc at a 1:1 ratio in the adjuvant system induced high and similar anti-HBc and HBs total Ig responses without the negative effects (Figure 26). Anti-HBc-specific total Ig responses were boosted after the third immunization (Figure 27). AS01 B-4 When HBs and HBc were co-formulated in a 1:1 ratio in the adjuvant system, HBs interference was observed on the level of HBc-specific antibody responses. The GMT ratio and 95% CI were 1.88 [1.44; 2.44], with a p value of <0.0001. Increasing the HBc-to-HBs ratio by 4 allowed for recovery of the HBc humoral response; the GMT ratio and 95% CI were 1.37 [1.04; 1.80], with a p value of 0.0262. As previously reported, HBc did not negatively affect the level of HBs-specific antibody responses (Figures 26 and 27).

[0230] conclusion The results of these experiments demonstrate that the HBc-specific CD4 + It was shown that the negative interference of HBs on T cell and humoral responses was overcome for formulations with an HBc / HBs ratio of ≥ 4. Consequently, a dose of 4 μg HBc and 1 μg HBs was selected for further preclinical studies. The present application provides the following: 1. An immunogenic composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector containing a polynucleotide encoding hepatitis B surface antigen (HBs), a polynucleotide encoding hepatitis B virus core antigen (HBc), and a polynucleotide encoding human invariant chain (hIi) fused to HBc. 2. The immunogenic composition described in 1 above, wherein the ChAd vector is selected from the group consisting of ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 and Pan 9. 3. The immunogenic composition according to 1 or 2 above, wherein the ChAd vector is ChAd155 and comprises polynucleotides encoding hIi, HBc, 2A and HBc. 4. The vector comprises a polynucleotide encoding an HBc comprising an amino acid sequence of SEQ ID NO: 11 or at least 98% homologous thereto, fused to a polynucleotide encoding an hIi comprising an amino acid sequence of SEQ ID NO: 7 or at least 98% homologous thereto, or an amino acid sequence of SEQ ID NO: 12 or at least 98% homologous thereto, and an HBs comprising an amino acid sequence of SEQ ID NO: 1 or at least 98% homologous thereto; The polynucleotides encoding HIi-HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3 incorporating the 2A cleavage region of foot-and-mouth disease virus or an amino acid sequence at least 98% homologous thereto. The immunogenic composition according to any one of 1 to 3 above. 5. The immunogenic composition according to any one of 1 to 4 above, wherein the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 9 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 15. 6. The immunogenic composition according to any one of 1 to 5 above, wherein the vector comprises the nucleotide sequence shown in SEQ ID NO: 10 or the nucleotide sequence shown in SEQ ID NO: 14. 7. An immunogenic composition comprising a modified vaccinia virus Ankara (MVA) vector containing a polynucleotide encoding hepatitis B surface antigen (HBs) and a polynucleotide encoding hepatitis B virus core antigen (HBc). 8. The immunogenic composition according to claim 7, wherein the polynucleotides encoding HBs and HBc are separated by a polynucleotide encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV). 9. The immunogenic composition described in 7 or 8 above, wherein the vector comprises a polynucleotide encoding HBc comprising SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto, and HBs comprising SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto, and the polynucleotides encoding HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto, which incorporates the 2A cleavage region of foot-and-mouth disease virus. 10. The immunogenic composition according to any one of 7 to 9 above, wherein the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:5. 11. The immunogenic composition according to any one of 7 to 10 above, wherein the vector comprises the polynucleotide sequence shown in SEQ ID NO:6. 12. An immunogenic composition comprising a recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant containing MPL and QS-21. 13. The immunogenic composition according to claim 12, wherein the truncated recombinant HBc comprises amino acids 1 to 145, 1 to 146, 1 to 147, 1 to 148 or amino acids 1 to 149 of the wild-type hepatitis B core antigen protein. 14. The immunogenic composition according to paragraph 12 or 13 above, comprising recombinant HBc and recombinant HBs in a ratio of 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1 or more (e.g., 3:1 to 5:1, e.g., 4:1). 15. The immunogenic composition according to any one of 12 to 14 above, comprising recombinant HBs (SEQ ID NO: 1), amino acids 1 to 149 of HBc (SEQ ID NO: 2), and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in phosphate-buffered saline solution. 16. The following: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant. 10. An immunogenic combination comprising: 17. The immunogenic combination according to claim 16, wherein the composition of component a) of the combination is a composition described in any one of claims 1 to 6 above, the composition of component b) of the combination is a composition described in any one of claims 7 to 11 above, and the composition of component c) of the combination is a composition described in any one of claims 12 to 15 above. 18. The following components: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and c) A composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant. together with instructions for administering the components sequentially or simultaneously for the treatment of CHB.

[0231] Sequence Listing SEQ ID NO: 1: Amino acid sequence of HBs TIFF0007795861000009.tif21167

[0232] SEQ ID NO: 2: Amino acid sequence of HBc truncation TIFF0007795861000010.tif13166

[0233] SEQ ID NO: 3: Amino acid sequence of the spacer incorporating the 2A cleavage region of foot-and-mouth disease virus TIFF0007795861000011.tif757

[0234] SEQ ID NO: 4: Nucleotide sequence encoding a spacer incorporating the 2A cleavage region of foot-and-mouth disease virus TIFF0007795861000012.tif6166

[0235] SEQ ID NO: 5: Amino acid sequence of HBc-2A-HBs TIFF0007795861000013.tif38167

[0236] SEQ ID NO: 6: Nucleotide sequence encoding HBc-2A-HBs TIFF0007795861000014.tif53167TIFF0007795861000015.tif72166

[0237] SEQ ID NO: 7: Amino acid sequence of hIi TIFF0007795861000016.tif24167

[0238] SEQ ID NO: 8: Nucleotide sequence encoding hIi TIFF0007795861000017.tif52166

[0239] SEQ ID NO: 9: Amino acid sequence of hIi-HBc-2A-HBs TIFF0007795861000018.tif46166TIFF0007795861000019.tif13166

[0240] SEQ ID NO: 10: Nucleotide sequence encoding hIi-HBc-2A-HBs TIFF0007795861000020.tif188166

[0241] SEQ ID NO: 11: Amino acid sequence of HBc TIFF0007795861000021.tif19166

[0242] SEQ ID NO: 12: Amino acid sequence of hIi alternative variant TIFF0007795861000022.tif25166

[0243] SEQ ID NO: 13: Nucleotide sequence encoding hI alternative variant TIFF0007795861000023.tif66167

[0244] SEQ ID NO: 14: Alternative nucleic acid sequence of hIi-HBc-2A-HBs TIFF0007795861000024.tif113167TIFF0007795861000025.tif73169

[0245] SEQ ID NO: 15: Alternative amino acid sequence of hIi-HBc-2A-HBs TIFF0007795861000026.tif64168

[0246] References: TIFF0007795861000027.tif243169TIFF0007795861000028.tif245170TIFF0007795861000029.tif237169TIFF0007795861000030.tif166168

Claims

1. 1. An immunogenic composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs), a polynucleotide encoding a hepatitis B core antigen (HBc), and a polynucleotide encoding a human invariant chain (hIi) fused to HBc, wherein the ChAd vector is selected from the group consisting of ChAd3, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7, and Pan 9; the vector comprises a polynucleotide encoding an HBc comprising an amino acid sequence of SEQ ID NO: 11 or at least 98% homologous thereto, fused to a polynucleotide encoding an hIi comprising an amino acid sequence of SEQ ID NO: 7 or at least 98% homologous thereto, or an amino acid sequence of SEQ ID NO: 12 or at least 98% homologous thereto, and an HBs comprising an amino acid sequence of SEQ ID NO: 1 or at least 98% homologous thereto; the polynucleotides encoding hIi-HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3 incorporating the 2A cleavage region of foot-and-mouth disease virus or an amino acid sequence at least 98% homologous thereto; Immunogenic composition.

2. The immunogenic composition of claim 1, wherein the ChAd vector is ChAd155 and comprises polynucleotides encoding hIi, HBc, 2A, and HBs.

3. The immunogenic composition of claim 1 or 2, wherein the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 9 or a polynucleotide encoding the amino acid sequence of SEQ ID NO:

15.

4. The immunogenic composition according to any one of claims 1 to 3, wherein the vector comprises the nucleotide sequence shown in SEQ ID NO: 10 or the nucleotide sequence shown in SEQ ID NO:

14.

5. 1. An immunogenic composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs) and a polynucleotide encoding a hepatitis B virus core antigen (HBc), wherein the polynucleotides encoding HBs and HBc are separated by a polynucleotide encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV); The vector comprises a polynucleotide encoding HBc comprising an amino acid sequence of SEQ ID NO: 11 or at least 98% homologous thereto, and HBs comprising an amino acid sequence of SEQ ID NO: 1 or at least 98% homologous thereto, and the polynucleotides encoding HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3 or at least 98% homologous thereto, which incorporates the 2A cleavage region of foot-and-mouth disease virus. Immunogenic composition.

6. The immunogenic composition of claim 5, wherein the vector comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:

5.

7. The immunogenic composition of claim 5 or 6, wherein the vector comprises the polynucleotide sequence shown in SEQ ID NO:

6.

8. An immunogenic composition comprising a recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant containing MPL and QS-21, wherein the ratio of recombinant HBc to recombinant HBs is 4:1 or greater.

9. 9. The immunogenic composition of claim 8, wherein the truncated recombinant HBc comprises amino acids 1-145, 1-146, 1-147, 1-148, or 1-149 of a wild-type Hepatitis B Core Antigen protein.

10. 10. The immunogenic composition of claim 8 or 9, comprising recombinant HBs (SEQ ID NO: 1), amino acids 1 to 149 of HBc (SEQ ID NO: 2), and an adjuvant comprising MPL, QS-21, and liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in phosphate buffered saline.

11. below: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs) and a nucleic acid encoding a hepatitis B core antigen (HBc), wherein the ChAd vector is selected from the group consisting of ChAd3, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 and Pan 9, and the vector comprises polynucleotides encoding HBc comprising SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto, and HBs comprising SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto, fused to a polynucleotide encoding an hIi comprising SEQ ID NO: 7 or an amino acid sequence at least 98% homologous thereto, or SEQ ID NO: 12 or an amino acid sequence at least 98% homologous thereto, wherein the polynucleotides encoding hIi-HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto, which incorporates the 2A cleavage region of foot-and-mouth disease virus; b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs) and a nucleic acid encoding a hepatitis B core antigen (HBc), wherein the polynucleotides encoding HBs and HBc are separated by a polynucleotide encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV), the vector comprising polynucleotides encoding HBc comprising SEQ ID NO: 11, or an amino acid sequence at least 98% identical thereto, and HBs comprising SEQ ID NO: 1, or an amino acid sequence at least 98% identical thereto, wherein the polynucleotides encoding HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3, or an amino acid sequence at least 98% identical thereto, which incorporates the 2A cleavage region of foot-and-mouth disease virus; and c) a composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant, wherein the ratio of recombinant HBc to recombinant HBs is 4:1 or greater; 10. An immunogenic combination comprising:

12. 12. The immunogenic combination of claim 11, wherein the composition of component a) of the combination is a composition according to any one of claims 1 to 4, the composition of component b) of the combination is a composition according to any one of claims 5 to 7, and the composition of component c) of the combination is a composition according to any one of claims 8 to 10.

13. Components include: a) a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs) and a nucleic acid encoding a hepatitis B core antigen (HBc), wherein the ChAd vector is selected from the group consisting of ChAd3, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 and Pan 9, and the vector comprises polynucleotides encoding HBc comprising SEQ ID NO: 11 or an amino acid sequence at least 98% homologous thereto, fused to a polynucleotide encoding an hIi comprising SEQ ID NO: 7 or an amino acid sequence at least 98% homologous thereto, or SEQ ID NO: 12 or an amino acid sequence at least 98% homologous thereto, and HBs comprising SEQ ID NO: 1 or an amino acid sequence at least 98% homologous thereto, wherein the polynucleotides encoding hIi-HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3 or an amino acid sequence at least 98% homologous thereto, which incorporates the 2A cleavage region of foot-and-mouth disease virus; b) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding a hepatitis B surface antigen (HBs) and a nucleic acid encoding a hepatitis B core antigen (HBc), wherein the polynucleotides encoding HBs and HBc are separated by a polynucleotide encoding a 2A cleavage region of foot-and-mouth disease virus (FMDV), the vector comprising polynucleotides encoding HBc comprising SEQ ID NO: 11, or an amino acid sequence at least 98% identical thereto, and HBs comprising SEQ ID NO: 1, or an amino acid sequence at least 98% identical thereto, wherein the polynucleotides encoding HBc and HBs are separated by a polynucleotide encoding an amino acid sequence comprising SEQ ID NO: 3, or an amino acid sequence at least 98% identical thereto, which incorporates the 2A cleavage region of foot-and-mouth disease virus; and c) a composition comprising a recombinant hepatitis B surface antigen (HBs), a recombinant hepatitis B core antigen (HBc), and an adjuvant, wherein the ratio of recombinant HBc to recombinant HBs is 4:1 or greater; together with instructions for administering the components sequentially or simultaneously for the treatment of CHB.

Images