Hepatitis B immune regimens and compositions

Abstract

The present invention relates to hepatitis B immune regimens and compositions. The present invention provides a method of treating chronic hepatitis B infection (CHB) in a human comprising the steps of: a) administering to the human a composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector, the replication-deficient chimpanzee adenovirus (ChAd) vector comprises a polynucleotide encoding a hepatitis B surface antigen (HBs) and a nucleic acid encoding a hepatitis B virus core antigen (HBc); b) administering to the human a composition comprising a modified vaccinia virus Ankara (MVA) vector, the 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 virus core antigen (HBc) and an adjuvant.

Description

This application is a divisional application of Chinese patent application 201880089468.9 “Hepatitis B immunization regimen and composition” with a filing date of December 14, 2018. Technical Field

[0001] The present invention relates to an immunization regimen particularly suitable for treating chronic hepatitis B, a method for treating chronic hepatitis B, and a composition for use in such regimens and methods. The regimens and methods involve administering a composition comprising a vector for delivering a hepatitis B antigen and a composition comprising a recombinant hepatitis B antigen protein. Background Art Hepatitis B virus (HBV) infection is a major public health problem. Approximately 257 million people are infected with HBV worldwide [WHO, 2017]. The clinical course and outcome of HBV infection depend largely on the age at infection and the complex interaction between viral and host immune responses [Ott, 2012; Maini, 2016]. Thus, exposure to HBV may result in acute hepatitis, which resolves spontaneously or may progress to various forms of chronic infection, including inactive hepatitis B surface antigen (HBsAg) carrier state, chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [Liaw, 2009]. The prevalence of HBsAg in the adult population is >2%, with rates of 5-8% in Southeast Asia and China and >8% in Africa. 15-40% of people with chronic hepatitis B infection (defined as serum HBsAg detectable for more than 6 months) will develop hepatic sequelae, with cirrhosis (LC), hepatic decompensation, and HCC being the major complications.

[0003] Although implementation of universal prophylactic hepatitis B immunization in infants has been highly effective in reducing the incidence and prevalence of hepatitis B in many endemic countries, it has not yet led to a strong reduction in the prevalence of chronic hepatitis B infection (CHB) in adolescents and adults, and is not expected to affect HBV-related deaths until decades after its introduction. In 2015, hepatitis B caused 887,000 deaths, mainly from cirrhosis and liver cancer [WHO, 2017].

[0004] Clinical management of chronic hepatitis B aims to improve survival and quality of life by preventing disease progression and, therefore, 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 cornerstone endpoint for all current treatment modalities. Achieving loss of (detectable) hepatitis B e-antigen (HBeAg) is another valuable biomarker, however, HBsAg loss is generally considered the best endpoint representing a “functional cure” as it indicates a profound suppression of HBV replication and viral protein expression, regardless of the presence of anti-HBs seroconversion [Block, 2017; Cornberg, 2017]. Currently, there are two main treatment options for patients with CHB: either by pegylated interferon α (PegIFNα) or by nucleoside (nucleotide) analogs (NA) [EASL, 2017]. PegIFNα, which aims to induce long-term immune control with limited duration treatment, can achieve sustained off-treatment control, but durable virological responses and hepatitis B surface antigen (HBsAg) loss are limited to a small fraction of patients. In addition, a significant number of patients are not suitable for this type of treatment due to its poor tolerability and long-term safety concerns.

[0005] NA works by inhibiting DNA replication by inhibiting HBV polymerase reverse transcriptase activity. NAs approved for HBV treatment in Europe include entecavir (ETV), tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) (which are associated with a high barrier to HBV resistance), as well as lamivudine (LAM), adefovir dipivoxil (ADV) and telbivudine (TBV) (which are associated with a low barrier to HBV resistance). The main advantage of treating with an effective NA with a high barrier to resistance is its predictable long-term high antiviral efficacy (leading to HBV DNA suppression in the vast majority of compliant patients) and its favorable safety profile. The disadvantage of NA treatment is its long-term treatment regimen, because NA usually does not achieve HBV eradication, and NA discontinuation can lead to HBV recurrence [Kranidioti, 2015]. HBsAg loss, representing functional cure, is now the gold standard treatment endpoint in CHB [Block, 2017; Cornberg, 2017], however, it is rarely achieved with NA treatment [Zoutendijk, 2011].

[0006] Due to the low HBsAg seroclearance rate [Zoutendijk, 2011] and the high risk of viral relapse outside of NA [Kranidioti, 2015], most patients are maintained on long-term or even unlimited NA therapy, which may be associated with reduced patient compliance to therapy, increased financial costs, and increased risk of drug toxicity and resistance mutations after long-term exposure [Terrault, 2015]. Therefore, new strategies are necessary to supplement NA therapy to achieve a “functional cure” with limited regimens.

[0007] New therapeutic strategies currently being explored include novel antiviral strategies as well as novel immunotherapeutic strategies that enhance HBV-specific adaptive immune responses or activate innate intrahepatic immunity [Durantel, 2016]. To date, none of these experimental treatments have been shown to be effective. None of the vaccination strategies evaluated has been able to induce robust multifunctional CD8 +T-cell responses are essential for restoring immune control of the virus [Lau, 2002; Li, 2011; Liang, 2011; Bertoletti, 2012; Boni, 2012]. Early efforts with recombinant vaccines based on HBV surface and / or PreS antigens initially induced antibody responses but no HBV-specific CD8+ T-cell responses, with no clinical or virological benefit [Jung, 2002; Vandepapelière, 2007]. DNA vaccines expressing the HBV envelope were unable to restore T-cell responses specific for HBsAg and HBcAg and therefore did not reduce the risk of relapse in patients after NA cessation [Fontaine, 2015]. Using a new delivery system, DNA vaccines encoding S, preS1 / S2 (priming vaccines) and MVA viral vector vaccines (boosting vaccines) did not show a reduction in T-cell induction or viremia, indicating that HBV PreS and surface antigens alone are not sufficient to cure patients [Cavenaugh, 2011]. More recently, vaccine strategies and novel delivery systems targeting multiple HBV antigens have been investigated. Recombinant HBsAg / HBcAg vaccines resulted in a reduction in viral load to very low levels (i.e., ~50 IU / ml) in only half of the patients [Al-Mahtab, 2013]. DNA vaccines encoding S, preS1 / S2, core, polymerase, and X proteins together with genetically adjuvanted IL-12 together with lamivudine induced multispecific T cell responses and >2 log10 reductions in viral load in half of the patients. However, no changes in quantitative detection of HBsAg, loss of HBsAg, or HBsAg seroconversion were observed in any patient [Yang, 2012]. GS-4774 vaccine, a yeast-based T cell vaccine expressing large S, core, and X proteins of HBV, did not provide significant reductions in HBsAg in virally suppressed CHB patients [Lok, 2016].

[0008] There remains an unmet need for treatments that can clear HBsAg to allow patients to safely discontinue NA therapy without virological or clinical relapse. Summary of the invention In one aspect, a method of treating chronic hepatitis B infection (CHB) in a human is provided, comprising the steps of: a) administering to said 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 core antigen (HBc); b) administering to said human 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) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0010] In one embodiment, the steps of the method are performed sequentially, wherein step a) precedes step b), and step b) precedes step c). Optionally, step c) may be repeated. In another embodiment, step c) is performed simultaneously with step a) and / or with step b).

[0011] Thus, in another aspect, there is provided a method of treating chronic hepatitis B infection (CHB) in a human comprising the steps of: a) administering to said 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 virus core antigen (HBc), and, simultaneously, ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; and b) administering to said 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 virus core antigen (HBc), and, simultaneously, a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0012] In one embodiment, the steps of the method are performed sequentially, wherein step a) precedes step b). Optionally, step b) may be repeated.

[0013] In another aspect, an immunogenic combination product is provided for use in a method of treating chronic hepatitis B infection (CHB) in a human, the immunogenic combination product 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 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 recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, wherein the method comprises administering the composition to the human sequentially or simultaneously.

[0014] In another aspect, an immunogenic composition is provided for use in a method for treating chronic hepatitis B infection (CHB) in a human, the immunogenic composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs), a nucleic acid encoding hepatitis B virus core antigen (HBc), and a nucleic acid encoding a human constant chain (hIi) fused to the HBc, wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In certain embodiments, the immunogenic composition in the method for treating chronic CHB further comprises one or more recombinant HBV protein antigens.

[0015] In a further aspect, an immunogenic composition is provided for use in a method for treating chronic hepatitis B infection (CHB) in a human, the immunogenic 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), wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In certain embodiments, the immunogenic composition in the method for treating chronic CHB further comprises one or more recombinant HBV protein antigens.

[0016] In a further aspect, an immunogenic composition is provided for use in a method for treating chronic hepatitis B infection (CHB) in a human, the immunogenic composition comprising recombinant hepatitis B surface antigen (HBs), a C-terminally truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant containing MPL (3-D monophosphoryl lipid A) and QS-21 (a triterpene glycoside purified from the bark of a soap tree (Quillaja saponaria)), wherein the method comprises administering the composition in a prime-boost regimen with at least one other immunogenic composition. In certain embodiments, the immunogenic composition in the method for treating chronic CHB further comprises one or more vectors encoding one or more HBV antigens. The present invention relates to the following scheme: 1. A method for treating chronic hepatitis B infection (CHB) in a human, comprising the steps of: a) administering to said 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 core antigen (HBc); b) administering to said human 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) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant. 2. The method according to scheme 1, wherein the steps of the method are implemented sequentially, wherein step a) precedes step b), and step b) precedes step c). 3. The method according to scheme 2, wherein step c) of the method is repeated. 4. A method according to any preceding embodiment, wherein the time period between each step is 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 6 months or 12 months, such as 4 weeks or 8 weeks. 5. The method according to Scheme 1, wherein step c) is performed simultaneously with step a) and / or with step b). 6. A method for treating chronic hepatitis B infection (CHB) in a human, comprising the steps of: a) administering to said 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 virus core antigen (HBc), and, simultaneously, ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; and b) administering to said 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 virus core antigen (HBc), and, simultaneously, a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant. 7. An immunogenic combination product for use in a method of treating chronic hepatitis B infection (CHB) in humans, the immunogenic combination product 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 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 recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, wherein the method comprises administering the composition to the human sequentially or simultaneously. 8. An immunogenic composition for use in a method for treating chronic hepatitis B infection (CHB) in humans, the immunogenic composition comprising a replication-defective chimpanzee adenovirus (ChAd) vector, the replication-defective chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs), a nucleic acid encoding hepatitis B virus core antigen (HBc), and a nucleic acid encoding a human constant chain (hIi) fused to the HBc, wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. 9. The immunogenic composition used according to Scheme 7, further comprising one or more recombinant HBV protein antigens. 10. An immunogenic composition for use in a method of treating chronic hepatitis B infection (CHB) in humans, the 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 virus core antigen (HBc), wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. 11. The immunogenic composition for use according to Scheme 9, further comprising one or more recombinant HBV protein antigens. 12. An immunogenic composition for use in a method for treating chronic hepatitis B infection (CHB) in humans, the immunogenic composition comprising recombinant hepatitis B surface antigen (HBs), C-terminally truncated recombinant hepatitis B virus core antigen (HBc) and an adjuvant comprising MPL and QS-21, wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. 13. An immunogenic composition for use according to Scheme 12, wherein the ratio of HBc to HBs in the composition is greater than 1. 14. An immunogenic composition for use according to Scheme 13, wherein the ratio of HBc to HBs in the composition is 4:1. 15. The immunogenic composition for use according to any one of schemes 12 to 14, further comprising one or more vectors encoding one or more HBV antigens. 16. Use of an immunogenic composition in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in humans, the immunogenic composition comprising a replication-defective chimpanzee adenovirus (ChAd) vector, the replication-defective chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs), a nucleic acid encoding hepatitis B virus core antigen (HBc), and a nucleic acid encoding human invariant chain (hIi) fused to the nucleic acid encoding HBc, wherein the method for treating chronic hepatitis B infection comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. 17. Use of an immunogenic composition in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in humans, the immunogenic composition comprising a modified vaccinia virus Ankara (MVA) vector, the 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), wherein the method for treating chronic hepatitis B infection comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. 18. Use of an immunogenic combination product in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in humans, the immunogenic combination product 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 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 recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, wherein the method for treating chronic hepatitis B infection comprises administering the composition to the human sequentially or simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 – HBc-specific (A) and HBs-specific (B) CD8 14 days after primary immunization with ChAd155-HBV (with and without hIi) and 7 days after booster immunization with MVA-HBV + T-cell responses (represented individu- al animals with median values).

[0018] Figure 2 - HBc-specific antibody responses (represented from individual animals with geometric mean titers (GMTs)) 14 days after primary immunization with ChAd155-HBV (with and without hIi) and 7 days after booster immunization with MVA-HBV.

[0019] Figure 3 - in 50 μl AS01 B-4 HBc and HBs-specific CD4 7 days after the third dose of NaCl, HBc, HBs or HBc-HBs formulated in + T-cell responses (represents pool of 5 animals / group with median values).

[0020] Figure 4 - in 50 μl AS01 B-4 HBs-specific CD8 7 days after the third administration of NaCl, HBc, HBs or HBc-HBs formulated in + T-cell responses (represents pool of 5 animals / group with median values).

[0021] Figure 5 - in 50 μl AS01 B-4 Anti-HBc and anti-HBs antibody responses 14 days after the third dose of NaCl, HBc, HBs, or HBc-HBs formulated in (represents individual animals with geometric means and 95% CI).

[0022] Figure 6 - In NaCl, HBc-HBs, HBc-HBs plus alum, HBc-HBs plus AS01 B-4 or HBc-HBs plus AS01 E-4 HBs- (A) and HBc- (B) specific CD4+ and HBs-specific CD8+ (C) T-cell responses 7 days after the third dose of (represents the pool of 5 animals / group with median value).

[0023] Figure 7 - In NaCl, HBc-HBs, HBc-HBs plus alum, HBc-HBs plus AS01 B-4 or HBc-HBs plus AS01 E-4HBs-(A) and HBc-(B) specific antibody responses 14 days after the third dose of HBs-(A) and HBc-(B) (represents individual animals with geometric means and 95% CI).

[0024] Figure 8 - HBc-(A) and HBs-(B) specific CD8 7 days after the second and fourth administration of NaCl, heterologous vector prime-boost with subsequent recombinant proteins or heterologous vector prime-boost with simultaneous recombinant proteins + T-cell responses (individual animals with median values). Group 1: mice immunized with 108 vp ChAd155-hIi-HBV on day 0, 107 pfu MVA-HBV on day 28, and HBc-HBs 4-1 / AS01B-4 on days 42 and 56. At 7 dp IV, one data point was lost due to a technical problem. Group 2: mice immunized with 108 vp ChAd155-hIi-HBV+HBc-HBs 4-1 / AS01B-4 on day 0 and 107 pfu MVA-HBV+HBc-HBs 4-1 / AS01B-4 on days 28, 42, and 56. Group 3: mice injected with NaCl on days 0, 28, 42, and 56.

[0025] Fig. 9 - HBc-(A) or HBs-(B) specific CD4 7 days after the second and fourth administration of NaCl, prime-boost with subsequent heterologous vector of recombinant protein or prime-boost with simultaneous heterologous vector of recombinant protein + T-cell responses (individual animals with median values). Group 1: mice immunized with 108 vp ChAd155-hIi-HBV on day 0, 107 pfu MVA-HBV on day 28, and HBc-HBs 4-1 / AS01B-4 on days 42 and 56. At 7 dp IV, one data point was lost due to a technical problem. Group 2: mice immunized with 108 vp ChAd155-hIi-HBV+HBc-HBs 4-1 / AS01B-4 on day 0 and 107 pfu MVA-HBV+HBc-HBs 4-1 / AS01B-4 on days 28, 42, and 56. Group 3: mice injected with NaCl on days 0, 28, 42, and 56.

[0026] Fig.10- HBc- and HBs-specific CD4 in liver-infiltrating lymphocytes 7 days after the fourth dose in NaCl, prime-boost with subsequent heterologous vector of recombinant protein or prime-boost with simultaneous heterologous vector of recombinant protein + (A) and CD8 + (B) T-cells (pooled from 3 or 4 animals with a median value). Group 1: mice immunized with 108 vp ChAd155-hIi-HBV on day 0, 107 pfu MVA-HBV on day 28, and HBc-HBs 4-1 / AS01B-4 on days 42 and 56. At 7 dp IV, one data point was lost due to a technical problem. Group 2: mice immunized with 108 vp ChAd155-hIi-HBV+HBc-HBs 4-1 / AS01B-4 on day 0 and 107 pfu MVA-HBV+HBc-HBs 4-1 / AS01B-4 on days 28, 42, and 56. Group 3: mice injected with NaCl on days 0, 28, 42, and 56.

[0027] Fig.11 - HBc-specific (A) and HBs-specific (B) antibody responses after a prime-boost vaccine regimen (represents individual animals with geometric means). Group 1: On day 0, 10 8 vp ChAd155-hIi-HBV immunization, 10 7 pfu MVA-HBV immunization, HBc-HBs 4-1 / AS01 on days 42 and 56 B-4 Immunized mice. Group 2: On day 0, 10 8 vpChAd155-hIi-HBV+HBc / HBs 4-1 / AS01 B-4 Immunization, 10 on days 28, 42, and 56 7 pfu MVA-HBV+HBc-HBs 4-1 / AS01 B-4 Immunized mice. Group 3: mice injected with NaCl on days 0, 28, 42, and 56.

[0028] Fig.12 – PBS or ChAd155-mIi-HBV vector (10 9 vp) at 2 weeks after the first or second injection of mIi-, HBc-, and HBs-specific IFNγ ELISpot responses. ChAd155-mIi-HBV (10 9 mIi-, HBc- and HBs-specific IFN-γ ELISpot responses elicited by 2 administrations of 5 animals / group / time point. The red dashed line is the cutoff value of the assay.

[0029] Fig.13 - ChAd155-mIi-HBV (10 9 vp) elicited anti-mIi antibody responses (ELISA). ChAd155-mIi-HBV (10 9 vp) (representative of 10 animals after the first injection and 5 animals after the second injection).

[0030] Fig.14 - HBc-specific spleen (A) or liver (B) CD8+ T cells (individual animals with median values) 7 days after the second dose and 7 days after the fourth dose in NaCl, prime-boost with a heterologous vector of subsequent recombinant protein or prime-boost with a heterologous vector of simultaneous recombinant protein.

[0031] Fig.15 - HBc-specific spleen (A) or liver (B) CD4+ T cells (individual animals with median values) 7 days after the second dose and 7 days after the fourth dose in NaCl, prime-boost with a heterologous vector of subsequent recombinant protein or prime-boost with a heterologous vector of simultaneous recombinant protein.

[0032] Fig.16 - HBs-specific spleen (A) or liver (B) CD8+ T cells (individual animals with median values) 7 days after the second dose and 7 days after the fourth dose in NaCl, prime-boost with a heterologous vector of subsequent recombinant protein or prime-boost with a heterologous vector of simultaneous recombinant protein.

[0033] Fig.17 - HBs-specific spleen (A) or liver (B) CD4+ T cells (individual animals with median values) 7 days after the second and fourth doses of NaCl, prime-boost with subsequent heterologous vectors of recombinant proteins or prime-boost with simultaneous heterologous vectors of recombinant proteins.

[0034] Fig.18- Anti-HBs (A) and anti-HBc (B) binding antibody responses on Days 23, 65 and 93 (before dosing, in NaCl, 7 days after the second dose prime-boost with subsequent recombinant protein or prime-boost with simultaneous recombinant protein).

[0035] Fig.19 - AST (A) and ALT (B) levels measured in serum from mice (Groups 1, 2, 3 and 4) on days 38, 65 and 93 (7 days after the first, second and fourth administration of NaCl, prime-boost with subsequent heterologous vector of recombinant protein or prime-boost with heterologous vector of simultaneous recombinant protein, Groups 1, 2, 3) or on day 93 (Group 4). ALT and AST levels were measured in 21 animals / group on days 38 and 65, and in 12 animals from Group 1, in 11 animals from Groups 2 and 3 and in 8 animals from Group 4 on day 93. * represents statistical significance.

[0036] Fig. 20 - HBs antigen levels in sera from AAV2 / 8-HBV injected mice before dosing, 7 days after the second dose in NaCl, prime-boost with subsequent heterologous vectors of recombinant proteins or prime-boost with heterologous vectors of simultaneous recombinant proteins, and 7 days after the fourth dose.

[0037] Fig.21 -Structure of the HBc-2A-HBs construct.

[0038] Fig. 22 - Structure of the hIi-HBc-2A-HBs construct.

[0039] Fig.23 - Frequencies of HBc- and HBs-specific CD4+ T-cells in the leukocytes of CB6F1 mice 7 days after the second immunization with various ratios of adjuvanted HBc, HBs and HBc / HBs.

[0040] Fig.24 - Frequencies of HBc- and HBs-specific CD4+ T-cells in the leukocytes of CB6F1 mice 7 days after the third immunization with various ratios of adjuvanted HBc, HBs and HBc / HBs.

[0041] Fig.25 - Frequencies of HBs-specific CD8+ T-cells in leukocytes of CB6F1 mice 7 days after the 2nd and 3rd immunizations with various ratios of adjuvanted HBc, HBs and HBc / HBs.

[0042] Fig.26 - Anti-HBc and HBs humoral responses induced in CB6F1 mice 14 days after the second immunization with various ratios of adjuvanted HBc, HBs and HBc / HBs.

[0043] Fig. 27 - Anti-HBc and HBs humoral responses induced in CB6F1 mice 14 days after the third immunization with various ratios of adjuvanted HBc, HBs and HBc / HBs.

[0044] Sequence Listing SEQ ID NO: 1: Amino acid sequence of HBs SEQ ID NO:2: Amino acid sequence of HBc truncate SEQ ID NO: 3: Amino acid sequence of the spacer region incorporating the 2A cleavage region of foot-and-mouth disease virus SEQ ID NO: 4: Nucleotide sequence encoding the spacer region of 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 substitution variant SEQ ID NO: 13: Nucleotide sequence encoding hI substitution 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. DETAILED DESCRIPTION definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those 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 Klb1, H. ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0046] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or groups of integers or steps but not the exclusion of any other integer or step or groups of integers or steps.

[0047] 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 above or below, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to precede such disclosures due to prior invention. All definitions provided in the context of one aspect of the present invention also apply to other aspects of the present invention.

[0048] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein and refer to any peptide-linked amino acid chain, regardless of length, co-translational or post-translational modification. A fusion protein (or "chimeric protein") is a recombinant protein comprising two or more peptide-linked proteins. A fusion protein is produced by connecting two or more genes that originally encoded separate proteins. Translation of the fusion gene produces a single fusion protein. With respect to a protein or polypeptide, recombinant means that the protein is expressed from a recombinant polynucleotide.

[0049] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymer macromolecules made from nucleotide monomers. Suitably, the polynucleotides of the present invention are recombinant. Recombinant means that the polynucleotide is the product of at least one of a cloning, restriction or ligation step or other procedure that produces a polynucleotide different from a polynucleotide found in nature.

[0050] 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 is not synthetically prepared or modified. A sequence is "derived from" a source when it is isolated from the source but is suitably modified (e.g., by deletion, substitution (mutation), insertion, or other modification) so as not to disrupt the normal function of the source gene.

[0051] Suitably, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is a polynucleotide 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. For example, a polynucleotide is considered isolated if it is cloned into a vector that is not part of its natural environment or if it is contained within a cDNA.

[0052] As used herein, the term "treatment" related to chronic hepatitis B infection refers to the administration of a suitable composition with the purpose 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 can include preventing the onset of liver disease or stabilizing pre-existing liver disease, as indicated by ALT (alanine aminotransferase) levels, liver fibrosis, or other suitable detectable markers. Other markers of CHB include serum HBV DNA levels (which are indicators of viral replication) and serum HBs antigen levels (which are indicators of viral load), so treating CHB can include reducing serum HBsAg (e.g., as determined by quantitative immunoassay) or HBV DNA (e.g., as determined by HBV DNA) to undetectable levels (“cleared” HBsAg or HBV DNA) as determined by the HBV assay (Roche) or equivalent.

[0053] As used herein, "simultaneous" administration refers to administration during the same ongoing immune response, and "simultaneously" should be understood accordingly. Preferably, two components are administered simultaneously (such as a composition comprising a vector and a composition comprising a protein are administered simultaneously), however, one component may be administered within a few minutes (e.g., at the same medical appointment or doctor's visit) or hours of the other component. Such administration is also referred to as co-administration. The simultaneous administration of separate components may be performed via the same route of administration (e.g., intramuscular injection). Alternatively, the simultaneous administration of separate components may be performed via different routes of administration (e.g., intramuscular injection and intradermal injection, intramuscular and intranasal administration, inhalation and subcutaneous administration, etc.). In some embodiments, simultaneous administration may refer to the administration of an adenoviral vector and a protein component. In other embodiments, co-administration refers to the administration of an adenoviral vector and another viral vector (e.g., a poxvirus such as MVA). In other embodiments, co-administration refers to the administration of an adenoviral vector and a protein component (wherein the protein component is optionally adjuvanted).

[0054] "Sequential" administration refers to the administration of a first composition followed by administration of a second composition after a substantial period of time. The time period between two sequential administrations is between 1 week and 12 months, such as between 2 weeks and 12 weeks, such as 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks or 12 weeks, 6 months or 12 months. More specifically, it is between 4 weeks and 8 weeks, such as the time period between sequential administrations can be 4 weeks. Thus, sequential administration encompasses the first 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 caused by the first administration.

[0055] As used herein, an "immunogenic combination" refers to multiple separately formulated immunogenic compositions administered sequentially and / or simultaneously in a single immunization regimen (ie, a prime-boost regimen), each separately formulated immunogenic composition being a component of the immunogenic combination.

[0056] Regarding percent homology, looking at a pairwise alignment of two sequences, the aligned identical residues ('identities') between the two sequences can be observed. The percent identity (or homology) can be calculated by multiplying the quotient between (a) the number of identities and the full length of the reference sequence by 100 (i.e., percent identity = (number of identities x 100) / length of the reference sequence).

[0057] plan The present disclosure encompasses a vaccine regimen that provides a heterologous prime-boost schedule with two viral vectors encoding hepatitis B core (HBc) and hepatitis B surface (HBs) antigens to induce a robust CD8 +T-cell responses in which adjuvanted recombinant HBc and HBs proteins are administered sequentially or simultaneously to induce strong antigen-specific CD4 + The disclosed vaccine regimen successfully restored HBs- and HBc-specific antibodies and CD8 T-cell responses in a mouse model that recapitulates the virological and immunological features of human chronic HBV infection. + T cell responses and HBs-specific CD4 + T cell responses without signs of associated liver-altering side effects.

[0058] More specifically, provided is a method of treating chronic hepatitis B infection (CHB) in a human comprising the steps of: a) administering to said 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 core antigen (HBc); b) administering to said human 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) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0059] In one embodiment, the steps of the method are sequentially implemented, wherein step a) is before step b), and step b) is before step c). Optionally, step c can be repeated. In certain embodiments, the time period between the steps of the method is 2 to 12 weeks, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In one embodiment, the time period between the steps of the method is 4 to 8 weeks. In one embodiment, the time period between the sequential application of the composition according to the method is 4 weeks. In another embodiment, step c) is implemented simultaneously with step a) and / or with step b). In certain embodiments, simultaneous steps b) and c) can be repeated. In one embodiment, the steps of the method are sequentially implemented, wherein step b) is before step a), and step c) is after step a), or is implemented simultaneously with step a) and / or with step b). In one embodiment, the steps of the method are sequentially implemented, wherein step c) is before step a), and step a) is before step b). In another embodiment, the steps of the method are sequentially implemented, wherein step c) is before step b), and step b) is before step a). In a further embodiment, step c is repeated, and the steps of the method are implemented in the following order: step a), step b), step c), step c). In certain embodiments, the time period between the steps of the method is 2 to 12 weeks, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In one embodiment, the time period between the steps of the method is 4 to 8 weeks. In one embodiment, the time period between sequentially applying the composition according to the method is 4 weeks.

[0060] In certain embodiments, the composition administered in step a) of the method comprises a ChAd vector selected from the group consisting of ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also known as C7) and Pan9, in particular ChAd63 or ChAd155. In certain embodiments, the ChAd vector comprises a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto) that is combined with 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 having at least 98% homology thereto) is fused to hIi (e.g., SEQ ID NO: 7 or an amino acid sequence having at least 98% homology thereto or SEQ ID NO: 12 or an amino acid sequence having at least 98% homology thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 12). In a specific embodiment, the composition administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs, e.g., encoding a vector having Fig. 22In one embodiment, the composition administered in step a) of the method comprises a ChAd vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the composition administered in step a) of the method comprises a ChAd vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 10 or the nucleotide sequence given in SEQ ID NO: 14. In a specific embodiment, the vector is a ChAd155 vector. Thus, in certain embodiments, the composition administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 9. In other embodiments, the composition administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 15. In one embodiment, the composition administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 10. In other embodiments, the composition administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO:14.

[0061] In one embodiment, the composition administered in step b) of the method comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc and HBs separated by a sequence encoding a spacer region that is coupled to the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, the composition administered in step b) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A, and HBs, e.g., encoding a polynucleotide having Fig.21In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto) that is coupled to the 2A cleavage region of foot-and-mouth disease virus. In one embodiment, the composition administered in step b) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition administered in step b) of the method comprises an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 6.

[0062] In one embodiment, the composition administered in step c) of the method comprises recombinant HBc and recombinant HBs in a ratio of 1:1. In another embodiment, the ratio of HBc to HBs in the composition is greater than 1, for example, the ratio of HBc to HBs can 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, especially 3:1 to 5:1, such as 3:1, 4:1 or 5:1, especially a ratio of 4:1. In a specific embodiment, the composition administered in step c) of the method comprises recombinant HBc and recombinant HBs in a ratio of 4:1 or greater. In certain embodiments, the composition administered in step c) of the method comprises full-length recombinant hepatitis B surface antigen (HBs) (e.g., SEQ ID NO:1 or an amino acid sequence having at least 98% homology thereto), a recombinant hepatitis B virus core antigen (HBc) truncated at the C-terminus, and an adjuvant. 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 having at least 98% homology thereto). In one embodiment, the composition administered in step c) of the method comprises full-length recombinant HBs, amino acids 1-149 of HBc, and an adjuvant comprising MPL and QS-21. For example, the composition administered in step c) of the method comprises full-length recombinant HBs (SEQ ID NO: 1), amino acids 1-149 of HBc (SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21. In certain embodiments, the recombinant protein HBs and HBc antigens are in the form of virus-like particles.

[0063] In a further embodiment, there is provided a method of treating CHB in a human comprising the steps of: a) administering to said 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 core antigen (HBc); b) administering to said human 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); c) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; and d) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0064] In another aspect of the present invention, there is provided a method for treating chronic hepatitis B infection (CHB) in a human, comprising the steps of: a) administering to said 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 virus core antigen (HBc), and, simultaneously, ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; and b) administering to said 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 virus core antigen (HBc), and, simultaneously, a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0065] In one embodiment of this aspect of the invention, the steps of the method are performed sequentially, wherein step a) precedes step b). Optionally, step a may be repeated. In one embodiment, the method steps are performed in the following order: step a), followed by step a), followed by step b). In an alternative embodiment, the method steps are performed in the following order: 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 following order: step a), followed by step b), followed by step b). In an alternative embodiment, the method steps are performed in the following order: step b), followed by step a), followed by step b). Optionally, step b may be repeated more than once. Optionally, both steps a) and b) may be repeated. In one embodiment of this aspect of the invention, the method steps are performed in the following order: step a), followed by step b), followed by step b), followed by step b). In an alternative embodiment, the method steps are performed in the following order: step b), followed by step a), followed by step b), followed by step b). In a further embodiment, the method steps are implemented 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 time period between the steps of the method is 2 to 12 weeks, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In one embodiment, the time period between the steps of the method is 4 to 8 weeks. In one embodiment, the time period between sequentially applying the composition according to the method is 4 weeks.

[0066] Therefore, in another embodiment of this aspect of the invention, there is provided a method of treating CHB in a human comprising the steps of: a) administering to said 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 virus core antigen (HBc), and, simultaneously, ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; b) administering to said human i) a composition comprising a modified vaccinia virus Ankara (MVA) vector comprising a polynucleotide encoding an HBs antigen and a nucleic acid encoding an HBc antigen, and, simultaneously, ii) a composition comprising a recombinant HBs protein antigen, a recombinant HBc protein antigen and an adjuvant; c) administering to said human i) a composition comprising an MVA vector comprising a polynucleotide encoding an HBs antigen and a nucleic acid encoding an HBc antigen, and, simultaneously, ii) a composition comprising a recombinant HBs protein antigen, a recombinant HBc protein antigen and an adjuvant; and d) administering to said human i) a composition comprising an MVA vector comprising a polynucleotide encoding an HBs antigen and a nucleic acid encoding an HBc antigen, and, simultaneously, ii) a composition comprising a recombinant HBs protein antigen, a recombinant HBc protein antigen and an adjuvant.

[0067] In certain embodiments, the time period between the steps of the method is 2 to 12 weeks, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In one embodiment, the time period between the steps of the method is 4 to 8 weeks. In one embodiment, the time period between the sequential administration of the composition according to the method is 4 weeks. In one embodiment, the composition i) administered in step a) of the method comprises a ChAd vector selected from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also known as C7) and Pan9, in particular ChAd63 or ChAd155. In certain embodiments, the ChAd vector comprises a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc and HBs separated by a sequence encoding a spacer region that is incorporated into the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, HBc is fused to hIi. In a specific embodiment, the composition i) administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A and HBs, e.g., encoding a vector having Fig. 22In some embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a spacer region encoding the 2A cleavage region of foot-and-mouth disease virus (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto). In some embodiments, HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) is fused to hIi (e.g., SEQ ID NO: 7 or an amino acid sequence having at least 98% homology thereto or SEQ ID NO: 12 or an amino acid sequence having at least 98% homology thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 12). In one embodiment, the composition i) administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 9. In another embodiment, the composition i) administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 15. In one embodiment, the composition i) administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 10. In another embodiment, the composition i) administered in step a) of the method comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID No: 14. In certain embodiments, the composition ii) administered in step a) of the method comprises full-length recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) truncated at the C-terminus, and an adjuvant. In certain embodiments, the truncated recombinant HBc comprises the assembly domain of HBc, e.g., amino acids 1-149 of HBc. In one embodiment, the composition ii) administered in step a) of the method comprises full-length recombinant HBs (e.g., SEQ ID NO: 1), amino acids 1-149 of HBc (e.g., SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21. In certain embodiments, the recombinant protein HBs and HBc antigen are in the form of virus-like particles.

[0068] In one embodiment, the composition i) administered in step b) of the method comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region encoding the 2A cleavage region of foot-and-mouth disease virus (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto). In a specific embodiment, the composition i) administered in step b) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A and HBs, e.g., encoding a polynucleotide having Fig.21 In one embodiment, the composition i) administered in step b) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition i) administered in step b) of the method comprises an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 6. In certain embodiments, the composition ii) administered in step b) of the method comprises full-length recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) truncated at the C-terminus, and an adjuvant. In certain embodiments, the truncated recombinant HBc comprises an assembly domain of HBc, such as amino acids 1-149 of HBc. In one embodiment, the composition ii) administered in step b) of the method comprises full-length recombinant HBs (e.g., SEQ ID NO: 1), amino acids 1-149 of HBc (e.g., SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21. In certain embodiments, the recombinant protein HBs and HBc antigens are in the form of virus-like particles.

[0069] In one embodiment, the composition i) administered in step c) of the method comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region encoding the 2A cleavage region of foot-and-mouth disease virus (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto). In a specific embodiment, the composition i) administered in step c) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A and HBs, e.g., encoding a sequence having Fig.21 In one embodiment, the composition i) administered in step c) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition i) administered in step c) of the method comprises an MVA vector comprising a polynucleotide vector insert having a nucleotide sequence given in SEQ ID NO: 6. In certain embodiments, the composition ii) administered in step c) of the method comprises full-length recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) truncated at the C-terminus, and an adjuvant. In certain embodiments, the truncated recombinant HBc comprises an assembly domain of HBc, such as amino acids 1-149 of HBc. In certain embodiments, the recombinant protein HBs and HBc antigen are in the form of virus-like particles. In one embodiment, the composition ii) administered in step c) of the method comprises full-length recombinant HBs (e.g., SEQ ID NO: 1), amino acids 1-149 of HBc (e.g., SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21.

[0070] In one embodiment, the composition i) administered in step c) of the method comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region encoding the 2A cleavage region of foot-and-mouth disease virus (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto). In a specific embodiment, the composition i) administered in step d) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A and HBs, e.g., encoding a sequence having Fig.21 In one embodiment, the composition i) administered in step d) of the method comprises an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition i) administered in step d) of the method comprises an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 6. In certain embodiments, the composition ii) administered in step d) of the method comprises full-length recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) truncated at the C-terminus, and an adjuvant. In certain embodiments, the truncated recombinant HBc comprises an assembly domain of HBc, such as amino acids 1-149 of HBc. In one embodiment, the composition ii) administered in step d) of the method comprises full-length recombinant HBs (e.g., SEQ ID NO: 1), amino acids 1-149 of HBc (e.g., SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21. In certain embodiments, the recombinant protein HBs and HBc antigens are in the form of virus-like particles.

[0071] The present invention also provides a method for inducing a cellular immune response and a humoral immune response, particularly a CD4+ response and a CD8+ response, and an antibody response in a person with CHB, the method comprising the following steps: a) administering to said 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 core antigen (HBc); b) administering to said human 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) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0072] In one embodiment, the steps of the method are performed sequentially, wherein step a) precedes step b), and step b) precedes step c). Optionally, step c) may be repeated). In another embodiment, step c) is performed simultaneously with step a) and / or with step b). In a further embodiment, a method for inducing a cellular immune response and a humoral immune response, in particular a CD4+ response and a CD8+ response, and an antibody response in a person with CHB comprises the following steps: a) administering to said 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 virus core antigen (HBc), and, simultaneously, ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; and b) administering to said 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 virus core antigen (HBc), and, simultaneously, a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0073] In one embodiment, the steps of the method are performed sequentially, wherein step a) precedes step b). Optionally, step b) may be repeated.

[0074] The present invention also provides a method for reducing the level of serum HBsAg and / or the level of serum HBV DNA in a person with CHB, the method comprising the steps of: a) administering to said 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 core antigen (HBc); b) administering to said human 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) administering to the human a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0075] In one embodiment, the steps of the method are performed sequentially, wherein step a) precedes step b), and step b) precedes step c). Optionally, step c) may be repeated. In another embodiment, step c) is performed simultaneously with step a) and / or with step b).

[0076] In a further embodiment, the method of reducing the level of serum HBsAg and / or the level of serum HBV DNA in a human with CHB comprises the following steps: a) administering to said 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 virus core antigen (HBc), and, simultaneously, ii) a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant; and b) administering to said 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 virus core antigen (HBc), and, simultaneously, a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant.

[0077] In one embodiment, the steps of the method are performed sequentially, wherein step a) precedes step b). Optionally, step b) may be repeated). In a further embodiment, the level of serum HBsAg is reduced to an undetectable level as determined by a quantitative immunoassay. In another embodiment, the level of serum HBV DNA is reduced to an undetectable level as determined by a quantitative immunoassay. HBV assay or equivalent. In another embodiment, the level of serum HBsAg and / or the level of serum HBV DNA is reduced to and maintained at an undetectable level for at least 6 months. In another embodiment, the level of serum HBsAg and / or the level of serum HBV DNA is reduced to and maintained at an undetectable level, and the ALT level is maintained within the normal range for at least 6 months.

[0078] Antigens have identified at least nine genotypes of HBV (A to I), with genome differences exceeding 8%. Within a given HBV genotype, multiple genotypes have been identified, with differences of 4-8%. The antigens used in the disclosed method are suitably selected to provide immunological coverage across multiple, preferably all HBV genotypes. Hepatitis B core protein antigen (HBc) is highly conserved across genotypes and genotypes, and hepatitis B surface protein antigen (HBs) sequences are suitably selected to include key B-cell epitopes retained across genotypes, which allow induction of a wide range of neutralization responses. Suitably, the sequences of HBc and HBs used in the disclosed methods and compositions are based on those from genotype / subtype A2.

[0079] Suitably, the HBs antigen used in the disclosed methods and compositions is derived from a small, medium or large surface antigen protein. Specifically, a suitable HBs antigen comprises a small (S) protein of HBV adw2 strain, genotype A. For example, a suitable HBs antigen has 226 amino acids of the amino acid sequence SEQ ID NO:1. When used as a recombinant protein, the HBs antigen is preferably assembled into virus-like particles. The antigen is included in a well-studied commercially available hepatitis B preventive vaccine (Engerix B, Fendrix, Twinrix and others) and has been shown to be protective against hepatitis B across genotypes. Preferably, the recombinant HBs protein antigen is expressed and purified from yeast for use in the vaccine composition and method of the present invention. Suitable methods for expression and purification are known, for example, from EP1307473B1.

[0080] Hepatitis B core protein (HBc) is the main component of the nucleocapsid that packages the viral genome. The protein (183-185aa long) is expressed in the cytoplasm of infected cells and remains unglycosylated. HBc contains an assembly domain of 149 residues and an RNA-binding domain of 34-36 residues at the C-terminus. The HBc antigen used in the disclosed methods and compositions can be full-length, or can contain a C-terminally truncated protein (lacking an RNA-binding C-terminus), such as 145-149 amino acids including the assembly domain of the wild-type core antigen protein, such as amino acids 1-145, 1-146, 1-147, 1-148 or amino acids 1-149 of the wild-type hepatitis B core antigen protein. The truncated protein retains the ability to assemble into nucleocapsid particles. Suitable HBc antigens used in the disclosed methods and compositions have amino acid sequences from HBV adw2 strains, genotype A. When used as a recombinant protein, the HBc antigen is suitably truncated from the wild type at the C-terminus, and in particular, the antigen may have the amino acid sequence of SEQ ID NO: 2. Preferably, the recombinant HBc protein antigen is expressed and purified from E. coli for use in the vaccine composition and method of the present invention. Methods for recombinantly expressing viral proteins in E. coli are well known in the art.

[0081] When used as a recombinant protein, the HBc antigen is preferably assembled into virus-like particles. When expressed from a viral vector, the HBc antigen can be full-length or truncated, for example, suitably a full-length HBc antigen (e.g., SEQ ID NO: 11). A suitable dose of the recombinant HBs antigen used in the methods disclosed herein is 10ug to 100ug per dose, such as 10ug, 15ug, 20ug, 25ug, 30ug, 35ug, 40ug, 45ug, 50ug, 55ug, 60ug, 65ug, 70ug, 75ug, 80ug, 85ug, 90ug, 95ug or 100ug per dose. A suitable dosage of the recombinant HBc antigen for use in the methods disclosed herein is 10 ug to 100 ug per dose, such as 10 ug, 15 ug, 20 ug, 25 ug, 30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, 75 ug, 80 ug, 85 ug, 90 ug, 95 ug or 100 ug per dose.

[0082] Antigen is a substance that induces immune response, especially the production of antibodies in vivo. Antigens can be of foreign origin, i.e. pathogen origin, or from the organism itself, the latter being referred to as self or autoantigens. Antigens can be presented on the surface of antigen presenting cells by MHC molecules. There are two types 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. In order to prevent endogenous peptides (i.e., self-antigens) from binding to MHC-II molecules and presenting to generate immune responses, the newly formed MHC-II molecules are combined with another protein (constant chain) (which blocks the peptide-binding cleft of the MHC-II molecule). Human constant chain (hIi, also known as CD74 when expressed on the plasma membrane) is an evolutionarily conserved type II membrane protein that has several functions in cells and throughout the immune system [Borghese, 2011]. When the MHC class II compartment fuses to the late endosome containing the engulfed and degraded foreign protein, the invariant chain is cleaved, leaving only the CLIP region bound to the MHC-II molecule. In a second step, CLIP is removed by HLA-DM molecules, leaving the MHC-II molecule free to bind fragments of foreign proteins. Once the MHC class II compartment fuses with the plasma membrane, the fragments are presented on the surface of antigen-presenting cells, thus presenting the foreign antigen to other cells, mainly T-helper cells.

[0083] It is known that when an adenovirus expression system encoding a fusion of a constant chain and the antigen is used for vaccination, the immune response to the antigen is increased (see WO2007 / 062656, which is also disclosed as US2011 / 0293704 and is incorporated by reference for the purpose of disclosing constant chain sequences), that is, the constant chain enhances the immunogenicity of the antigen, and constant chains such as hIi are sometimes referred to as "genetic adjuvants" in identifying this effect. In addition, it has been demonstrated that the adenovirus construct can be used to elicit an immune response in the context of a prime-boost vaccination regimen (see WO2014 / 141176, which is also disclosed as US2016 / 0000904; and WO2010 / 057501, which is also disclosed as US2010 / 0278904 and is incorporated by reference for the purpose of disclosing constant chain sequences and adenovirus vectors encoding constant chain sequences). Specifically, the hIi sequence and hIi have the ability to increase CD8 +Potential for T-cell response [Spencer, 2014; Capone, 2014]. In certain embodiments, the nucleotide sequence included in the vector used in the methods, uses and compositions disclosed herein may include a nucleotide sequence encoding hIi. The amino acid sequence of hIi that may be included in the disclosed adenoviral vector ChAd155-hIi-HBV is shown in SEQ ID NO:7, and the alternative sequence is shown in SEQ ID NO:12. The nucleotide sequences encoding these amino acid sequences are shown in SEQ ID NO:8 and SEQ ID NO:13. Suitably, the nucleotide sequence encoding hIi is fused to the nucleotide sequence encoding the HBc antigen to produce a fusion protein in which the hIi polypeptide is N-terminally fused to the HBc antigen.

[0084] Carrier In addition to the polynucleotides encoding the antigenic protein (also referred to herein as "inserts"), the vectors used in the methods and compositions disclosed herein may also include conventional control elements that are operably linked to the encoding polynucleotides in a manner that allows them to be transcribed, translated, and / or expressed in cells transfected with the vector. Thus, the vector insert polynucleotides encoding the protein antigen are incorporated into an expression cassette with appropriate control elements.

[0085] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals, including rabbit β-globin poly A; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product.

[0086] Promoter is a nucleotide sequence that allows the binding of RNA polymerase and guides the transcription of a gene. Typically, a promoter is located in the 5' non-coding region of a gene near the transcription start site of the gene. Sequence elements in the promoter that function in the initiation of transcription are often characterized by a common nucleotide sequence. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses and mammals (including humans). A large number of expression control sequences (including internal, natural, constitutive, inducible and / or tissue-specific promoters) are known in the art and can be utilized.

[0087] Examples of constitutive promoters include TBG promoter, retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter (optionally with CMV enhancer, see, e.g., Boshart et al., Cell, 41:521-530 (1985)), CASI promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1a promoter (Invitrogen). Suitably, the promoter is CMV promoter or a variant thereof, more suitably human CMV (HCMV) promoter or a variant thereof.

[0088] Adenovirus vectors Adenoviruses have been widely used for gene transfer applications due to their ability to achieve highly efficient gene transfer in a variety of target tissues and their large transgene capacity. Conventionally, the E1 gene of the adenovirus is deleted and replaced with a transgene cassette consisting of a selected promoter, the cDNA sequence of the gene of interest, and a polyadenylation signal, thereby generating a replication-defective recombinant virus. Human adenovirus vectors have been shown to be useful for inducing CD8 T cells to respond to transgenes in animal models as well as in humans. + An effective carrier of T-cell response. Adenovirus has a wide range of tropism and has the ability to infect replicating and non-replicating cells. The main limitation of the clinical application of vectors based on human adenovirus is the general prevalence of neutralizing antibodies in the general population. Adenoviruses isolated from alternative species have been considered as potential vaccine vectors to bypass the problem of pre-existing anti-adenovirus immunity in humans. Among them, simian adenoviruses derived from chimpanzees, gorillas or bonobos can be used to deliver antigens and induce targeted T cells and / or humoral responses to those antigens in humans. Simian adenoviruses, including those derived from chimpanzees, have been tested in clinical studies. Chimpanzee adenovirus vectors have low seropositivity / no seropositivity in the population, are known to not cause pathological diseases in humans, and some ChAd vectors can be grown to high titers in cell lines previously used to produce clinical grade materials, such as human embryonic kidney cells 293 (HEK 293).

[0089] A replication-incompetent or replication-defective adenovirus is an adenovirus that is replication-incompetent because it has 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, etc., or a complete removal of a gene encoding a gene product essential for viral replication, such as complete removal of one or more of the adenoviral genes selected from E1A, E1B, E2A, E2B, E3 and E4 (such as E3 ORF1, E3 ORF2, E3 ORF3, E3ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3 ORF9, E4 ORF7, E4 ORF6, E4ORF4, E4 ORF3, E4 ORF2 and / or E4ORF1). Suitably, the E1 and E3 genes are deleted. More suitably, the E1, E3 and E4 genes are deleted.

[0090] 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 known 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 the purpose of disclosing ChAd155 vector sequences and methods) belongs to the same phylogenetic adenovirus group as the ChAd3 vector (Group C). In one embodiment, the vector used in the methods and compositions disclosed herein is a ChAd vector of phylogenetic group C, such as ChAd3 or ChAd155. In a specific embodiment, the method for treating chronic hepatitis B disclosed herein comprises the following steps: 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 the ChAd vector for use in the method disclosed herein is 1×10 8 – 1x10 11 Virus particles (vp), for example, about 1x10 8 , 5x10 8 , 1x10 9 , 5x10 9 , 1x10 10 , 5x10 10 or 1x10 11Virus particles (vp).

[0091] More specifically, in one embodiment, the vector used in the methods and compositions disclosed herein is a replication-deficient chimpanzee adenovirus vector ChAd155, which encodes a fusion of sequences derived from the following two HBV proteins: HBc (core, nucleocapsid protein) and HBs (small surface antigen). In certain specific embodiments, the vector is ChAd155, which encodes HBc and HBs, which are separated by SEQ ID NO: 3 (a spacer region incorporating a sequence encoding the 2A cleavage region of foot-and-mouth disease virus (FMDV)) [Donnelly et al. 2001] (generating 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) for processing HBc and HBs into separate proteins. Cleavage of the core from the surface antigen allows for the correct folding of HBs, allowing the generation of an antibody response to the surface antigen. Alternatively, the adenovirus vector can be a dual promoter (bicistronic) vector to allow independent expression of the HBs and HBc antigens.

[0092] In certain embodiments, the N-terminal portion of the gene encoding the HBc protein can be fused to the gene encoding the human major histocompatibility complex (MHC) class II-associated invariant chain, p35 isoform (i.e., hIi or CD74). Thus, a specific ChAd155 vector for use in the methods and compositions disclosed herein comprises a gene encoding a protein having Fig. 22 The polynucleotide vector insert of the construct of the structure shown in , the construct comprising hIi, HBc, 2A and HBs. The amino acid sequence of this construct is given in SEQ ID NO:9, and the nucleotide sequence encoding the amino acid sequence of the construct is given in SEQ ID NO:10. The amino acid sequence of this alternative construct is given in SEQ ID NO:15, and the nucleotide sequence encoding the amino acid sequence of the construct is given in SEQ ID NO:14.

[0093] Modified vaccinia virus Ankara (MVA) vector Modified vaccinia virus Ankara (MVA) with replication defects in humans and other mammals is derived from vaccinia virus. It belongs to the poxvirus family and was initially developed to pass vaccinia virus in chicken embryo fibroblast (CEF) cells for more than 570 times, resulting in multiple deletions, after which the virus was highly attenuated and replication-defective in humans and other mammals to improve the safety of smallpox vaccination. Replication defects occur in the late stage of virion assembly, so that viral and recombinant gene expression is not impaired, making MVA an effective single-round expression vector that cannot cause infection in mammals. MVA has subsequently been widely used as a viral vector to induce antigen-specific immunity against transgenics in both animal models and humans. The description of MVA can be found in Mayr A, et al. (1978) and Mayr, A., et al. (1975).

[0094] In one embodiment, the MVA is derived from virus seed batch 460MG obtained from the 571st passage of vaccinia virus on CEF cells. In another embodiment, the MVA is derived from virus seed batch MVA 476MG / 14 / 78. In a further embodiment, the MVA was derived or produced before December 31, 1978 and is free of viral contamination. A suitable dose of the MVA vector for use in the methods disclosed herein is 1×10 6 – 1x10 9 Plaque forming units (pfu), for example, about 1x10 per dose 6 , 2x10 6 , 5x10 6 , 1x10 7 , 2x10 7 , 5x10 7 , 1x10 8 , 2x10 8 , 5x10 8 or 1x10 9 pfu.

[0095] In a specific embodiment, the method for treating chronic hepatitis B disclosed herein comprises the following steps: administering to a human a composition comprising an MVA vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc).

[0096] More specifically, in one embodiment, the vector used 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 (small surface antigen). In certain embodiments, the vector used in the methods and compositions disclosed herein is an MVA encoding HBc and HBs separated by SEQ ID NO: 3 (a spacer region incorporating a sequence encoding the 2A cleavage region of foot-and-mouth disease virus) (generating 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) for processing HBc and HBs into separate proteins. Thus, a specific MVA vector used in the methods and compositions disclosed herein comprises a vector encoding a fusion of sequences derived from two HBV proteins: HBc (core nucleocapsid protein) and HBs (small surface antigen). In certain embodiments, the vector used in the methods and compositions disclosed herein is an MVA ... Fig.21 The polynucleotide vector insert of the construct of the structure shown in , the construct comprising HBc, 2A and HBs. The amino acid sequence of this construct is given in SEQ ID NO:5, and the nucleotide sequence encoding the amino acid insert construct is given in SEQ ID NO:6.

[0097] Pharmaceutical composition In certain embodiments, the composition comprising a replication-deficient chimpanzee adenoviral vector for use in the method for treating CHB comprises a ChAd vector selected from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan6, Pan 7 (also known as C7) and Pan 9, in particular ChAd63 or ChAd155. In certain embodiments, the ChAd vector comprises a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto) that is incorporated into 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 having at least 98% homology thereto) is fused to hIi (e.g., SEQ ID NO: 7 or an amino acid sequence having at least 98% homology thereto or SEQ ID NO: 12 or an amino acid sequence having at least 98% homology thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 12). In a specific embodiment, a composition comprising a replication-defective chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs, e.g., encoding a polynucleotide having Fig. 22In one embodiment, the composition comprising a replication-deficient chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the composition comprising a replication-deficient chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 10 or the nucleotide sequence given in SEQ ID NO: 14. In a specific embodiment, the vector is a ChAd155 vector. Therefore, in certain embodiments, the composition comprising a replication-deficient chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 9. In other embodiments, the composition comprising a replication-deficient chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd155 vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 15. In one embodiment, the composition comprising a replication-defective chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 10. In other embodiments, the composition comprising a replication-defective chimpanzee adenoviral vector for use in a method for treating CHB comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 14.

[0098] In one embodiment, a composition comprising an MVA vector for use in a method for treating CHB comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In certain embodiments, the vector insert encodes HBc and HBs separated by a sequence encoding a spacer region that is coupled to the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, a composition comprising an MVA vector for use in a method for treating CHB comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A, and HBs, e.g., encoding a polynucleotide having Fig.21In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), which are separated by a sequence encoding a spacer region (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto) that is coupled to the 2A cleavage region of foot-and-mouth disease virus. In one embodiment, the composition comprising an MVA vector used in the method for treating CHB comprises such an MVA vector comprising a polynucleotide vector insert encoding the amino acid sequence of SEQ ID NO: 5. In one embodiment, the composition comprising an MVA vector used in the method for treating CHB comprises such an MVA vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 6.

[0099] In one embodiment, the composition comprising recombinant HBs antigen, recombinant HBc antigen and adjuvant used in the method for treating CHB comprises recombinant HBc and recombinant HBs in a ratio of 1:1. In another embodiment, the ratio of HBc to HBs in the composition is greater than 1, for example, the ratio of HBc to HBs can 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, especially 3:1 to 5:1, such as 3:1, 4:1 or 5:1, especially 4:1. In a specific embodiment, the composition comprising recombinant HBs antigen, recombinant HBc antigen and adjuvant used in the method for treating CHB comprises recombinant HBc and recombinant HBs in a ratio of 4:1 or greater. In certain embodiments, the composition comprising recombinant HBs antigen, recombinant HBc antigen and adjuvant used in the method for treating CHB comprises full-length recombinant hepatitis B surface antigen (HBs) (e.g., SEQ ID NO: 1), recombinant hepatitis B virus core antigen (HBc) truncated at the C-terminus and an adjuvant. In certain embodiments, the truncated recombinant HBc comprises an assembly domain of HBc, such as amino acids 1-149 of HBc (e.g., SEQ ID NO: 2). In one embodiment, the composition comprising recombinant HBs antigen, recombinant HBc antigen and adjuvant used in the method for treating CHB comprises full-length recombinant HBs, amino acids 1-149 of HBc and an adjuvant comprising MPL and QS-21. For example, the composition comprising recombinant HBs antigen, recombinant HBc antigen and adjuvant used in the method for treating CHB comprises full-length recombinant HBs (SEQ ID NO: 1), amino acids 1-149 of HBc (SEQ ID NO: 2) and an adjuvant comprising MPL and QS-21. In certain embodiments, the recombinant protein HBs and HBc antigens are in the form of virus-like particles.

[0100] The compositions disclosed herein that can be used in the disclosed methods are suitably pharmaceutically acceptable compositions. Suitably, the pharmaceutical composition will include a pharmaceutically acceptable carrier.

[0101] Compositions comprising ChAd or MVA vectors can be prepared for administration by suspending the viral vector particles in a pharmaceutically or physiologically acceptable carrier, such as isotonic saline or other isotonic salt solutions. Suitable carriers will be apparent to those skilled in the art and will largely depend on the route of administration.

[0102] Compositions comprising recombinant protein antigens can be prepared as follows: from expressing their cell culture separation and purification of proteins, suspended in a formulation buffer comprising one or more salts, surfactants and / or cryoprotectants and lyophilized. For example, suitable formulation buffers may include a mixture of sugar or sugar (e.g., sucrose, trehalose or sucralose) as cryoprotectants, and a nonionic copolymer as a surfactant, such as poloxamer. For administration, the lyophilized recombinant protein formulation is reconstructed in a pharmaceutically or physiologically acceptable carrier, such as isotonic saline or other isotonic saline solutions for injection or suction. Suitable carriers will be apparent to those skilled in the art, and will depend largely on the route of administration. The reconstructed composition may also include an adjuvant or a mixture of adjuvants, and in one embodiment, the lyophilized recombinant protein is reconstructed in a liquid adjuvant system formulation.

[0103] The term "carrier" as used herein refers to a pharmacologically inactive substance, such as but not limited to a diluent, excipient or vehicle used together with a therapeutically active ingredient. Liquid carriers include, but are not limited to, sterile liquids, such as saline solutions in water and oils, the oils including those of petroleum, animal, plant 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 EW Martin.

[0104] In addition to the vector or recombinant protein of the composition, the composition used in the method disclosed herein may include an adjuvant system. The term "adjuvant" refers to an agent that strengthens, stimulates, activates, enhances or regulates the immune response to the antigen of the composition at the cellular or body fluid level, for example, an immune adjuvant stimulates the immune system to respond to the antigen, but has no immunological effect itself. Whether or not the vector included in the composition also encodes a "genetic adjuvant", such as hIi, the composition disclosed herein may include an adjuvant as a separate component in the formulation.

[0105] Suitable adjuvants are those that can enhance the immune response in subjects with chronic conditions and subverted immune competence. CHB patients are characterized by their inability to produce effective innate and adaptive immune responses to the virus, which makes effective vaccine development challenging. In these patients, a key function of adjuvanted vaccine formulations should be intended to guide cell-mediated immune responses to the T-assisted 1 (Th1) profile that is recognized as being crucial to removing intracellular pathogens.

[0106] 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., immunostimulatory complexes (ISCOMS), liposomes or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A (MPL), such as 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 such as QS21 or squalene) and / or a bacterial adjuvant (e.g. a monophosphoryl lipid A (MPL) such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL) A suitable adjuvant is monophosphoryl lipid A (MPL), especially 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 method taught in GB 2122204B, which also discloses the preparation of diphosphoryl lipid A and its 3-O-deacylated variants. Other purified and synthetic lipopolysaccharides have been described [U.S. Patent No. 6,005,099 and EP0729473B1; Hilgers, 1986; Hilgers, 1987; and EP0549074B1].

[0107] 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) and its fractions are described in U.S. Pat. No. 5,057,540 and Kensil, 1996; and EP 0362279 B1. Purified fractions of Quil A are also known as immunostimulants, such as QS21 and QS17; their production methods are disclosed in U.S. Pat. No. 5,057,540 and EP 0362279 B1. The use of QS21 is further described in Kensil, 1991. Combinations of QS21 and polysorbates or cyclodextrins are also known (WO 99 / 10008). Microparticle adjuvant systems comprising fractions of Quil A (such as QS21 and QS7) are described in WO 96 / 33739 and WO 96 / 11711.

[0108] Adjuvants such as those described above can be formulated with carriers such as liposomes, oil-in-water emulsions and / or metal salts (including aluminum salts such as aluminum hydroxide). For example, 3D-MPL can be formulated with aluminum hydroxide (EP 0689454) or oil-in-water emulsions (WO95 / 17210); QS21 can be formulated with cholesterol-containing liposomes (WO 96 / 33739), oil-in-water emulsions (WO95 / 17210) or alum (WO 98 / 15287).

[0109] Combinations of adjuvants may 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 combinations of QS21 and 3D-MPL as disclosed in WO 94 / 00153, or compositions in which QS21 is quenched in cholesterol-containing liposomes (DQ) (as disclosed in WO 96 / 33739). Effective adjuvant formulations involving QS21, 3D-MPL and tocopherol in oil-in-water emulsions are described in WO 95 / 17210 and are another formulation that can be used in the disclosed compositions. Thus, suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A (preferably 3D-MPL) together with an aluminum salt (e.g. as described in WO 00 / 23105). Another exemplary adjuvant comprises QS21 and / or MPL and / or CpG. QS21 may be quenched in liposomes containing cholesterol, as disclosed in WO 96 / 33739.

[0110] Therefore, a suitable adjuvant for use in the disclosed composition is AS01, a liposome-based adjuvant containing MPL and QS-21. The liposomes as a vehicle for the MPL and QS-21 immunopotentiator are composed of dioleoylphosphatidylcholine (DOPC) and cholesterol in a phosphate-buffered saline solution. AS01 B-4 A particularly preferred variant of the AS01 adjuvant is composed of the immunopotentiators QS-21 (triterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A) with DOPC / cholesterol liposomes (as a vehicle for these immunopotentiators) and sorbitol in a PBS solution. Specifically, a single human dose of AS01 B-4 (0.5mL) contains 50μg QS-21 and 50μg MPL. AS01 E-4 Corresponds to AS01 B-4 A two-fold dilution of , i.e., each human dose contains 25 μg QS-21 and 25 μg MPL.

[0111] In one embodiment, an immunogenic combination product is provided for use in a method of treating chronic hepatitis B infection (CHB) in a human, the immunogenic combination product comprising a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc), and an adjuvant. In one embodiment, the immunogenic combination product comprises a composition comprising recombinant hepatitis B surface antigen (HBs), truncated recombinant hepatitis B virus core antigen (HBc), and an adjuvant. In one embodiment, the immunogenic combination product comprises a composition comprising recombinant HBs, truncated recombinant HBc, and AS01 adjuvant. In a specific embodiment, the immunogenic combination product comprises a composition comprising truncated recombinant HBc and recombinant HBs at a ratio of 4:1 or greater, and AS01 adjuvant, such as AS01 B-4 or AS01 E-4 .

[0112] In one embodiment, an immunogenic combination product is provided for use in a method of treating chronic hepatitis B infection (CHB) in a human, the immunogenic combination product 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 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 recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, wherein the method comprises administering the composition to the human sequentially or simultaneously.

[0113] In another aspect, an immunogenic composition is provided for use in a method for treating chronic hepatitis B infection (CHB) in a human, the immunogenic composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs), a nucleic acid encoding hepatitis B virus core antigen (HBc), and a nucleic acid encoding a human constant chain (hIi) fused to the HBc, wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In one embodiment, the composition comprises a ChAd vector selected from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan5, Pan 6, Pan 7 (also known as C7), and Pan 9, in particular ChAd63 or ChAd155. In certain embodiments, the ChAd vector comprises a vector insert encoding HBc and HBs separated by a spacer region incorporating a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A and HBs, e.g., encoding a Fig. 22 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 another 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 given in SEQ ID NO: 10. In another embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 14.

[0114] In a further aspect, an immunogenic composition is provided for use in a method of treating chronic hepatitis B infection (CHB) in a human, the 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 virus core antigen (HBc), wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In one embodiment, the composition comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a spacer region incorporating a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, the composition comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A, and HBs, e.g., encoding a vector having Fig.21 In one embodiment, the composition comprises an MVA vector comprising 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 given in SEQ ID NO: 6.

[0115] In a further aspect, an immunogenic composition is provided for use in a method of treating chronic hepatitis B infection (CHB) in a human, the immunogenic composition comprising 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, wherein the method comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In one embodiment, the composition comprises a truncated recombinant HBc comprising an assembly domain of HBc, e.g., amino acids 1-149 of HBc. In one embodiment, the composition comprises full-length recombinant HBs, amino acids 1-149 of HBc, and an adjuvant comprising MPL and QS-21. More specifically, the composition used in the method for treating chronic hepatitis B infection (CHB) in humans comprises full-length recombinant HBs (e.g., SEQ ID NO: 1), amino acids 1-149 of HBc (e.g., SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21, and liposomes comprising dioleoylphosphatidylcholine (DOPC) and cholesterol. In certain embodiments, the recombinant protein HBs and HBc antigens are in the form of virus-like particles. In a specific embodiment, the composition comprises a truncated recombinant HBc and full-length recombinant HBs and an AS01 adjuvant at a ratio of 4: 1 or greater. In certain embodiments, the composition comprises a truncated core antigen consisting of amino acids 1-149 of HBc (e.g., SEQ ID NO: 2) and full-length recombinant HBs (e.g., SEQ ID NO: 1) and an AS01 at a ratio of 4: 1. B-4 .

[0116] In another aspect, there is provided a use of an immunogenic composition in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in humans, the immunogenic composition comprising a replication-deficient chimpanzee adenovirus (ChAd) vector, the replication-deficient chimpanzee adenovirus (ChAd) vector comprising a polynucleotide encoding hepatitis B surface antigen (HBs), a nucleic acid encoding hepatitis B virus core antigen (HBc), and a nucleic acid encoding a human constant chain (hIi) fused to the HBc, wherein the method for treating chronic hepatitis B infection comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In one embodiment, the composition comprises a ChAd vector selected from ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan 5, Pan 6, Pan 7 (also known as C7), and Pan 9, in particular ChAd63 or ChAd155. In certain embodiments, the ChAd vector comprises a vector insert encoding HBc and HBs separated by a spacer region incorporating a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert encoding hIi, HBc, 2A, and HBs, e.g., encoding a vector having Fig. 22In some embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), which are separated by a spacer region encoding the 2A cleavage region of foot-and-mouth disease virus (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto). In some embodiments, HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) is fused to hIi (e.g., SEQ ID NO: 7 or an amino acid sequence having at least 98% homology thereto or SEQ ID NO: 12 or an amino acid sequence having at least 98% homology thereto). For example, HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 7), or HBc (e.g., SEQ ID NO: 11) is fused to hIi (e.g., SEQ ID NO: 12). 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 an alternative 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 given in SEQ ID NO: 10. In an alternative embodiment, the composition comprises a ChAd155 vector comprising a polynucleotide vector insert having the nucleotide sequence given in SEQ ID NO: 14.

[0117] In a further aspect, there is provided a use of an immunogenic composition in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in a human, the 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 virus core antigen (HBc), wherein the method for treating chronic hepatitis B infection comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In one embodiment, the composition comprises an MVA vector comprising a vector insert encoding HBc and HBs separated by a spacer region incorporating a sequence encoding the 2A cleavage region of foot-and-mouth disease virus. In a specific embodiment, the composition comprises an MVA vector comprising a polynucleotide vector insert encoding HBc, 2A, and HBs, e.g., encoding a vector having Fig.21In certain embodiments, the vector insert encodes HBc (e.g., SEQ ID NO: 11 or an amino acid sequence having at least 98% homology thereto) and HBs (e.g., SEQ ID NO: 1 or an amino acid sequence having at least 98% homology thereto), separated by a sequence encoding a spacer region (e.g., SEQ ID NO: 3 or an amino acid sequence having at least 98% homology thereto) that is coupled to the 2A cleavage region of foot-and-mouth disease virus. In one embodiment, the composition comprises an MVA vector comprising 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 given in SEQ ID NO: 6.

[0118] In a further aspect, there is provided a use of an immunogenic composition in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in a human, the immunogenic composition comprising 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, wherein the method of treating chronic hepatitis B infection comprises administering the composition with at least one other immunogenic composition in a prime-boost regimen. In one embodiment, the composition comprises a truncated recombinant HBc comprising an assembly domain of HBc, e.g., amino acids 1-149 of HBc. In one embodiment, the composition comprises full-length recombinant HBs (e.g., SEQ ID NO: 1), amino acids 1-149 of HBc (e.g., SEQ ID NO: 2), and an adjuvant comprising MPL and QS-21 (e.g., AS01 adjuvant, e.g., AS01 B-4 or AS01 E-4 ). In certain embodiments, the recombinant protein HBs and HBc antigens are in the form of virus-like particles.

[0119] In one embodiment, there is provided the use of an immunogenic combination product in the preparation of a medicament for treating chronic hepatitis B infection (CHB) in humans, the immunogenic combination product comprising: 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); ii. a composition comprising a modified vaccinia virus Ankara (MVA) vector, wherein the modified vaccinia virus Ankara (MVA) vector comprises a polynucleotide encoding hepatitis B surface antigen (HBs) and a nucleic acid encoding hepatitis B virus core antigen (HBc); and iii. a composition comprising recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, wherein the method for treating chronic hepatitis B infection comprises administering the composition to the human sequentially or simultaneously.

[0120] In a specific embodiment, the use of the immunogenic combination product in the preparation of a medicament for treating CHB comprises: i. A composition comprising a replication-deficient ChAd vector, wherein the replication-deficient ChAd vector comprises a polynucleotide encoding HBs, a nucleic acid encoding HBc and a polynucleotide encoding hIi; ii. a composition comprising an MVA vector comprising a polynucleotide encoding HBs and a nucleic acid encoding HBc; and iii. a composition comprising recombinant HBs, truncated HBc and an adjuvant comprising MPL and QS-21, The method for treating CHB comprises the following steps: a) administering the composition i. to said human; b) administering the composition ii. to said human; and c) administering the composition iii. to said human, wherein the steps of the method are performed sequentially, wherein step a) precedes step b), and step b) precedes step c). In a further embodiment, step c) is repeated, and the steps of the method are performed sequentially in the order a), b), c), c). In another embodiment, step c) is performed simultaneously with step a) and / or with step b).

[0121] In a further aspect, the invention provides a kit 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 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 recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, and instructions for sequentially or simultaneously administering the components for treating CHB.

[0122] Application In one embodiment of the disclosed method, the disclosed composition is administered via intranasal, intramuscular, subcutaneous, intradermal or topical routes. Preferably, it is administered via the intramuscular route.

[0123] Intranasal administration is to apply the composition to the mucous membrane of the entire respiratory tract including the lungs. More specifically, the composition is applied to the mucous membrane of the nose. In one embodiment, intranasal administration is achieved by means of spray or aerosol. Intramuscular administration refers to the injection of the composition into any muscle of an individual. Exemplary intramuscular injections are applied to the deltoid muscle, vastus lateralis muscle or ventral buttocks and dorsal buttocks regions. Preferably, it is applied to the deltoid muscle. Subcutaneous administration refers to the injection of the composition into the lower skin. Intradermal administration refers to the injection of the composition into the dermis between the skin layers. Topical application is to apply the composition to any part of the skin or mucous membrane without penetrating the skin with a needle or a suitable device. The composition can be topically applied to the mucous membrane of the mouth, nose, genital area and / or rectum. Topical application includes modes of application, such as sublingual and / or buccal administration. Sublingual administration is to apply the composition sublingually (e.g., using an oral thin film (OTF)). Buccal administration is to apply the carrier via the buccal mucosa of the cheek.

[0124] The method disclosed herein can take the form of a priming-boosting immunization scheme. Therefore, the composition used in the method for treating CHB is disclosed herein, and the method for treating CHB is a priming-boosting immunization method. In many cases, a single administration of an immunogenic composition is not enough to generate many long-acting immune cells required for effective protection or therapeutic treatment of the disease. Therefore, in order to establish a lasting and protective immunity or treatment or functional cure of a given disease for a specific pathogen or disease, it may be necessary to use repeated attacks of biological agents specific to the pathogen or disease. The administration scheme including repeated administration of an immunogenic composition or vaccine for the same pathogen or disease is referred to as a "priming-boosting scheme". In one embodiment, the priming-boosting scheme involves at least two administrations of an immunogenic composition for hepatitis B. The first administration of an immunogenic composition is referred to as "priming", and any subsequent administration of the same immunogenic composition or an immunogenic composition for the same pathogen is referred to as "boosting". It should be understood that 2, 3, 4 or even 5 administrations are also considered for boosting immune responses. The time period between priming and boosting is optionally 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks or 12 weeks. More specifically, it is 4 weeks or 8 weeks. If more than one boost is performed, 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 previous boost. For example, the interval between any two boosts may be 4 weeks or 8 weeks.

[0125] The composition used in the disclosed method is applied in a treatment regimen, and the treatment regimen involves the application of other immunogenic components, which are each formulated in different compositions. The composition is advantageously applied in the same position or near the same position in a co-position. For example, the component can be intramuscularly applied to the same side or limb ("same side" application) or the opposite side or limb ("contralateral" application). For example, in the contralateral application, the first composition can be applied to the left deltoid muscle, and the second composition can be applied to the right deltoid muscle sequentially or simultaneously. Alternatively, in the same side application, the first composition can be applied to the left deltoid muscle, and the second composition can also be applied to the left deltoid muscle sequentially or simultaneously.

[0126] General preparation method ChAd155-hIi-HBV: The DNA fragment inserted as a transgene into the recombinant replication-defective 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, which are separated by the self-cleaving 2A region of foot-and-mouth disease virus (FMDV) [Donnelly et al. 2001]. The 2A region of FMDV allows the HBc-HBs fusion to be processed into separate protein antigens. In addition, the N-terminal portion of the gene encoding the HBc protein has been fused to the gene encoding the human major histocompatibility complex (MHC) class II-associated invariant chain p35 isotype (hIi). A schematic diagram of the hIi-HBV transgene sequence is provided at ( Fig. 22 )middle.

[0127] The 2A region (18 amino acids) has been supplemented with a 6 amino acid spacer at its N-terminus; a spacer of this nature has been reported to increase the efficiency of 2A-mediated cleavage. Region 2A-mediated protease cleavage occurs at the C-terminus of 2A just before the last proline in the 2A amino acid sequence. Proline is retained at the N-terminus of the HBs protein, while the hIi-HBc-2A polypeptide retains the 23 amino acids preceding the proline cleavage site.

[0128] Thus, after protease processing, expression of the transgene results in the production of two separate polypeptides: hIi-HBc-Spacer-2A and HBs. 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.

[0129] An expression cassette encoding an antigenic protein operably linked to a regulatory component in a manner that allows expression in a host cell is assembled into a ChAd155 vector plasmid construct as described previously (see WO2016 / 198621, which is incorporated by reference for the purpose of disclosing ChAd155 vector sequences and methods) to obtain ChAd155-hIi-HBV. The hIi-HBV transgene is under the transcriptional control of the human cytomegalovirus (hCMV) promoter and the bovine growth hormone polyadenylation signal (BGH pA). The expression cassette encodes HBs, HBc, and hIi amino acid sequences, wherein the hIi sequence is fused to the HBc N-terminus of HBc, and the HBs and HBc sequences are separated by a spacer region that incorporates the 2A cleavage region of foot-and-mouth disease virus for processing HBc and HBs into separate proteins.

[0130] In order to generate a replication-deficient recombinant ChAd155 adenovirus, the function of the gene region required for the replication and infectivity of the deleted adenovirus must be provided 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 Procell 92 cell line. The Procell 92 cell line is based on HEK293 cells expressing the adenoviral E1 gene (transfected with Tet repressor under the control of the human phosphoglycerate kinase-1 (PGK) promoter) and the G418-resistance gene (Vitelli et al. PLOS One (2013) 8 (e55435): 1-9). Procell 92.S is suitable for growth in suspension conditions and can be used to produce adenoviral vectors expressing toxic proteins.

[0131] Production of ChAd155-hIi-HBV API: The preparation of ChAd155-hIi-HBV viral particles (API) involves culturing Procell-92.S cells at a cell density of 5e5 cells / ml at the time of infection. The cells are then infected with ChAd155-hIi-HBV master virus seeds (MVS) using a multiplicity of infection of 200 vp / cell. The ChAd155-hIi-HBV viral harvest is purified after cell lysis, lysate clarification and concentration (filtration step) by a multistep process including anion exchange chromatography.

[0132] Vaccine formulation and filling The purified ChAd155-hIi-HBV bulk drug substance was then processed as follows: - Dilute purified ChAd155-hIi-HBV drug substance in formulation buffer. -Sterile filtration. -Fill the final container.

[0133] The ChAd155-hIi-HBV vaccine is a liquid formulation contained in a vial. The formulation buffer includes Tris (10 mM), L-histidine (10 mM), NaCl (75 mM), MgCl (1 mM) and EDTA (0.1 mM) with sucrose (5% w / v), polysorbate-80 (0.02% w / v) and ethanol (0.5% w / v), which is adjusted to pH 7.4 with HCl (water for injection to final volume).

[0134] MVA-HBV: MVA-HBV is a recombinant modified vaccinia virus Ankara (MVA) carrying two different HBV proteins: core and S protein, which are separated by a 2A peptide. The MVA-HBV construct was generated by the MVA-Red vector system [Di Lullo et al. 2010], which was derived from an MVA virus seed batch from attenuated passage 571 (referred to as MVA-571) described by Professor Anton Mayr [Mayr, A. et al. 1978].

[0135] The MVA-HBV transgene encodes the core nucleocapsid protein HBc and the small surface antigen HBs of HBV. The HBc-HBs sequence is separated by the self-cleaving 2A region of foot-and-mouth disease virus, which allows the fusion protein to be processed into separate HBc and HBs antigens as described above for adenoviral vectors. A schematic diagram of the transgene is provided at Fig.21 middle.

[0136] After protease processing, expression of the transgene results in the production of two separate polypeptides: HBc-Spacer-2A and HBs. For brevity, the HBc-Spacer-2A polypeptide is referred to as the HBc protein.

[0137] The expression cassette was subcloned into the MVA shuttle vector p94-elisaRen to generate the transfer vector p94-HBV. p94-HBV contains the antigen expression cassette under the control of the vaccinia P7.5 early / late promoter and flanked by the FlankIII-2 region and the FlankIII-1 region to allow insertion into the delIII of MVA by homologous recombination.

[0138] The generation of recombinant viruses is based on two events of in vivo recombination in CEF cells.

[0139] In brief, primary chicken embryo fibroblasts (CEF) are infected with MVA-Red, and then transfected with the p94-HBV carrying antigen transgene (and the EGFP marker gene under the control of synthetic promoter sP). The first recombination event occurs between the homologous sequences (FlankIII-1 and -2 regions) present in both the MVA-Red genome and the transfer vector p94-HBV, and causes the Hcred protein gene to be replaced by transgenic / eGFP boxes. The infected cells containing the MVA-Green intermediate are separated by FACS sorting, and are used to infect fresh CEF. The intermediate recombinant MVA produced by the first recombination carries both transgenic and eGFP boxes, but is unstable due to the presence of repeated Z regions.

[0140] Therefore, a spontaneous second recombination event involving the Z region occurs and removes the eGFP cassette. The resulting recombinant MVA is colorless and carries the transgene cassette.

[0141] Finally, cells infected with the untagged recombinant virus (MVA-HBV) were sorted by FACS, and MVA-HBV was cloned by terminal dilution and amplified in CEFs by conventional methods.

[0142] Production of MVA-HBV API MVA-HBV viral particles (API) were prepared in primary cell cultures of chicken embryo fibroblast (CEF) cells to a cell density between 1E6 and 2E6 cells / ml and then infected with MHC-HBV master virus seeds (MVS) at a multiplicity of infection between 0.01 and 0.05 PFU / cell. The MVA-HBV viral harvest was purified by a multistep process based on precipitation by centrifugation, resuspension and graded gradient centrifugation steps.

[0143] Vaccine formulation and filling The purified MVA-HBV bulk drug substance was then processed as follows: - Dilute purified MVA-HBVDS in formulation buffer. -Fill the final container.

[0144] The MVA-HBV vaccine is a liquid formulation contained in vials. The formulation buffer includes Tris(hydroxymethyl)aminomethane pH 7.7 (10 mM), NaCl (140 mM) and water for injection to the final volume.

[0145] HBs-HBc recombinant protein mixture: Production of HBc API The HBc recombinant protein (API) preparation process consists of inoculating pre-culture flasks with recombinant E. coli working seeds, followed by a fermentation process and a multi-step purification process, including harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0146] Production of HBs API The HBs recombinant protein (API) preparation process consists of inoculating pre-culture flasks with recombinant Saccharomyces cerevisiae working seeds, followed by a fermentation process and a multi-step purification process, including harvesting, extraction, clarification, and multiple chromatography and filtration steps.

[0147] Vaccine formulation and filling Purified HBs drug substance and HBc drug substance were diluted in a formulation buffer including sucrose as a cryoprotectant and poloxamer as a surfactant, filled and lyophilized in 4 mL clear glass vials. Example

[0148] Nonclinical development goals : Strong and functional CD8+ and CD4 + T cell responses, especially to HBcAg, have been associated with HBV clearance and resolution of infection [Boni, 2012; Li, 2011; Liang, 2011; Lau, 2002; Bertoletti, 2012]. In addition, anti-S antibodies prevent HBV transmission to uninfected hepatocytes and may be critical for controlling HBV replication rebound after treatment [Rehermann 2005; Neumann 2010]. The proposed vaccination regimen involves a heterologous prime-boost schedule with two viral vectored vaccines (ChAd155-hIi-HBV and MVA-HBV) encoding hepatitis B core (HBc) and hepatitis B surface (HBs) antigens to induce a strong CD8 + T-cell responses, together with sequential or simultaneous administration of AS01 B-4 -Adjuvanted HBc-HBs protein to induce strong antigen-specific CD4 in CHB patients + T-cell and antibody responses. This vaccine-induced immune response should ultimately translate into a substantial decrease in HBsAg concentrations or HBsAg loss (ie, HBsAg concentrations below detectable levels), which is considered a sign of complete and durable control of HBV infection.

[0149] The main objectives of nonclinical development are to: Demonstration of investigational vaccine components (e.g., ChAd155-hIi-HBV, MVA-HBV, and HBc-HBs / AS01 B-4 ) in naive and HBV-tolerant mice to guide the choice of vector construct, the inclusion of hIi, the composition of the protein formulation (including the adjuvant system), and the schedule of immunization. Demonstrate safety of the complete vaccine regimen in HBV-tolerant mice (non-GLP studies) and in a repeat-dose GLP toxicity study in NZW rabbits • Documentation of the biodistribution of the vector in vaccinated animals (GLP studies).

[0150] Nonclinical strategies and rationale for selecting animal models Immunogenicity packages are first generated in healthy mice to guide the choice of vector construct, protein formulation (including adjuvant system), and schedule of immunization.

[0151] Most preclinical experiments were performed in inbred CB6F1 (a cross between C57Bl / 6 and BALB / c mice) mice (a model previously used to evaluate T-cell responses elicited by AS01-adjuvanted vaccine candidates and adenoviral vectors [Lorin, 2015]) to support the selection of vector constructs, protein formulation (including adjuvant system), and schedule of immunization.

[0152] Use HLA.A2 / DR1 mice (transgenic for human HLA-A2 and HLA-DR1 molecules) to evaluate candidate vaccines for induction of HBc-specific CD8 + The ability of T-cell responses to be expressed was unknown, as no such responses were detected against this antigen in inbred CB6F1 mice. This is most likely due to the absence of H2-K in the HBc sequence of the studied vaccine. b An MHC-I-restricted immunodominant epitope (MGLKFRQL) based on the sequence of HBV genotype A / subtype adw with one amino acid difference, where isoleucine (I) replaces phenylalanine (F) in the epitope (MGLKIRQL), as reported by Riedl et al. [Riedl, 2014]. HBV-specific CD4 + T-cells and antibodies.

[0153] Animal models that can be used to evaluate the efficacy of therapeutic vaccines are limited because HBV only naturally infects chimpanzees and humans. Mouse models have been developed in which the entire HBV genome is expressed by integrating the viral genome into the host genome (HBV transgenic mice) or by infection with replicative HBV DNA or a vector expressing the HBV genome. Although these do not reproduce chronic HBV pathogenesis, viral replication intermediates and proteins can be detected in the liver, and immune tolerance is observed.

[0154] The AAV2 / 8-HBV-transduced HLA.A2 / DR1 mouse model recapitulates the virological and immunological features of chronic HBV infection and is selected [Dion, 2013; Martin, 2015] To demonstrate that the vaccine regimen can overcome tolerance to HBs and HBc antigens. To evaluate the effects of liver-infiltrating HBc-specific CD8+ T-cells that may target HBcAg-expressing hepatocytes on liver histology (hematoxylin-eosin [H&E] staining) and ALT / AST levels.

[0155] Finally, standard animal models for biodistribution (Sprague-Dawley rats) and toxicology studies (NZW rabbits) have been selected to evaluate the candidate vaccines because, although they are not infection models, they are able to generate pharmacologically relevant immune responses to vector-expressed and recombinant proteins and are recognized species for toxicity testing of vaccines. These species have also been previously used for AS01 B In the toxicology testing program of adjuvants and their immunopotentiators MPL and QS-21.

[0156] Nonclinical Pharmacology Many preclinical studies have been conducted to demonstrate the efficacy of investigational vaccine components (e.g., ChAd155-hIi-HBV, MVA-HBV, and HBc-HBs / AS01 B-4 ) in untreated and HBV-tolerant animals after intramuscular administration. First, the investigational vaccines adjuvanted with viral vectors (ChAd155-hIi-HBV and MVA-HBV) and HBV recombinant proteins (HBc-HBs / AS01 B-4 ) to assess the antigen-specific immunogenicity profile. In the second phase the immunogenicity and safety profile of the intended full vaccine regimen in FTiH (first in humans) was assessed.

[0157] Material Doses of AS01 Adjuvant System Used in Nonclinical Immunogenicity Studies AS01 B-4 The adjuvant system consists of the immunopotentiators QS-21 (triterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A) with liposomes as vehicles for these immunopotentiators and sorbitol. B-4 (0.5 mL) contains 50 μg QS-21 and 50 μg MPL. 1 / 10 of the human dose (ie 50 μl) is the volume injected in mice (corresponding to 5 μg QS-21 and MPL).

[0158] AS01 E-4 Adjuvant system corresponds to AS01 B-4 Two-fold dilutions. 1 / 10 of the human dose (ie 50 μl) is the volume injected in mice (corresponding to 2.5 μg QS-21 and MPL).

[0159] Cellular immune response - intracellular cytokine staining (ICS) Fresh pools of peripheral blood leukocytes (PBL), splenocytes, or liver infiltrating lymphocytes collected at different time points were stimulated ex vivo for 6 h with a pool of 11aa overlapping 15-mers covering the HBc or HBs sequence. CD4+ cells expressing IFN-γ and / or IL-2 and / or tumor necrosis factor (TNF)-α were measured by ICS. + or CD8 + The number of T cells was used to assess HBc- and HBs-specific cellular responses. Technical acceptance criteria for considering ICS results included the acquisition of CD8 + T or CD4 + Minimum number of T cells >3000 events. Alternatively, IFN-γ-ELISpot was performed after restimulation of splenocytes with the same peptide as for ICS.

[0160] Humoral immune response - Enzyme-linked immunosorbent assay (ELISA) HBc- and HBs-specific antibody responses were measured by ELISA in serum from immunized mice at different time points. In short, 96-well plates were coated with HBc or HBs antigens. Each serum sample was then added in serial dilutions and incubated for 2 hours. Biotinylated anti-mouse F(ab)'2 fragments were then added and the antigen-antibody complex was revealed by incubation with streptavidin horseradish peroxidase complex and peroxidase substrate o-phenylenediamine dihydrochloride / H2O2. For each time point and each antigen (HBc, HBs), an analysis of variance (ANOVA) model was fitted on log10 titers (including groups, studies and interactions as fixed effects) and using a heterogeneous variance model (assuming that there were no identical variances between groups). The model was used to estimate the geometric mean (and its 95% CI) and the geometric mean ratio and its 95% CI. Since no predefined criteria were set, the analysis was descriptive and the 95% CI of the ratio between groups was calculated without adjusting for multiplicity.

[0161] ALT / AST measurement Quantify the levels of ALT and AST in mouse serum using the following commercial kits: Alanine aminotransferase activity assay kit SigmaAldrich catalog number MAK052 Aspartate aminotransferase activity assay kit SigmaAldrich catalog number MAK055.

[0162] Serum HBs antigen quantification Circulating HBs antigen in mouse sera was quantified using Monolisa Anti-HBs PLUS from BIO-RAD (Cat. No. 72566) and international standards (Abbott Diagnostics).

[0163] Histopathological analysis The livers were collected (one lobe per liver) and stored in 10% formaldehyde fixative. All samples for microscopic examination were trimmed based on the RITA guidelines [Ruehl-Fehlert, 2003; Kittel 2004; Morawietz 2004], embedded in paraffin, sectioned at approximately 4 micrometers thickness, and stained with H&E. The histological activity (necroinflammatory lesions) and fibrosis were graded according to the METAVIR scoring system [Bedossa, 1996; Mohamadnejad, 2010; Rammeh, 2014]. The inflammatory cell foci were graded according to the Desmet score as described by Buchmann et al. [Buchmann, 2013].

[0164] The statistical analyses performed in each study are detailed in the section covering each individual study.

[0165] Example 1 - Evaluation of ChAd155-HBV (with and without hIi) priming in the HLA.A2 / DR1 transgenic mouse model and MVA-HBV boost Target The primary goal of this experiment was to determine whether priming with a dose of ChAd155-HBV (with or without hIi) followed by a booster dose of MVA-HBV could induce a robust CD8 response to HBc in HLA.A2 / DR1 mice transgenic for human MHC-I / II molecules. + In addition, a head-to-head comparison between ChAd155-HBV with and without hIi was performed to investigate whether the hIi sequence further increased HBc-specific CD8 + The potential of T-cell responses was assessed as previously reported for other antigens [Spencer, 2014; Capone, 2014]. HBs-specific CD8 + T-cell responses and HBc- and HBs-specific CD4 + T-cell and antibody responses.

[0166] Study Design HLA.A2 / DR1 mice (11 mice per group) were treated with 10 8 vp of ChAd155-HBV (with and without hIi) and on day 28, 10 7 pfu of MVA-HBV (without hIi) were used for boosting (Table 1). Mice were sacrificed 14 days after the first immunization (14dpI) (priming) or 7 days after the second immunization (7dpII) (boosting) to measure HBc- and HBs-specific humoral and cellular immune responses in serum and spleen, respectively.

[0167] Table 1 Study design of ChAd155-HBV (with and without hIi) prime / MVA-HBV (without hIi) boost experiments in HLA.A2 / DR1 mice.

[0168] Statistical analysis In the case of including 2 groups (with or without hIi) and experiments (results from 3 experiments after priming and results from 2 experiments after boosting) as fixed parameters, log10 CD8 + ANOVA models were fitted on T-cell frequencies using a heterogeneous variance model. This model was used to estimate geometric means (and their 95% CIs) and geometric mean ratios and their 95% CIs.

[0169] result HBc- and HBs-specific T-cell responses Both ChAd155-HBV and ChAd155-hIi-HBV vectors induce HBc-specific CD8 + T-cell response ( Figure 1 A) The presence of hIi tends to induce higher CD8 + T-cell responses. MVA-HBV boost in mice immunized with ChAd155-hIi-HBV induces HBc-specific CD8 + A 3-fold increase in T-cell responses was observed in the ChAd155-HBV group, whereas no increase was observed for the ChAd155-HBV group.

[0170] Both vectors induced HBs-specific CD8 + T-cell responses, and the MVA-HBV boosting effect was more pronounced in mice primed with the ChAd155-HBV construct ( Figure 1 B).

[0171] HBc- and HBs-specific CD4 + T-cell responses were low (data not shown).

[0172] The results of this experiment were consistent with those of two other similar independent experiments (data not shown). Although each individual study was not statistically powered to compare groups due to limitations related to animal availability, a macro-analysis of the three studies was performed to compare HBc-specific CD8 induced by ChAd155-HBV vs. ChAd155-hIi-HBV after priming and after MVA-HBV boost. + T-cell responses.

[0173] Statistical analysis showed that HBc-specific CD8 + T-cell responses were significantly higher than those elicited by the prime-boost regimen using ChAd155-HBV, supporting the use of human constant chain sequences fused to HBc sequences.

[0174] a. 14 days after dose I, the geometric mean ratio (hIi / no hIii) was estimated to be 3.27 (95% CI: 1.57–6.79) b. 7 days after dose II, the geometric mean ratio (hIi / no hIii) was estimated to be 6.25 (95% CI: 2.11–18.51).

[0175] HBc- and HBs-specific antibody responses Immunization with ChAd155-HBV induced HBc-specific antibodies, but immunization with ChAd155-hIi-HBV did not. After MVA-HBV boost, anti-HBc antibody responses were comparable between groups primed with ChAd155-HBV or ChAd155-hIi-HBV ( Figure 2 No HBs-specific antibody response was detected (data not shown).

[0176] in conclusion ChAd155-hIi-HBV induced the highest CD8 against HBc when compared with ChAd155-HBV + T-cell responses were induced and further increased after MVA-HBV boost.

[0177] Example 2 - HBc-HBs / AS01 in inbred mice (CB6F1) B-4 Immunogenicity studies Target The primary goal of this immunogenicity study was to determine the B-4 Whether HBc and HBs proteins are able to induce HBc- and HBs-specific humoral and T cell responses when co-formulated in the medium.

[0178] Study Design CB6F1 mice (30 mice per group) aged 6 to 8 weeks were treated with 50 μl AS01 on days 0, 14, and 28. B-4 HBc, HBs or HBc-HBs (listed in Table 2 below) formulated in 30% HBc were immunized intramuscularly 3 times. HBc- and HBs-specific T cell responses were measured on 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.

[0179] Table 2: Treatment groups Group antigen 1 <![CDATA[1μgHBc / AS01 B-4 (HBc / AS01 B-4 )]]> 2 <![CDATA[1μgHBs / AS01 B-4 (HBs / AS01 B-4 )]]> 3 <![CDATA[1μgHBc+1μgHBs / AS01 B-4 (HBc-HBs / AS01 B-4 )]]> 4 NaCl .

[0180] Statistical analysis Statistical analysis was performed using ANOVA on log10 values ​​with 1 factor (group) using a heterogeneous variance model, i.e., equal variance was not assumed for the different levels of the factor. + and CD8 + The analysis was performed for T cells) and antigen specificity (HBs and HBc). Back-transformation of log10 values ​​was used to obtain estimates of geometric mean ratios between groups and their 95% confidence intervals (CI). Tukey's method was used to adjust for multiplicity. Multiplicity-adjusted 95% confidence intervals are provided.

[0181] result HBc- and HBs-specific T-cell responses Use HBc / AS01 B-4 、HBs / AS01 B-4 or HBc-HBs / AS01 B-4 Immunization of mice induces effective CD4 against two antigens + T-cell response ( Figure 3 ). and HBc / AS01 B-4 Compared with HBc-HBs / AS01 B-4 Preparation of HBc-specific CD4 + The magnitude of the T-cell response was significantly lower (2.3-fold, P-value = 0.0468), indicating interference of HBs with HBc-specific T-cell responses. + The levels of T-cells were still considered strong and much higher than those in the control group. + No such interference was observed at the level of T-cell responses.

[0182] HBs / AS01 B-4 or HBc-HBs / AS01 B-4 The formulation induces strong HBs-specific CD8 + T-cell response ( Figure 4 None of the candidate vaccines induced detectable HBc-specific CD8 + T-cell responses (data not shown) were as expected using this mouse model, as the H2-K b The MHC-I restricted immunodominant epitope - MGLKFRQL - was reported by others [Riedl, 2014].

[0183] HBc- and HBs-specific antibody responses Each of the three preparations induced high levels of anti-HBc and / or anti-HBs antibodies (see Figure 5 ). and HBc / AS01 B-4 Compared with HBc-HBs / AS01 B-4 A significantly lower level of anti-HBc antibody response was observed in the HBc-HBs / AS01 formulation (2.35-fold, P < 0.0001). B-4 The presence of HBc in the combination did not negatively affect the anti-HBs antibody response ( Figure 5 ).

[0184] in conclusion All preparations (HBs / AS01 B-4 、HBc / AS01 B-4 and HBc-HBs / AS01 B-4 ) are immunogenic and induce cellular and humoral responses against both antigens, except for the HBc-specific CD8 + T cell response (as expected in this model). B-4 The anti-HBc response induced by the preparation was lower than that caused by HBc / AS01 B-4 The response elicited by HBc-HBs was 4:1, indicating interference associated with 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 was able to restore anti-HBc immune responses (antibodies and specific CD4 + T-cells) without affecting the anti-HBs antibody response. The preparation HBc-HBs 4-1 / AS01 was selected B-4 For use in subsequent nonclinical immunogenicity studies with adjuvanted protein formulations.

[0185] Example 3 - Adjuvant comparison experiment in inbred mice (CB6F1) Target The main purpose of this experiment was to compare the use of different adjuvants (alum, AS01 B-4 or AS01 E-4 HBc and HBs antigens formulated together or without adjuvant induce strong CD4 + Capacity of T-cell and humoral responses.

[0186] Study Design CB6F1 mice (35 mice for Groups 1-4 and 25 mice for Group 5) aged 6 to 8 weeks were treated with alum, AS01 B-4 or AS01 E-4The prepared HBc-HBs antigen (4 μg-1 μg) (listed in Table 3 below) was intramuscularly immunized three times. B-4 Compared with AS01 E-4 The adjuvant system contained half the amount of the immunopotentiators QS-21 and MPL. HBc- and HBs-specific T cell responses were measured on fresh PBL 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.

[0187] Table 3: Treatment groups Group antigen 1 HBc-HBs4-1 2 HBc-HBs4-1 / Alum 3 <![CDATA[HBc-HBs4-1 / AS01 B-4 ]]> 4 <![CDATA[HBc-HBs4-1 / AS01 E-4 ]]> 5 NaCl .

[0188] Statistical analysis For statistical analysis, ANOVA models were fitted on log2 T cell frequencies and on log10 antibody titers (including group as a fixed effect) using a heterogeneous variance model (identical variance was not assumed between groups, and the NaCl group was excluded from the analysis). This model was used to estimate the geometric means (and their 95% CIs) as well as the geometric mean ratios (AS01 B Compared with the 3 other groups) and their 95% CI. HBc- and HBs-specific CD4 + Dunnett's adjustment was applied for T cell frequency (primary endpoint) and anti-HBs antibody titer (secondary endpoint). For other responses / time points, the analysis was descriptive and no adjustment was applied.

[0189] result HBc- and HBs-specific T-cell responses AS01-adjuvanted formulations elicited significantly higher HBc-specific CD4 + T-cell response ( Figure 6 B) In AS01 B-4 and AS01 E-4 No statistically significant differences were observed between the preparations. As previously observed in CB6F1 mice, HBc-specific CD8 + T-cell responses (data not shown).

[0190] HBs-specific CD4 + ( Figure 6 A) and CD8 + ( Figure 6 C) T-cell responses were significantly higher in AS01B-4 and AS01 E-4 No statistically significant differences were observed between formulations.

[0191] HBc and HBs-specific antibody responses AS01-adjuvanted formulations elicited significantly higher anti-HBc and anti-HBs total IgG responses compared to alum-adjuvanted and non-adjuvanted formulations ( Figure 7 ). By AS01 B-4 and AS01 E-4 - There was no statistical difference in the total IgG antibody responses elicited by the adjuvanted formulations.

[0192] in conclusion Overall, the AS01 adjuvant system (AS01 E-4 or AS01 B-4 ) induced the highest humoral and cellular responses against HBc and HBs.

[0193] Example 4 - ChAd155-hIi-HBV / MVA-HBV / HBs-HBc / in HLA.A2 / DR1 transgenic mice AS01 B-4 Immunogenicity assessment of vaccine regimens Target The goal of this study was to evaluate the immunogenicity of different vaccine regimens consisting of: priming / boosting with ChAd155-hIi-HBV / MVA-HBV viral vectors followed by either or two doses of HBc-HBs 4-1 / AS01 B-4 protein, or with two doses of HBc-HBs 4-1 / AS01 B-4 Protein co-administration.

[0194] Study Design The first group of mice (N=16) were immunized with ChAd155-hIi-HBV on day 0, followed by MVA-HBV 28 days later. This prime / boost viral vector regimen was followed by two doses of HBc-HBs 4-1 μg / AS01 injected 14 days apart. B-4 (Table 4) The second group of mice (N=16) were treated with ChAd155-hIi-HBV and HBc-HBs 4-1 / AS01 on day 0. B-4 Immunization, followed by HBc-HBs 4-1 / AS01 28 days later B Co-administered MVA-HB boost. Two subsequent MVA-HBV and HBc-HBs 4-1 / AS01 were administered 14 days apart. BThe mice of the third group (N=8) were injected with NaCl as a negative control. The mice were sacrificed 7 days after the second immunization (7dpII) and 7 days after the fourth immunization (7dpIV) to measure the HBc- and HBs-specific humoral (serum) and cellular immune responses (to spleen cells and liver infiltrating lymphocytes).

[0195] The study was descriptive, and no statistical sample size justification and analysis were performed.

[0196] Table 4: Treatment groups

[0197] result HBc- and HBs-specific CD8 + T-cell response (splenocytes) When compared with ChAd155-hli-HBV / MVA-HBV alone injected at 7 dpII (Group 1), HBc-HBs 4-1 / AS01 B-4 Co-administration with ChAd155-hIi-HBV vector as priming and with MVA-HBV vector as boosting (Group 2) induced HBc-specific CD8 + A 4-fold increase in T-cell responses ( Figure 8 ). Similar CD8 serotypes against HBs were induced in both groups. + T-cell response ( Figure 8 ).

[0198] At 7 dp IV, HBc-HBs / AS01 was then administered B-4 Afterwards, HBc-, but not HBs-specific CD8 + T-cell responses were significantly enhanced (5-fold compared to 7dpII) (Group 1). When two additional doses of MVA-HBV / HBc-HBs4-1 / AS01 were co-administered B-4 No HBc- or HBs-specific CD8 + Further increase of T-cells (Group 2).

[0199] HBc- and HBs-specific CD4 + T-cell response (splenocytes) Low levels of HBc- and HBs-specific CD4 were detected after prime-boost ChAd155-hIi-HBV / MVA-HBV immunization + T-cells (median values ​​were 0.17% and 0.11%, respectively) (Group 1), while HBc-HBs 4-1 / AS01 B-4When co-administered with prime-boost ChAd155-hIi-HBV / MVA-HBV (Group 2), effective responses were observed against both antigens ( Fig. 9 ).

[0200] At 7 dp IV, HBc-HBs 4-1 / AS01 was then administered after ChAd155-hIi-HBV / MVA-HBV prime-boost B-4 (Group 1) Significantly enhanced HBc- and HBs-specific CD4 + Finally, when two additional doses of MVA-HBV and HBc-HBs / AS01 were added B-4 Co-administered with HBc-HBs / AS01 B-4 When the prime-boost ChAd155-hIi-HBV / MVA-HBV vaccinated mice were co-administered at the same time point (Group 2), HBs-specific CD4 + In this same group, HBc-specific CD4 + T-cells remained at the same level as 7dpost II.

[0201] HBc- and HBs-specific T-cell responses detected in liver infiltrating lymphocytes The presence of vaccine-induced T-cell responses in the liver was investigated by ICS 7 days after the last immunization. In order to have a sufficient number of liver infiltrating lymphocytes for in vitro restimulation and ICS, a pool of cells recovered after perfusion of 3 or 4 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.

[0202] Both vaccine regimens elicited HBc- and HBs-specific CD4 + T-cells ( Fig.10 Strong HBc-specific CD8 + T-cell responses, while much lower HBs-specific CD8 + Frequency of T-cells.

[0203] HBc- and HBs-specific antibody responses ChAd155-hIi-HBV / MVA-HBV and HBc-HBs 4-1 / AS01 B-4 Co-administration of (Group 2) induced the highest amount of anti-HBc antibodies at 7dpII ( Fig.11 ). MVA-HBV+HBc-HBs / AS01B-4 Subsequent injections of HBc-HBs / AS01 did not further increase the level of anti-HBc antibody response (7 dp IV). B-4 After that, a significant increase in anti-HBc-specific antibody response was observed at 7dpIV (Group 1). B-4 The presence of the components appears to be important in the timeline for eliciting effective anti-HBs antibodies, as no anti-HBs antibody responses were detected in animals after immunization with ChAd155-hIi-HBV / MVA-HBV ( Fig.11 ). After the last immunization, the highest response amplitude was observed in the co-administration group (Group 2).

[0204] in conclusion ChAd155-hIi-HBV / MVA-HBV elicits low but detectable HBc-specific CD4 + T-cell response, while HBc-HBs 4-1 / AS01 B-4 The initial prime-boost immunization with ChAd155-hIi-HBV / MVA-HBV induced potent HBc- and HBs-specific CD8 + T-cell response, in which HBc-HBs / AS01 B-4 After boosting, the HBc-specific response increased further.

[0205] Interestingly, when ChAd155-hIi-HBV / MVA-HBV was combined with HBc-HBs 4-1 / AS01 B-4 When co-administered, high levels of HBc- and HBs-specific CD4 + and CD8 + T-cells and antibodies. Using MVA-HBV+HBc-HBs / AS01 B-4 Further immunization with WT mice did not further increase the levels of these responses.

[0206] In addition, vaccine-induced HBc- and HBs-specific CD4 + and CD8 + T-cells.

[0207] Example 5 - Evaluation of T and B-cell tolerance of the "invariant chain" sequence Ii encoded by the ChAd155 vector: ChAd155 construct encoding mouse Ii sequence (mIi) was used in CB6F1 mice Target Immunogenicity studies were performed in CB6F1 mice using a ChAd155 construct encoding the mouse Ii sequence (mIi): ChAd155-mIi-HBV, to investigate T- and B-cell tolerance to the "invariant chain" sequence Ii in a syngeneic model.

[0208] Study Design With high doses (10 9 After two intramuscular immunizations (days 0 and 14) with ChAd155-mIi-HBV vectors containing 100 μg / mL of HBV-binding protein 2 (vp), the induction of autologous mIi-specific immune responses was assessed by IFN-γ ELISpot (in splenocytes) and by ELISA (in serum) (Table 5).

[0209] Table 5: Treatment groups Group preparation 1 <![CDATA[ChAd155-mIi-HBV(10 9 vp), on days 0 and 14]]> 2 PBS, on days 0 and 14 .

[0210] method For T-cell responses, 15-mer peptides overlapping by 11 amino acids, covering the mouse Ii sequence and arranged as a pool were used as antigens in the IFN-γ-ELISpot assay. For antibody responses, commercially available mouse Ii recombinant protein and a monoclonal antibody specific for mouse Ii were used to coat the ELISA plate and served as positive controls, respectively. As positive controls for "vaccine use", HBc- and HBs-specific T-cell responses were monitored in the IFN-γ-ELISpot assay.

[0211] result After the first and second immunizations with ChAd155-mIi-HBV, potent HBc-specific T-cell responses and lower, but detectable, HBs-specific T-cell responses were measured. Of note, the HBc Kb-restricted dominant class I epitope (MGLKFRQL) was added to this construct to monitor HBc-specific CD8 + T-cell responses were detected and spleen cells were restimulated with this specific sequence in an ELISpot assay. No anti-mIi antibodies were detected in any animal 2 weeks after the first or second immunization ( Fig.13 ) and mIi-specific T-cells ( Fig.12 ), indicating that immune tolerance to the mIi sequence is retained.

[0212] Example 6—ChAd155-hIi-HBV / MVA-HBV / HBc-HBs / AS01 B-4 Vaccine regimen in AAV2 / 8-HBV transfection Evaluation of immunogenicity and safety in HLA.A2 / DR1 mice Target The AAV2 / 8-HBV-transduced HLA.A2 / DR1 mouse model recapitulates the virological and immunological features 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.

[0213] 5x10 10 vg (viral genome) resulted 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 HBc antigen were detected in the liver up to 1 year after injection without associated significant liver inflammation. HBs and HBe antigens as well as HBV DNA could be detected in the serum up to 1 year. In addition, the establishment of immune tolerance to HBV antigens was observed in this alternative model of chronic HBV infection.

[0214] 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 both HBs and HBc antigens.

[0215] Evaluation of liver-infiltrating HBc-specific CD8 + Effects of T-cells on liver histology (H&E staining) and ALT and AST levels as surrogate parameters of liver function.

[0216] Study Design Two different vaccine regimens were tested, based on the use of HBc-HBs 4-1 / AS01 alone or in combination B-4 Sequential immunization with combined ChAd155-hIi-HBV and MVA-HBV (both encoding HBV core [HBc] and surface [HBs] antigens) followed by two additional doses of HBc-HBs 4-1 / AS01 B-4 (alone or in combination with MVA-HBV) (Table 6).

[0217] HLA.A2 / DR1 mice from groups 1, 2, and 3 were treated with 5x10 10 vg were transduced with AAV2 / 8-HBV vector (administered intravenously), while Group 4 served as a positive control for immunogenicity (no tolerance was established before vaccination).

[0218] Animals from group 1 (N=21) were immunized with ChAd155-hIi-HBV on day 31, followed by MVA-HBV on day 58. Two doses of HBc-HBs4-1 μg / AS01 were injected on days 72 and 86 after this prime / boost viral vector regimen. B-4 (Table 6).

[0219] Animals from group 2 (N=21) were immunized with ChAd155-hIi-HBV on day 31 and co-immunized with HBc-HBs 4-1 / AS01 B-4 Co-administered, followed by HBc-HBs 4-1 / AS01 on day 58 B Co-administered MVA-HBV boost. Two subsequent MVA-HBV and HBc-HBs 4-1 / AS01 were administered on days 72 and 86. B of co-immunity (Table 6).

[0220] Animals from Group 3 (N=21) were injected with NaCl on days 31, 58, 72, and 86 as negative controls.

[0221] Animals from Group 4 (N=8) received the same vaccine regimen as Group 2 (except they were not transduced with AAV2 / 8-HBV).

[0222] All vaccines are given intramuscularly.

[0223] Levels of HBs circulating antigen were measured in serum on days 23, 65 and 93 (Groups 1, 2 and 3).

[0224] HBs- and HBc-specific antibody responses were measured by ELISA in sera from 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+ T cells were evaluated in splenocytes and liver-infiltrating lymphocytes on days 65 (9 animals / group) and 93 (12 animals / group) after ex vivo restimulation and ICS (Groups 1, 2, and 3). + and CD8 + T cell responses. For animals from Group 4 (8 animals), these immunogenicity readouts were performed on day 93 only.

[0225] Regarding liver-related safety parameters, levels of AST and ALT were measured in serum on days 38, 65, and 93, and microscopic examination of liver sections stained with H&E was performed on days 65 and 93 to detect potential vaccine-related histopathological changes or inflammation (Groups 1, 2, and 3).

[0226] Table 6: Treatment groups *One mouse was found dead in Group 3 before Day 65, and one mouse was found dead in Group 2 before Day 93.

[0227] Statistical analysis AST and ALT levels Using an unstructured covariance structure, an ANOVA model of repeated measures (including sex, day, group and three pairwise interactions) was fitted on the log10-transformed enzymatic activity values. The model assumptions were verified. 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 the enzymatic activity of each group at each time point was derived from the model. The target group comparison was reported by the geometric mean ratio (GMR) also derived from the model. All of these statistical values ​​are presented with two-sided 95% confidence intervals. Multiplicity was not considered when calculating these GMRs.

[0228] All analyses were performed using SAS 9.2.

[0229] Humoral response Descriptive statistics were performed to calculate the number of responders. The cut-off values ​​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 not vaccinated).

[0230] Cellular response Descriptive analysis was performed to define HBc- and HBs-specific CD4 + or CD8 + Number of T cell responders. The cutoff value for responsiveness was defined as the 95th percentile of measurements performed in group 3 (AAV2 / 8-HBV transduced but not vaccinated).

[0231] 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 (Group 3).

[0232] Used alone (Group 1) or with HBc-HBs 4-1 / AS01 B-4Immunization with combined (group 2) ChAd155-hIi-HBV and MVA-HBV vectors induces HBc-specific CD8 + T cells (6 / 7 and 9 / 9 responders, respectively, at 7 days post-II), indicating bypass of tolerance to HBc antigen ( Fig.14 A) Two additional doses of HBc-HBs 4-1 / AS01 alone or in combination with MVA-HBV B-4 Only modest increases in these HBc-specific CD8 + T cell responses, as measured 7 days after the fourth dose, reached a median frequency of 1% in Group 1 and 1.45% in Group 2. HBc-specific CD8 + The frequency of T cells was higher in non-transduced HLA.A2 / DR1 mice from Group 4 (8 / 8 responders with a ~4-fold higher frequency 7 days post-IV), as expected due to immune tolerance to the HBc antigen. HBc-specific CD8 T cells were also detected in the livers of vaccinated mice. + T cells, where the profile is the same as in the spleen ( Fig.14 B).

[0233] HBc-specific CD4 elicited by two vaccine regimens in AAV2 / 8-HBV-transduced HLA-A2 / DR1 mice (Groups 1 and 2) + T cells were very low to undetectable, whereas robust responses were measured in non-transduced mice (Group 4), indicating that the vaccine regimen did not overcome CD4 T cells to HBc antigen under these experimental conditions. + T cell tolerance ( Fig.15 A, B).

[0234] HBs-specific CD8 + and CD4 + T cells In AAV2 / 8-HBV-transduced mice, HBs4-1 / AS01 was used alone (group 1) or in combination with HBc-HBs4-1 / AS01. B-4 Immunization with combined (group 2) ChAd155-hIi-HBV and MVA-HBV vectors elicits HBs-specific CD8 + T cells, in which two additional doses of HBc-HBs 4-1 / AS01 alone or in combination with MVA-HBV B-4 There is no further increase in strength after Fig.16 A). HBs-specific CD8 measured in animals from Groups 1 and 2 at the end of the vaccination schedule (7 days after the fourth dose). +The frequency of T cells was close to that detected in Group 4 (non-transduced HLA.A2 / DR1 mice, median = 0.62% 7 days after IV, 5 / 8 responders), indicating that T cell tolerance to HBs antigen was overcome. In most vaccinated animals, HBs-specific CD8 + T cells ( Fig.16 B).

[0235] When HBc-HBs 4-1 / AS01 is administered alone or in combination with a carrier B-4 After that, HBs-specific CD4+ T cells were induced starting from 7 days after the second vaccination in group 2 and starting from 7 days after the fourth vaccination in group 1. + T cells ( Fig.17 A). The vaccine schedule used in animals from Group 2 elicited HBs-specific CD4 + The frequency of T cells was approximately 3-fold higher (median = 3.7% 7 days after IV, 11 / 11 responders) and reached a level similar to that in Group 4 (non-transduced HLA.A2 / DR1 mice, median = 3% 7 days after IV, 8 / 8 responders), indicating that T cell tolerance to HBs antigen was completely overcome. + T cell responses were similar in all vaccinated animals, with HBs-specific CD4 + T cells ( Fig.17 B).

[0236] HBs- and HBc-specific antibody responses No anti-HBs antibody response was detected in HLA.A2 / DR1 mice 23 days after injection of AAV2 / 8-HBV vector, indicating strong humoral tolerance to HBs antigen. Immunization with ChAd155-hIi-HBV and MVA-HBV vector alone (Group 1) did not disrupt this tolerance, whereas immunization with HBc-HBs 4-1 / AS01 B-4 Immunization with the combined vector resulted in the induction of anti-HBs antibody responses in 15 of 21 animals on day 65 (Group 2) ( Fig.18 A) Two further doses of HBc-HBs 4-1 / AS01 were administered to group 1. B-4 elicited detectable anti-HBs antibodies (geometric mean titer (GMT) of 116.8 and 8 / 12 responders on day 93), and 2 additional doses of HBc-HBs 4-1 / AS01 in group 2 B-4Combined MVA-HBV further increased the magnitude of anti-HBs antibody responses up to a GMT of 775 (11 / 11 responders), while remaining -5-fold lower at day 93 than in non-AAV2 / 8-HBV transduced animals from Group 4 (GMT=3933).

[0237] Similarly, only when HBc-HBs 4-1 / AS01 is present in the vaccine regimen B-4 Anti-HBc antibody responses were induced only when the combination was added, with levels measured at day 93 in animals from group 2 being 3-fold higher (GMT=1335.5; 11 / 11 responders) compared to group 1 (GMT=442.8; 12 / 12 responders) ( Fig.18 B) Using the same vaccine regimen as in Group 2, higher anti-HBc antibody titers were induced in non-transduced mice (Group 4) (-27-fold, GMT = 357822).

[0238] These results show that the presence of the adjuvanted protein component in the vaccine regimen is crucial for breaking humoral tolerance to both HBc and HBs antigens. B-4 4 administrations) elicited the highest anti-HBc and anti-HBs antibody responses, but were still lower than those in non-AAV2 / 8-HBV-transduced mice (Group 4).

[0239] AST / ALT levels As liver-related inflammatory parameters, serum activities of AST and ALT were measured 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 vaccination) (all groups). Overall, AST and ALT levels were stable during the course of the vaccine regimen in AAV2 / 8-HBV-transduced HLA.A2 / DR1 mice (Groups 1 and 2) and were similar to the levels measured in mice that did not receive the vaccine (Group 3) ( Fig.19 ). On day 65, AST levels were found to be statistically significantly higher in animals from the vaccine groups (Groups 1 and 2) compared to the control group 3. However, on day 65, AST levels were surprisingly low in animals from Group 3 compared to the rest of the kinetics, suggesting that these differences were rather due to the particularly unexpectedly low values ​​obtained in the control group 3 at this time point, rather than to an increase in AST levels in the vaccine groups (Groups 1 and 2) ( Fig.19 A).

[0240] On day 38, slightly lower ALT levels were measured in animals from group 1 compared to control animals from group 3, but this difference was not considered clinically relevant ( Fig.19 B).

[0241] Microscopic examination of the liver Microscopic examination of liver sections stained with H&E was performed on days 65 and 93 to detect potential vaccine-related histopathological changes or inflammation (Groups 1, 2, and 3) (Table 7).

[0242] On day 65 (after the second viral vectored vaccine, with or without HBc-HBs 4-1 / AS01 B-4 There were no microscopic findings associated with the test items in AAV2 / 8-HBV-transduced HLA-A2 / DR mice at day 93 (7 days after the last injection of MVA-HBV) or day 93 (7 days after the last injection), i.e., no microscopic findings that could be associated with the vaccine components ChAd155-hIi-HBV, MVA-HBV, and HBc-HBs4-1 / AS01. B-4 Histopathological changes associated with the use of

[0243] Additionally, with the exception of control animal 3.13, which exhibited focal grade 1 fragmented necrosis, none of the animals exhibited morphological signs of chronic hepatitis.

[0244] Other microscopic findings noted in treated animals were considered to be incidental changes because they also occurred in controls, were of low incidence / magnitude, and / or were common background findings in mice of similar age [McInnes, 2012].

[0245] HBs antigen levels in sera from AAV2 / 8-HBV injected mice.

[0246] As already reported by Dion et al. [Dion, 2013], HBs antigen levels were higher in males compared with females 23 days after injection with the AAV2 / 8-HBV vector. These levels remained stable in all groups, with no detectable effect of the vaccination regimen ( Fig. 20 ). However, AAV2 / 8-HBV injected mice are not an animal model for studying the efficacy of vaccines against HBsAg.

[0247] in conclusion In an alternative 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 activated HBc- and HBs-specific IgG and CD8 + T cell responses and HBs-specific CD4 +T cell responses bypassed tolerance, although at lower levels than in untransduced mice, as expected due to strong immune tolerance. B-4 When co-administered, the magnitude of the antibody and T cell responses induced by the vaccine was higher than in vaccine regimens in which the vector and adjuvanted proteins were administered sequentially. In addition, while evaluating vaccine-associated liver inflammation by measuring serum activity of AST and ALT and by performing liver histopathological assessments, no increase in liver enzymes was detected in the vaccine group when compared to the unvaccinated group, and no microscopic findings could be associated with vaccine treatment. Altogether, these results show that under these experimental conditions, the tested vaccine candidates successfully restored HBs- and HBc-specific antibodies and CD8 + T cell responses and HBs-specific CD4 + T cell responses, without detectable associated signs of liver changes.

[0248] Summary of Nonclinical Immunology Data of Examples 1-6 Used in AS01 B-4 Studies in CB6F1 mice vaccinated with vaccine proteins formulated in an adjuvant system (HBc-HBs) showed that HBs antigens exerted significant effects on HBc-induced antibodies and CD4 + However, HBc-HBs / AS01 B-4 The combination vaccine was able to generate robust CD4 specificity for both vaccine antigens + T-cell and antibody responses.

[0249] - AS01 family-based formulations induced significantly higher CD4 + T-cell and antibody responses.

[0250] Administration of ChAd155-hIi-HBV in HLA.A2 / DR1 transgenic mice induces strong CD8 + T-cell responses and, to a lesser extent, CD8 + T-cell responses. The presence of hIi in the construct significantly enhanced the response to HBc antigen. Subsequent administration of MVA-HBV further increased CD8 + T-cell responses: After MVA boost, HBc-specific CD8 + The frequency of T-cells was higher, and HBs-specific CD8+ T-cell responses were not further enhanced.

[0251] When administered to HLA.A2 / DR1 transgenic mice, the complete vaccination regimen (i.e., sequential or simultaneous administration of viral vector and adjuvanted protein) induced robust CD4 + T-cells, CD8 + T-cell and antibody responses. In addition, vaccine-induced HBs- and HBc-specific CD4 + and CD8 + T-cells.

[0252] Immunogenicity studies were performed in CB6F1 using a ChAd155 construct encoding the mouse Ii sequence (mIi): ChAd155-mIi-HBV, to investigate T and B cell tolerance to the “invariant chain” sequence (Ii) in a syngeneic model. 9 vp) after two immunizations (days 0 and 14) with ChAd155-mIi-HBV vectors, the induction of autologous mIi-specific immune responses was assessed. No anti-mIi antibodies and mIi-specific T-cells were detected in any animal 2 weeks after the first or second immunization, indicating that immune tolerance to the mIi sequence was retained.

[0253] In a preclinical HBV-persistent mouse model (AAV2 / 8-HBV-transduced HLA.A2 / DR1 mice) in which immune tolerance to HBV antigens was observed, the vaccine regimen was able to induce HBc- and HBs-specific CD8 + T cells, HBs-specific CD4 + T cell and antibody responses to both HBs and HBc antigens break tolerance, although no HBc-specific CD4 + T cell responses. However, the levels of vaccine-induced responses in AAV-transduced mice were (and as expected) lower than those detected in untreated HLA.A2 / DR1 mice. In addition, while evaluating vaccine-associated liver inflammation by measuring serum activity of aspartate aminotransferase (AST) and ALT and by performing liver histopathological assessments, no increase in liver enzymes was detected in the vaccine groups when compared to the unvaccinated groups, and no microscopic findings could be associated with vaccine treatment. In summary, these results show that under these experimental conditions, the tested vaccine candidates successfully restored HBs- and HBc-specific antibodies and CD8 + T cell responses and HBs-specific CD4 + T cell responses, without detectable associated signs of liver changes.

[0254] Example 7 - Evaluation of the immunogenicity of different adjuvanted recombinant protein HBc / HBs ratios Target The purpose of this experiment is to confirm the effect of HBs antigen on HBc-induced CD4 + Negative interference of T cell responses, as seen in Example 2 at day 7 after the third immunization, where HBs and HBc antigens were mixed in a 1:1 ratio. A further objective was to evaluate various ratios of HBs / HBc to limit this interference and ensure at least effective HBc-specific CD4 + T cell responses, while generating robust HBc- and HBs-specific antibody responses.

[0255] Study Design On days 0, 14, and 28, 6-8 week-old CB6F1 mice (30 mice per group) were treated with 50 μl AS01 B or AS01 B-4 Various preparations (listed in Table 8) containing HBc and HBs antigens were immunized intramuscularly (gastrocnemius) 3 times. CB6F1 mice were randomly assigned to one of the study groups. The evaluation of HBc and HBs-specific T cell responses by intracellular cytokine staining (ICS) was performed by using leukocytes collected from 6 pools of 5 mice / group 7 days after the second and third immunizations. Serum was collected from individual mice 14 days after the second and third immunizations, and only sera from 20 randomized mice were tested due to statistical sample size analysis to evaluate HBc- and HBs-specific antibody total Ig responses.

[0256] Table 8: Study design for HBc / HBs ratio experiment in CB6F1 mice. Groups 6 and 7 were included in the experiment to address another objective and are omitted for clarity purposes. HD: human dose.

[0257] Statistical analysis The non-inferiority of the HBc-HBs group compared to the corresponding HBc group was evaluated. If, 7 days after administration of III, the HBc-specific CD4+ cells expressing at least one cytokine (IL-2 and / or IFN-γ and / or IFN-α) in the HBc group were higher than those in the corresponding HBc-HBs group, the HBc-HBs group was significantly lower than those in the corresponding HBc-HBs group. + This non-inferiority will be achieved if the UL of the 95% CI of the geometric mean ratio of the frequencies of T-cells (in %) is below 2. As this was the first assessment and as the criteria were not predefined, no adjustment for multiplicity was applied.

[0258] ANOVA (analysis of variance) model was used to answer two primary objectives. + The model was fit on frequencies (including group (4 to 12), interaction as fixed effects) and using a heterogeneous variance model (identical variance was not assumed between groups). The model was used to estimate geometric means (and their 95% CIs) and geometric mean ratios and their 95% CIs using back transformations of log10 means and differences.

[0259] result Antigen-specific CD4+ T cell responses All preparations induced strong anti-HBc and HBs-specific CD4 + T cell response (±1%). Although B-4 When compared with the AS01 B-4 HBc-specific CD4 + The magnitude of T-cell responses tends to be lower ( Fig.23 ), but these differences were not statistically significant as the ratio was ±1.4 (p=0.143) ( Fig.23 ). After the third immunization, when combined with HBc / AS01 alone B-4 When compared with the AS01 B-4 HBc-specific CD4 + The magnitude of the T-cell response was statistically lower (GMR 0.391 and 95% CI [0.184-0.828]) ( Fig.24 ). This confirms that HBs in Example 2 has an effect on HBc-specific CD4 + Interference with T cell responses was observed. When the antigens were formulated at a 4:1 ratio of HBc to HBs antigens, the interference was less pronounced. + This negative effect was not seen in T cell responses ( Fig.23 and Fig.24 ).

[0260] By further increasing the ratio of HBc to HBs, there is this HBc-specific CD4 + There was a trend toward additional restoration of T cell responses, with median values ​​up to total HBc-specific CD4 expressing IFN-γ and / or IL-2 and / or TNFα + 1.7% of T cells, although this was not statistically significant.

[0261] Evaluation of HBs-specific CD8 after the second and third immunizations + T cell response ( Fig.25 ). HBs-specific dose-range responses were observed after the second and third immunizations. After the third immunization, HBs-specific CD8 + T cell responses tended to decrease (GMR 0.66 and 95% CI [0.337-1.295]), however, this ratio was not statistically significant (p=0.1717). HBc-specific CD8 + T cell responses were low to undetectable (less than 0.1%, data not shown).

[0262] Antigen-specific antibody response When in AS01 B-4 When HBs and HBc were co-formulated in a 1:1 ratio in the adjuvant system, all formulations induced high and similar anti-HBc and HBs total Ig responses after the second immunization without negative effects ( Fig.26 After the third immunization, the anti-HBc specific total Ig response was enhanced ( Fig. 27 ). When in AS01 B-4 When HBs and HBc were co-formulated in a 1:1 ratio in the adjuvant system, HBs interference was seen in the level of HBc-specific antibody responses. The GMT ratio and 95% confidence interval were 1.88 [1.44; 2.44] with a p-value < 0.0001. Increasing the HBc to HBs ratio by 4 allowed restoration of the HBc humoral response with a GMT ratio and 95% confidence interval of 1.37 [1.04; 1.80] with p = 0.0262. HBc had no negative impact on the level of HBs-specific antibody responses, as previously reported ( Fig.26 and 27 ).

[0263] in conclusion The results of these experiments indicate that formulations with HBc / HBs ratios ≥ 4 overcome the observed HBs-to-HBc-specific CD4 + Negative interference of T cell and humoral responses. As a result, a dose of 4 μg HBc and 1 μg HBs was selected for further preclinical experiments.

[0264] Sequence Listing SEQ ID NO: 1: Amino acid sequence of HBs SEQ ID NO:2: Amino acid sequence of HBc truncate SEQ ID NO: 3: Amino acid sequence of the spacer region incorporating the 2A cleavage region of foot-and-mouth disease virus SEQ ID NO: 4: Nucleotide sequence encoding the spacer region of 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 substitution variant SEQ ID NO: 13: Nucleotide sequence encoding hI substitution 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 References: Al-Mahtab M, Akbar SM, Aguilar JC, Uddin MH, Khan MS, Rahman S. Therapeutic potential of a combined hepatitis B virus surface and core antigen vaccine in patients with chronic hepatitis B. Hepatol Int. 2013;7(4):981-9. Bedossa P, Poyrard T. An algorithm for the greding of activity in chronic hepatits C. The METAVIR Cooperative Study Group. Hepatology. 1996;24(2):289-93. Bertoletti A, Ferrari C. Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection. Gut. 2012;61(12):1754-64. Block TM, Locamini S, McMahon BJ, Rehermann B, Peters MG. Use of Current and New Endpoints in the Evaluation of Experimental Hepatitis B Therapeutics. Clin Infect Dis. 2017;64(9):1283-1288. Boni C, Laccabue D, Lampertico P, et al. Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues. Gastroenterology. 2012;143(4):963-73.e9. Borghese F, Clanchy FI. CD74: an emerging opportunity as a therapeutic target in cancer and autoimmune disease. Expert Opin Ther Targets. 2011;15(3):237-51. Buchmann P, Dembek C, Kuklick L, et al. Novel therapeutic hepatitis B vaccine induces cellular and humoral immune responses and breaks tolerance in hepatitis B virus (HBV) transgenic mice. Vaccine. 2013;31(8):1197-203. Cavenaugh JS, Awi D, Mendy M, et al. Partially Randomized, Non-Blinded Trial of DNA and MVA Therapeutic Vaccines Based on Hepatitis B Virus Surface Protein for Chronic HBV Infection. PLoS ONE. 2011;6:1-14. Capone S, Naddeo M, D′Alise AM, et al. Fusion of HCV nonstructural antigen to MHC class II-associated invariant chain enhanoes T-cell responses induced by vectored vaccines in nonhuman primates. Mol Ther. 2014;22(S):1039-47. Cornberg M, Wong VW, Locarnini S, Brunetto M, Janssen HL, Chan HL. The role of quantitative hepatitis B surface antigen revisited. J Hepatol. 2017;66(2):398-411. Di Lullo, G., E. Soprana, M. Panigada, A. Palini, A. Agresti, C. Comunian, A. Milani, I. Capua, V. Erfle and A. G. Siccardi (2010). The combination of marker genes swapping and fluoresoenoe-activated cell sorting improves the efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use. Journal of Virological Methods 163(2): 195 - 204 Dion S, Bourgine M, Godon O, Levillayer F, Michel ML. Adeno-associated virus-mediated gene transfer leads to persistent hepatitis B virus replication in mice expressing HLA-A2 and HLA-DR1 molecules. J Virol. 2013;87(10):5554 - 63. Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD. Analysis of the aphthovirus 2A / 2B polyprotein′clesvage′mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal′skip′. J Gen Virol. 2001 May;82(Pt 5):1013 - 25 Durantel D, Zoulim F. New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus. J Hepatol 2016;64;S117 - S131. EASL 2017. Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017; 67(2), 370 - 398. Fontaine H, Kahi S, Chazallon C, et al. Anti-HBV DNA vaccination does not prevent relapse after discontinuation of analogues in the treatment of chronic hepatitis B: a randomised trial - ANRS HB02 VAC-ADN. Gut. 2015; 64: 139 - 147. Hilgers et al., Synergistic Effects of Synthetic Adjuvants on the Humoral Immune Response. Int. Arch. Allergy. Immunol. 1986, 79(4): 392 - 6; Hilgers et al., Novel adjuvants for humoral immune responses. Immunology, 1987, 60(1): 141 - 6 Jung MC, Grüner N, Zachoval R, et al. Immunological monitoring during therapeutic vaccination as a prerequisite for the design of new effective therapies: induction of a vaccine-specific CD4+ T-cell proliferative response in chronic hepatitis B carriers. Vaccine. 2002; 20(29 - 30): 3598 - 612. Kensil C R et al. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J. Immunology (1991) 146: 431-437. Kensil C R, Saponins as Vaccine Adjuvants. Crit. Rev. Ther. Drug Carrier Syst., 1996, 13: 1-55; Kittel B, Ruehl-Fehlert C, Morawietz G, et al. Revised guides for organ sampling and trimming in rats and mice--Part 2. A joint publication of the RITA and NACAD groups. Exp Toxicol Pathol. 2004;55(6):413-31. Kranidioti H, Manolakopoulos S, Khakoo SI. Outcome after discontinuation of nucleot(s)ide analogues in chronic hepatitis B: relapse rate and associated factors. Ann Gastroenterol. 2015;28(2):173-181. Lau GK, Suri D, Liang R, et al. Resolution of Chronic Hepatitis B and Anti-HBs Seroconversion in Humans by Adoptive Transfer of Immunity to Hepatitis B Core Antigen. Gastroenterology 2002;122:614-24. Lacaille-Dubois, M and Wagner H, A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363-386 (1996). Li J, Han Y, Jin K, et al. Dynamic changes of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and natural killer T (NKT) cells in patients with acute hepatitis B infection. Virol J. 2011;8:199. Liang M, Ma S, Hu X, et al. Cellular immune responses in patients with hepatitis B surface antigen serodearance induced by antiviral therapy. Virol J. 2011;8:69. Liaw YF. Impact of therapy on the long-term outcome of chronic hepatitis B. Clin Liver Dis. 2013;17:413-423. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009;373, 582-592. Lok AS, Pan CQ, Han SH, et al. Randomized phase II study of GS-4774 as a therapeutic vaccine in virally suppressed patients with chronic hepatitis B. J Hepatol. 2016;65(3):509-16. Lorin C, Vanloubbeeck Y, Baudart S, et al. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques. PLoS One. 2015;10(4):e0122835. Maini M, Gehring A. The role of innate immunity in the immunopathology and treatment of HBV infection. J Hepatol 2016;64;S60-S70. Martin P, Furman RR, Rutherford S, et al. Phase I study of the anti-CD74 monoclonal antibody milatuzumab (hLL1) in patients with previously treated B-cell lymphomas. Leuk Lymphoma. 2015;12:1-6. Mayr A, Stiokl H, Muller HK, Danner K, Singer, H. The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defense mechanism. Zentralblatt fur Bekteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene Erste Abteilung Originale Reihe B: Hygiene, Betriebshygiene, praventive Medizin. 1978;167:375-90. Mayr, A., Hochstein-Mintzel, V. & Stickl, H. (1975). Infection 3, 6 - 14. McInnes KJ, Smith LB, Hunger NI, Saunders PT, Andrew R, Walker BR. Deletion of the androgen receptor in adipose tissue in male mice elevates retinol binding protein 4 and reveals independent effects on visceral fat mass and on glucose homeostasis. Diabetes. 2012;61(5):1072 - 81. Mohamadnejad M, Tavangar SM, Sotoudeh M, et al. Histopathological Study of Chronic Hepatitis B: A Comparative Study of Ishak and METAVIR Scoring Systems. Int J organ Transplant Med 2010;1(4):171 - 6. Morawietz G, Ruehl-Fehlert C, Kittel B, et al. Revised guides for organ sampling and trimming in rats and mice--Part 3. A joint publication of the RITA and NACAD groups. Exp Toxicol Pathol. 2004;55(6):433 - 49. Neumann AU, Phillips S, Levine I, et al. Novel mechanism of antibodies to hepatitis B virus in blocking viral partide release from cells. Hepatolpgy. 2010;52(3):875 - 85. Ott JJ, Stevens GA, Groeger J, Wiersma ST. Global epidemiology of hepatitis B virus infection: new estimates of age - specific HBsAg seroprevalence and endemicity. Vaccine 2012;30:2212 - 19. Rammeh S, Khadra HB, Znaidi NS, et al. Inter - observers agreement of Ishak and Metavir scores in histological evaluation of chronic viral hepatitis B and C. Ann Biol Clin(Paris). 2014;72(1):57 - 60. Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol. 2005;5(3):215 - 29. Riedl P, Reiser M, Stifter K, Krieger J, Schirmbeck R. Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope - specific manner. Eur J Immunol. 2014;44(7):1981 - 91. Ruehl - Fehlert C, Kittel B, Morawietz G, et al. Revised guides for organ sampling and trimming in rats and mice - - part 1. Exp Toxicol Pathol. 2003;55(2 - 3):91 - 106. Spencer AJ, Cottingham MG, Jenks JA, et al. Enhanced vaccine-induced CD8+ T cell responses to malaria antigen ME-TRAP by fusion to MHC class II invariant chain. PLoS One. 2014;9(6):e100538. Terrault NA, Bzowej NH, Chang K-M, et al. AASLD Guidelines for Treatment of Chronic Hepatitis B. Hepatology. 2015; DOI: 10.1002 / hep.28156. World Health Organization (WHO). Global Hepatitis report, 2017. http: / / apps.who.int / iris / bitstream / 10665 / 255016 / 1 / 9789241565455-eng.pdf?ua=1 . Registered in November 2017. Yang FQ, Yu YY, Wang GQ, et al. A pilot randomized controlled trial of dual-plasmid HBV DNA vaccine mediated by in vivo electroporation in chronic hepatitis B patients under lamivudine chemotherapy. J Viral Hepat. 2012;19:581-593. Zoutendijk R, Hansen BE, van Vuuren AJ, Boucher CA, Janssen HL. Serum HBsAg decline during long-term potent nucleos(t)ide analogue therapy for chronic hepatitis B and prediction of HBsAg loss. J Infect Dis. 2011;204(3):415-8.

Claims

1. Use of an immunogenic combination product in the preparation of a medicament for use in a method for treating chronic hepatitis B infection (CHB) in humans, the immunogenic combination product 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 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 recombinant hepatitis B surface antigen (HBs), recombinant hepatitis B virus core antigen (HBc) and an adjuvant, The method of treating chronic hepatitis B infection comprises administering the composition to the human sequentially or simultaneously.

2. The use according to claim 1, wherein the steps of the method are performed sequentially, wherein step a) precedes step b), and step b) precedes step c).

3. Use according to claim 2, wherein step c) of the method is repeated.

4. Use according to any preceding claim, wherein the time period between each step is 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 6 months or 12 months, such as 4 weeks or 8 weeks.

5. The use according to claim 1, wherein step c) is performed simultaneously with step a) and / or with step b).

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