Adenoviral vectors encoding hepatitis b viral antigens fused to herpes virus glycoprotein d and methods of using the same

Abstract

To provide non-naturally occurring variants of the hepatitis B virus (HBV) Core protein, the HBV polymerase N-terminal domain, the HBV polymerase C-terminal domain, and immunogenic fragments thereof.SOLUTION: Provided herein are non-naturally occurring variants of the hepatitis B virus (HBV) Core protein, the HBV polymerase N-terminal domain, the HBV polymerase C-terminal domain, and immunogenic fragments thereof. Also provided are HBV variants fused to a herpes simplex virus (HSV) glycoprotein (gD) sequence, as well as methods of using the fusion proteins.SELECTED DRAWING: Figure 25B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 958,809, filed January 9, 2020; U.S. Provisional Patent Application No. 62 / 958,827, filed January 9, 2020; U.S. Provisional Patent Application No. 62 / 967,242, filed January 2 ... This application claims priority to U.S. Provisional Patent Application No. 67,104, filed August 12, 2020, U.S. Provisional Patent Application No. 63 / 064,506, filed August 12, 2020, U.S. Provisional Patent Application No. 63 / 064,571, filed August 12, 2020, U.S. Provisional Patent Application No. 63 / 112,202, filed November 11, 2020, and U.S. Provisional Patent Application No. 63 / 112,219, filed November 11, 2020.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on January 7, 2021 is named 111876_000035_SL.txt and is 151,446 bytes in size.

[0003] Disclosed herein are non-naturally occurring variants of the hepatitis B virus (HBV) core protein, the HBV polymerase N-terminal domain, and the HBV polymerase C-terminal domain, as well as immunogenic fragments thereof and fusion proteins comprising the same. [Background technology]

[0004] The World Health Organization estimates that in 2015, 257 million people were living with chronic hepatitis B infection (defined as hepatitis B surface antigen-positive), and that hepatitis B caused an estimated 887,000 deaths, primarily due to cirrhosis and hepatocellular carcinoma (i.e., primary liver cancer). Assuming that women of reproductive age represent 25.3% of the world's population (UN data), chronically infected adults could include 65 million women of childbearing age who can potentially transmit HBV to their children (WHO Global Hepatitis Report 2017. Available at: apps_who_int / iris / bitstream / handle / 10665 / 255016 / 9789241565455-eng.pdf;jsessionid=D78616700ED7322D4109CA4541FB94EA?sequence=1). The overall incidence rate in 2016 was 1.0 cases per 100,000 people (Centers for Disease Control and Prevention. Viral Hepatitis Surveillance-United States, 2017. Atlanta: US Department of Health and Human Services, Centers for Disease Control and Prevention; 2019. Available at: www_cdc_gov / hepatitis / statistics / 2017surveillance / index.htm.). In 2017 alone, a total of 3,407 cases of acute hepatitis B were reported to the Centers for Disease Control and Prevention (CDC).

[0005] Despite the availability of a preventive HBV vaccine, the burden of chronic HBV infection remains a significant unmet global medical problem due to suboptimal treatment options and a persistent rate of new infections in most parts of the developing world. Summary of the Invention

[0006] Provided herein is a Hepatitis B virus (HBV) core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof.

[0007] Also provided is an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof.

[0008] Also disclosed is an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof.

[0009] Also provided are fusion proteins comprising an N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or a variant thereof, a disclosed HBV core protein, an HBV polymerase N-terminal domain, an HBV polymerase C-terminal domain, or an immunogenic fragment thereof, and a C-terminal HSV gD sequence or a variant thereof.

[0010] Also provided herein are fusion proteins comprising an N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or variants thereof, a combination of the disclosed HBV core protein, the HBV polymerase N-terminal domain, the HBV polymerase C-terminal domain, and / or immunogenic fragments thereof, and a C-terminal HSV gD sequence or variants thereof.

[0011] Disclosed herein are nucleic acid molecules encoding the disclosed proteins or fusion proteins, vectors comprising the nucleic acid molecules, and vaccines comprising the disclosed vectors.

[0012] Also provided herein is a method for inducing an immune response against HBV in a subject, the method comprising providing to the subject an effective amount of any of the disclosed fusion proteins, nucleic acid molecules, vectors, or vaccines, thereby inducing an immune response against HBV.

[0013] This summary, as well as the following detailed description, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the disclosed proteins, vaccines, and methods, preferred embodiments of the proteins, vaccines, and methods are shown in the drawings, but the proteins, vaccines, and methods are not limited to the specific embodiments disclosed. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the frequency of epitope-optimized core amino acids, with the amino acid residue shown on the X-axis and the percentage of sequence similarity across all analyzed genomes on the Y-axis. [Figure 2A] Figures 2A, 2B, 2C, 2D, 2E, and 2F show vaccine insert-specific T cell frequencies in C57Bl / 6 mice after intramuscular (i.m.) injection of the indicated amounts of a chimpanzee serotype 6 replication-deficient adenoviral vector (AdC6) containing an epitope-optimized core sequence (SEQ ID NO: 15) genetically fused to gD (AdC6-gDCore) (Figures 2A and 2D); an AdC6 containing an epitope-optimized polymerase C-terminal domain sequence (SEQ ID NO: 19) genetically fused to gD (AdC6-gDPolC) (Figures 2B and 2E); and an AdC6 containing an epitope-optimized polymerase N-terminal domain sequence (SEQ ID NO: 17) genetically fused to gD (AdC6-gDPolN) (Figures 2C and 2F). Mice were bled 14 days after injection, and the frequencies of T cells specific to various HBV inserts were analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. Graphs show results for individual mice with medians indicated by straight lines. Figures 2A-2C show insert-specific CD8+ T cell frequencies, and Figures 2D-2F show insert-specific CD4+ T cell frequencies. [Figure 2B]Figures 2A, 2B, 2C, 2D, 2E, and 2F show vaccine insert-specific T cell frequencies in C57Bl / 6 mice after intramuscular (i.m.) injection of the indicated amounts of a chimpanzee serotype 6 replication-deficient adenoviral vector (AdC6) containing an epitope-optimized core sequence (SEQ ID NO: 15) genetically fused to gD (AdC6-gDCore) (Figures 2A and 2D); an AdC6 containing an epitope-optimized polymerase C-terminal domain sequence (SEQ ID NO: 19) genetically fused to gD (AdC6-gDPolC) (Figures 2B and 2E); and an AdC6 containing an epitope-optimized polymerase N-terminal domain sequence (SEQ ID NO: 17) genetically fused to gD (AdC6-gDPolN) (Figures 2C and 2F). Mice were bled 14 days after injection, and T cell frequencies against various HBV inserts were analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. Graphs show results for individual mice with medians indicated by straight lines. Figures 2A-2C show insert-specific CD8+ T cell frequencies, and Figures 2D-2F show insert-specific CD4+ T cell frequencies. [Figure 2C]Figures 2A, 2B, 2C, 2D, 2E, and 2F show vaccine insert-specific T cell frequencies in C57Bl / 6 mice after intramuscular (i.m.) injection of the indicated amounts of a chimpanzee serotype 6 replication-deficient adenoviral vector (AdC6) containing an epitope-optimized core sequence (SEQ ID NO: 15) genetically fused to gD (AdC6-gDCore) (Figures 2A and 2D); an AdC6 containing an epitope-optimized polymerase C-terminal domain sequence (SEQ ID NO: 19) genetically fused to gD (AdC6-gDPolC) (Figures 2B and 2E); and an AdC6 containing an epitope-optimized polymerase N-terminal domain sequence (SEQ ID NO: 17) genetically fused to gD (AdC6-gDPolN) (Figures 2C and 2F). Mice were bled 14 days after injection, and T cell frequencies against various HBV inserts were analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. Graphs show results for individual mice with medians indicated by straight lines. Figures 2A-2C show insert-specific CD8+ T cell frequencies, and Figures 2D-2F show insert-specific CD4+ T cell frequencies. [Figure 2D]Figures 2A, 2B, 2C, 2D, 2E, and 2F show vaccine insert-specific T cell frequencies in C57Bl / 6 mice after intramuscular (i.m.) injection of the indicated amounts of a chimpanzee serotype 6 replication-deficient adenoviral vector (AdC6) containing an epitope-optimized core sequence (SEQ ID NO: 15) genetically fused to gD (AdC6-gDCore) (Figures 2A and 2D); an AdC6 containing an epitope-optimized polymerase C-terminal domain sequence (SEQ ID NO: 19) genetically fused to gD (AdC6-gDPolC) (Figures 2B and 2E); and an AdC6 containing an epitope-optimized polymerase N-terminal domain sequence (SEQ ID NO: 17) genetically fused to gD (AdC6-gDPolN) (Figures 2C and 2F). Mice were bled 14 days after injection, and T cell frequencies against various HBV inserts were analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. Graphs show results for individual mice with medians indicated by straight lines. Figures 2A-2C show insert-specific CD8+ T cell frequencies, and Figures 2D-2F show insert-specific CD4+ T cell frequencies. [Figure 2E]Figures 2A, 2B, 2C, 2D, 2E, and 2F show vaccine insert-specific T cell frequencies in C57Bl / 6 mice after intramuscular (i.m.) injection of the indicated amounts of a chimpanzee serotype 6 replication-deficient adenoviral vector (AdC6) containing an epitope-optimized core sequence (SEQ ID NO: 15) genetically fused to gD (AdC6-gDCore) (Figures 2A and 2D); an AdC6 containing an epitope-optimized polymerase C-terminal domain sequence (SEQ ID NO: 19) genetically fused to gD (AdC6-gDPolC) (Figures 2B and 2E); and an AdC6 containing an epitope-optimized polymerase N-terminal domain sequence (SEQ ID NO: 17) genetically fused to gD (AdC6-gDPolN) (Figures 2C and 2F). Mice were bled 14 days after injection, and T cell frequencies against various HBV inserts were analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. Graphs show results for individual mice with medians indicated by straight lines. Figures 2A-2C show insert-specific CD8+ T cell frequencies, and Figures 2D-2F show insert-specific CD4+ T cell frequencies. [Figure 2F]Figures 2A, 2B, 2C, 2D, 2E, and 2F show vaccine insert-specific T cell frequencies in C57Bl / 6 mice after intramuscular (i.m.) injection of the indicated amounts of a chimpanzee serotype 6 replication-deficient adenoviral vector (AdC6) containing an epitope-optimized core sequence (SEQ ID NO: 15) genetically fused to gD (AdC6-gDCore) (Figures 2A and 2D); an AdC6 containing an epitope-optimized polymerase C-terminal domain sequence (SEQ ID NO: 19) genetically fused to gD (AdC6-gDPolC) (Figures 2B and 2E); and an AdC6 containing an epitope-optimized polymerase N-terminal domain sequence (SEQ ID NO: 17) genetically fused to gD (AdC6-gDPolN) (Figures 2C and 2F). Mice were bled 14 days after injection, and T cell frequencies against various HBV inserts were analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. Graphs show results for individual mice with medians indicated by straight lines. Figures 2A-2C show insert-specific CD8+ T cell frequencies, and Figures 2D-2F show insert-specific CD4+ T cell frequencies. [Figure 3A]Figures 3A, 3B, and 3C show T cell frequencies in different mouse strains (A: C57B1 / 6 mice; B: BALB / c mice; C: HLA-A2 transgenic (tg) mice) in response to pools of peptides representing the indicated HBV sequences. Results were obtained using splenocytes collected 4 weeks after immunization, and IFN-γ was assayed by ICS. Peptides were arranged in a matrix such that recognition of two pools resulted in the identification of a single peptide. Graphs show responses to the different pools; responses to the pool containing all peptides are shown on the right. Background frequencies obtained without peptide were subtracted. Pools deemed to elicit responses and peptides identified in response to the different pools are listed at the bottom of each panel. CD8+ and CD4+ T cell responses from BALB / c mice are shown, as are CD8+ T cell responses from HLA-A2tg mice bearing human MHC class I molecules but not mouse MHC class II molecules. T cells were gated on activated CD44+ cells. Each consecutively numbered "peptide" consists of 15 amino acids, beginning with amino acid 1, 6, 11, etc., of the Core, PolN, or PolC sequence. Thus, for example, Core peptide 1 corresponds to amino acids 1-15 of SEQ ID NO: 6 (i.e., the epitope-optimized Core amino acid sequence), Core peptide 2 corresponds to amino acids 6-20 of SEQ ID NO: 6, and Core peptide 3 corresponds to amino acids 11-25 of SEQ ID NO: 6. Similarly, PolN peptide 1 corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e., the epitope-optimized PolN amino acid sequence), PolN peptide 2 corresponds to amino acids 6-20 of SEQ ID NO: 8, and PolN peptide 3 corresponds to amino acids 11-25 of SEQ ID NO: 8. Similarly, PolC peptide 1 corresponds to amino acids 1 to 15 of SEQ ID NO: 10 (i.e., the epitope-optimized PolC amino acid sequence), PolC peptide 2 corresponds to amino acids 6 to 20 of SEQ ID NO: 10, and PolC peptide 3 corresponds to amino acids 11 to 25 of SEQ ID NO: 10. [Figure 3B]Figures 3A, 3B, and 3C show T cell frequencies in different mouse strains (A: C57B1 / 6 mice; B: BALB / c mice; C: HLA-A2 transgenic (tg) mice) in response to pools of peptides representing the indicated HBV sequences. Results were obtained using splenocytes collected 4 weeks after immunization, and IFN-γ was assayed by ICS. Peptides were arranged in a matrix such that recognition of two pools resulted in the identification of a single peptide. Graphs show responses to the different pools; responses to the pool containing all peptides are shown on the right. Background frequencies obtained without peptide were subtracted. Pools deemed to elicit responses and peptides identified in response to the different pools are listed at the bottom of each panel. CD8+ and CD4+ T cell responses from BALB / c mice are shown, as are CD8+ T cell responses from HLA-A2tg mice bearing human MHC class I molecules but not mouse MHC class II molecules. T cells were gated on activated CD44+ cells. Each consecutively numbered "peptide" consists of 15 amino acids, beginning with amino acid 1, 6, 11, etc., of the Core, PolN, or PolC sequence. Thus, for example, Core peptide 1 corresponds to amino acids 1-15 of SEQ ID NO: 6 (i.e., the epitope-optimized Core amino acid sequence), Core peptide 2 corresponds to amino acids 6-20 of SEQ ID NO: 6, and Core peptide 3 corresponds to amino acids 11-25 of SEQ ID NO: 6. Similarly, PolN peptide 1 corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e., the epitope-optimized PolN amino acid sequence), PolN peptide 2 corresponds to amino acids 6-20 of SEQ ID NO: 8, and PolN peptide 3 corresponds to amino acids 11-25 of SEQ ID NO: 8. Similarly, PolC peptide 1 corresponds to amino acids 1 to 15 of SEQ ID NO: 10 (i.e., the epitope-optimized PolC amino acid sequence), PolC peptide 2 corresponds to amino acids 6 to 20 of SEQ ID NO: 10, and PolC peptide 3 corresponds to amino acids 11 to 25 of SEQ ID NO: 10. [Figure 3C]Figures 3A, 3B, and 3C show T cell frequencies in different mouse strains (A: C57B1 / 6 mice; B: BALB / c mice; C: HLA-A2 transgenic (tg) mice) in response to pools of peptides representing the indicated HBV sequences. Results were obtained using splenocytes collected 4 weeks after immunization, and IFN-γ was assayed by ICS. Peptides were arranged in a matrix such that recognition of two pools resulted in the identification of a single peptide. Graphs show responses to the different pools; responses to the pool containing all peptides are shown on the right. Background frequencies obtained without peptide were subtracted. Pools deemed to elicit responses and peptides identified in response to the different pools are listed at the bottom of each panel. CD8+ and CD4+ T cell responses from BALB / c mice are shown, as are CD8+ T cell responses from HLA-A2tg mice bearing human MHC class I molecules but not mouse MHC class II molecules. T cells were gated on activated CD44+ cells. Each consecutively numbered "peptide" consists of 15 amino acids, beginning with amino acid 1, 6, 11, etc., of the Core, PolN, or PolC sequence. Thus, for example, Core peptide 1 corresponds to amino acids 1-15 of SEQ ID NO: 6 (i.e., the epitope-optimized Core amino acid sequence), Core peptide 2 corresponds to amino acids 6-20 of SEQ ID NO: 6, and Core peptide 3 corresponds to amino acids 11-25 of SEQ ID NO: 6. Similarly, PolN peptide 1 corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e., the epitope-optimized PolN amino acid sequence), PolN peptide 2 corresponds to amino acids 6-20 of SEQ ID NO: 8, and PolN peptide 3 corresponds to amino acids 11-25 of SEQ ID NO: 8. Similarly, PolC peptide 1 corresponds to amino acids 1 to 15 of SEQ ID NO: 10 (i.e., the epitope-optimized PolC amino acid sequence), PolC peptide 2 corresponds to amino acids 6 to 20 of SEQ ID NO: 10, and PolC peptide 3 corresponds to amino acids 11 to 25 of SEQ ID NO: 10. [Figure 4A]Figures 4A, 4B, and 4C show IFN-γ responses in C57Bl / 6 mice immunized with various doses of the indicated vectors and boosted with AdC6-gDCore (A), AdC6-gDPolC (B), and AdC6-gDPolN (C). The left graph shows responses measured in the blood 2 weeks after the primary inoculation with the AdC6 vector. Mice were boosted 8 weeks later with the same dose of the AdC7 vector expressing the same insert. The right graph shows responses in the blood 2 weeks after the booster inoculation. [Figure 4B] Figures 4A, 4B, and 4C show IFN-γ responses in C57Bl / 6 mice immunized with various doses of the indicated vectors and boosted with AdC6-gDCore (A), AdC6-gDPolC (B), and AdC6-gDPolN (C). The left graph shows responses measured in the blood 2 weeks after the primary inoculation with the AdC6 vector. Mice were boosted 8 weeks later with the same dose of the AdC7 vector expressing the same insert. The right graph shows responses in the blood 2 weeks after the booster inoculation. [Figure 4C] Figures 4A, 4B, and 4C show IFN-γ responses in C57Bl / 6 mice immunized with various doses of the indicated vectors and boosted with AdC6-gDCore (A), AdC6-gDPolC (B), and AdC6-gDPolN (C). The left graph shows responses measured in the blood 2 weeks after the primary inoculation with the AdC6 vector. Mice were boosted 8 weeks later with the same dose of the AdC7 vector expressing the same insert. The right graph shows responses in the blood 2 weeks after the booster inoculation. [Figure 5A]Figures 5A, 5B, and 5C show T cell frequencies in different mouse strains (A: C57B1 / 6 mice; B: BALB / c mice; C: HLA-A2tg mice) in response to pools of peptides representing the indicated HBV sequences. Mice were primed with AdC6 vectors expressing one of three inserts (i.e., Core, PolC, or PolN) and boosted 8 weeks later with AdC7 vectors expressing the same inserts. Results were obtained using splenocytes collected 4 weeks after immunization, and IFN-γ was assayed by ICS. Peptides were arranged in a matrix such that recognition of two pools resulted in the identification of one peptide. Graphs show responses to the different pools; responses to the pool containing all peptides are shown on the right. Background frequencies obtained without peptide were subtracted. Pools deemed to elicit responses and peptides identified in response to the different pools are listed at the bottom of each figure. Figure 1 shows CD8+ T cell and CD4+ T cell responses from BALB / c mice and HLA-A2tg mice bearing human MHC class I molecules but not mouse MHC class II molecules. T cells were gated on activated CD44+ cells. Each consecutively numbered "peptide" consists of 15 amino acids, beginning with the first, sixth, eleventh, etc. amino acid of the Core, PolN, or PolC sequence. Thus, for example, Core peptide 1 corresponds to amino acids 1-15 of SEQ ID NO:6 (i.e., the epitope-optimized Core amino acid sequence), Core peptide 2 corresponds to amino acids 6-20 of SEQ ID NO:6, and Core peptide 3 corresponds to amino acids 11-25 of SEQ ID NO:6. Similarly, Peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e., the epitope-optimized PolN amino acid sequence), Peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO: 8, and Peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO: 8. Similarly, Peptide 1 of PolC corresponds to amino acids 1-15 of SEQ ID NO: 10 (i.e., the epitope-optimized PolC amino acid sequence), Peptide 2 of PolC corresponds to amino acids 6-20 of SEQ ID NO: 10, and Peptide 3 of PolC corresponds to amino acids 11-25 of SEQ ID NO: 10. [Figure 5B] Figures 5A, 5B, and 5C show T cell frequencies in different mouse strains (A: C57B1 / 6 mice; B: BALB / c mice; C: HLA-A2tg mice) in response to pools of peptides representing the indicated HBV sequences. Mice were primed with AdC6 vectors expressing one of three inserts (i.e., Core, PolC, or PolN) and boosted 8 weeks later with AdC7 vectors expressing the same inserts. Results were obtained using splenocytes collected 4 weeks after immunization, and IFN-γ was assayed by ICS. Peptides were arranged in a matrix such that recognition of two pools resulted in the identification of one peptide. Graphs show responses to the different pools; responses to the pool containing all peptides are shown on the right. Background frequencies obtained without peptide were subtracted. Pools deemed to elicit responses and peptides identified in response to the different pools are listed at the bottom of each figure. Figure 1 shows CD8+ T cell and CD4+ T cell responses from BALB / c mice and HLA-A2tg mice bearing human MHC class I molecules but not mouse MHC class II molecules. T cells were gated on activated CD44+ cells. Each consecutively numbered "peptide" consists of 15 amino acids, beginning with the first, sixth, eleventh, etc. amino acid of the Core, PolN, or PolC sequence. Thus, for example, Core peptide 1 corresponds to amino acids 1-15 of SEQ ID NO:6 (i.e., the epitope-optimized Core amino acid sequence), Core peptide 2 corresponds to amino acids 6-20 of SEQ ID NO:6, and Core peptide 3 corresponds to amino acids 11-25 of SEQ ID NO:6. Similarly, Peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e., the epitope-optimized PolN amino acid sequence), Peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO: 8, and Peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO: 8. Similarly, Peptide 1 of PolC corresponds to amino acids 1-15 of SEQ ID NO: 10 (i.e., the epitope-optimized PolC amino acid sequence), Peptide 2 of PolC corresponds to amino acids 6-20 of SEQ ID NO: 10, and Peptide 3 of PolC corresponds to amino acids 11-25 of SEQ ID NO: 10. [Figure 5C]Figures 5A, 5B, and 5C show T cell frequencies in different mouse strains (A: C57B1 / 6 mice; B: BALB / c mice; C: HLA-A2tg mice) in response to pools of peptides representing the indicated HBV sequences. Mice were primed with AdC6 vectors expressing one of three inserts (i.e., Core, PolC, or PolN) and boosted 8 weeks later with AdC7 vectors expressing the same inserts. Results were obtained using splenocytes collected 4 weeks after immunization, and IFN-γ was assayed by ICS. Peptides were arranged in a matrix such that recognition of two pools resulted in the identification of one peptide. Graphs show responses to the different pools; responses to the pool containing all peptides are shown on the right. Background frequencies obtained without peptide were subtracted. Pools deemed to elicit responses and peptides identified in response to the different pools are listed at the bottom of each figure. Figure 1 shows CD8+ T cell and CD4+ T cell responses from BALB / c mice and HLA-A2tg mice bearing human MHC class I molecules but not mouse MHC class II molecules. T cells were gated on activated CD44+ cells. Each consecutively numbered "peptide" consists of 15 amino acids, beginning with the first, sixth, eleventh, etc. amino acid of the Core, PolN, or PolC sequence. Thus, for example, Core peptide 1 corresponds to amino acids 1-15 of SEQ ID NO:6 (i.e., the epitope-optimized Core amino acid sequence), Core peptide 2 corresponds to amino acids 6-20 of SEQ ID NO:6, and Core peptide 3 corresponds to amino acids 11-25 of SEQ ID NO:6. Similarly, Peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO: 8 (i.e., the epitope-optimized PolN amino acid sequence), Peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO: 8, and Peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO: 8. Similarly, Peptide 1 of PolC corresponds to amino acids 1-15 of SEQ ID NO: 10 (i.e., the epitope-optimized PolC amino acid sequence), Peptide 2 of PolC corresponds to amino acids 6-20 of SEQ ID NO: 10, and Peptide 3 of PolC corresponds to amino acids 11-25 of SEQ ID NO: 10. [Figure 6] Figure 6 shows the effect of vaccination on serum HBV genome copy number upon AAV-1.3HBV challenge. Groups of three mice were challenged with 1 x 10, 1 x 10, or 1.5 x 10 viral genomes (vg) of adeno-associated virus 8 (AAV8)-1.3HBV vector and vaccinated with AdC6-gDPolN 8 weeks later. Viral titers were determined 8 weeks after vaccination and compared with pre-vaccination titers. Changes in viral load from baseline for each treatment group are shown. [Figure 7A] Figures 7A, 7B, 7C, 7D, and 7E show exemplary HBV epitope shift experiments. Figure 7A—Mice were immunized with AdC6-gDPolN vaccine. Four weeks later, splenocytes were examined for IFN-γ responses to a peptide pool representing the PolN sequence by intracellular cytokine staining. Stimulated T cells were stained for T cell markers. Figure 7B—Results obtained by the same assay using splenocytes from mice challenged with 1 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figure 7C—Results obtained by the same assay using splenocytes from mice challenged with 1.5 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figures 7A, 7B, and 7C show the frequency of IFN-γ-producing CD44+CD8+ T cells among total CD44+CD8+ T cells. The background response obtained by splenocytes incubated without the peptide pool was subtracted. Figure 7D - Peptide pool. Figure 7E - Individual peptide sequences. Figure 7E discloses SEQ ID NOs: 55-68 and 189-233, respectively, in order of appearance. [Figure 7B]Figures 7A, 7B, 7C, 7D, and 7E show exemplary HBV epitope shift experiments. Figure 7A—Mice were immunized with AdC6-gDPolN vaccine. Four weeks later, splenocytes were examined for IFN-γ responses to a peptide pool representing the PolN sequence by intracellular cytokine staining. Stimulated T cells were stained for T cell markers. Figure 7B—Results obtained by the same assay using splenocytes from mice challenged with 1 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figure 7C—Results obtained by the same assay using splenocytes from mice challenged with 1.5 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figures 7A, 7B, and 7C show the frequency of IFN-γ-producing CD44+CD8+ T cells among total CD44+CD8+ T cells. The background response obtained by splenocytes incubated without the peptide pool was subtracted. Figure 7D - Peptide pool. Figure 7E - Individual peptide sequences. Figure 7E discloses SEQ ID NOs: 55-68 and 189-233, respectively, in order of appearance. [Figure 7C]Figures 7A, 7B, 7C, 7D, and 7E show exemplary HBV epitope shift experiments. Figure 7A—Mice were immunized with AdC6-gDPolN vaccine. Four weeks later, splenocytes were examined for IFN-γ responses to a peptide pool representing the PolN sequence by intracellular cytokine staining. Stimulated T cells were stained for T cell markers. Figure 7B—Results obtained by the same assay using splenocytes from mice challenged with 1 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figure 7C—Results obtained by the same assay using splenocytes from mice challenged with 1.5 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figures 7A, 7B, and 7C show the frequency of IFN-γ-producing CD44+CD8+ T cells among total CD44+CD8+ T cells. The background response obtained by splenocytes incubated without the peptide pool was subtracted. Figure 7D - Peptide pool. Figure 7E - Individual peptide sequences. Figure 7E discloses SEQ ID NOs: 55-68 and 189-233, respectively, in order of appearance. [Figure 7D]Figures 7A, 7B, 7C, 7D, and 7E show exemplary HBV epitope shift experiments. Figure 7A—Mice were immunized with AdC6-gDPolN vaccine. Four weeks later, splenocytes were examined for IFN-γ responses to a peptide pool representing the PolN sequence by intracellular cytokine staining. Stimulated T cells were stained for T cell markers. Figure 7B—Results obtained by the same assay using splenocytes from mice challenged with 1 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figure 7C—Results obtained by the same assay using splenocytes from mice challenged with 1.5 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figures 7A, 7B, and 7C show the frequency of IFN-γ-producing CD44+CD8+ T cells among total CD44+CD8+ T cells. The background response obtained by splenocytes incubated without the peptide pool was subtracted. Figure 7D - Peptide pool. Figure 7E - Individual peptide sequences. Figure 7E discloses SEQ ID NOs: 55-68 and 189-233, respectively, in order of appearance. [Figure 7E]Figures 7A, 7B, 7C, 7D, and 7E show exemplary HBV epitope shift experiments. Figure 7A—Mice were immunized with AdC6-gDPolN vaccine. Four weeks later, splenocytes were examined for IFN-γ responses to a peptide pool representing the PolN sequence by intracellular cytokine staining. Stimulated T cells were stained for T cell markers. Figure 7B—Results obtained by the same assay using splenocytes from mice challenged with 1 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figure 7C—Results obtained by the same assay using splenocytes from mice challenged with 1.5 x 10 vg of AAV8-1.3-HBV. Four weeks later, mice were vaccinated, and 10 weeks later, T cell responses were examined in the spleen. Figures 7A, 7B, and 7C show the frequency of IFN-γ-producing CD44+CD8+ T cells among total CD44+CD8+ T cells. The background response obtained by splenocytes incubated without the peptide pool was subtracted. Figure 7D - Peptide pool. Figure 7E - Individual peptide sequences. Figure 7E discloses SEQ ID NOs: 55-68 and 189-233, respectively, in order of appearance. [Figure 8A] Figures 8A, 8B, and 8C show data from the same experiment described above in Figure 7. Based on the response to the peptide pools, it was determined which individual peptides (both pools and peptides are shown in Figure 7) were positive. The graph shows the response to all of the peptides. Each peptide was present in two pools, therefore two frequency values ​​were obtained for each peptide, and only the lower data point is shown in this figure. [Figure 8B] Figures 8A, 8B, and 8C show data from the same experiment described above in Figure 7. Based on the response to the peptide pools, it was determined which individual peptides (both pools and peptides are shown in Figure 7) were positive. The graph shows the response to all of the peptides. Each peptide was present in two pools, therefore two frequency values ​​were obtained for each peptide, and only the lower data point is shown in this figure. [Figure 8C]Figures 8A, 8B, and 8C show data from the same experiment described above in Figure 7. Based on the response to the peptide pools, it was determined which individual peptides (both pools and peptides are shown in Figure 7) were positive. The graph shows the response to all of the peptides. Each peptide was present in two pools, therefore two frequency values ​​were obtained for each peptide, and only the lower data point is shown in this figure. [Figure 9A] Figures 9A, 9B, and 9C show the results of an exemplary immunogenicity experiment performed on C57B1 / 6 mice (n=5 per group) injected with various doses of exemplary AdC6-gDCore, AdC6-gDPolN, or AdC6-gDPolC vectors and boosted with an AdC7 vector containing the same insert (i.e., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC vector) two months after the first injection. Figure 9A shows antigen immunogenicity, Figure 9B shows the duration of response, and Figure 9C shows the prime-boost response. [Figure 9B] Figures 9A, 9B, and 9C show the results of an exemplary immunogenicity experiment performed on C57B1 / 6 mice (n=5 per group) injected with various doses of exemplary AdC6-gDCore, AdC6-gDPolN, or AdC6-gDPolC vectors and boosted with an AdC7 vector containing the same insert (i.e., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC vector) two months after the first injection. Figure 9A shows antigen immunogenicity, Figure 9B shows the duration of response, and Figure 9C shows the prime-boost response. [Figure 9C]Figures 9A, 9B, and 9C show the results of an exemplary immunogenicity experiment performed on C57B1 / 6 mice (n=5 per group) injected with various doses of exemplary AdC6-gDCore, AdC6-gDPolN, or AdC6-gDPolC vectors and boosted with an AdC7 vector containing the same insert (i.e., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC vector) two months after the first injection. Figure 9A shows antigen immunogenicity, Figure 9B shows the duration of response, and Figure 9C shows the prime-boost response. [Figure 10] Figure 10 shows CD8+ T cell peptide recognition of PolN epitopes in BALB / c, C57Bl / 6, and HLA-A2 transgenic mice after vaccination with a primary dose of AdC6-gDPolN and a booster dose of AdC7-gDPolN. CD8+ T cell peptide recognition was calculated as the fraction of the number of positive peptides recognized 2 weeks after either the primary or booster dose divided by the total number of overlapping peptides derived from PolN (59 peptides in total). [Figure 11A] Figures 11A and 11B show vaccine-induced HBV-specific CD8+ T cell responses in the livers of C57B1 / 6 mice injected with the indicated vectors. *p-values ​​between 0.01 and 0.05, ***p-values ​​between 0.0001 and 0.001 by one-way analysis of variance. [Figure 11B] Figures 11A and 11B show vaccine-induced HBV-specific CD8+ T cell responses in the livers of C57B1 / 6 mice injected with the indicated vectors. *p-values ​​between 0.01 and 0.05, ***p-values ​​between 0.0001 and 0.001 by one-way analysis of variance. [Figure 12A] Figures 12A, 12B, 12C, 12D, 12E, and 12F show hematoxylin and eosin staining of liver samples from C57B1 / 6 mice injected with the indicated vectors. Magnification: 20x. Arrows indicate areas of lymphocytic infiltrate. [Figure 12B]Figures 12A, 12B, 12C, 12D, 12E, and 12F show hematoxylin and eosin staining of liver samples from C57B1 / 6 mice injected with the indicated vectors. Magnification: 20x. Arrows indicate areas of lymphocytic infiltrate. [Figure 12C] Figures 12A, 12B, 12C, 12D, 12E, and 12F show hematoxylin and eosin staining of liver samples from C57B1 / 6 mice injected with the indicated vectors. Magnification: 20x. Arrows indicate areas of lymphocytic infiltrate. [Figure 12D] Figures 12A, 12B, 12C, 12D, 12E, and 12F show hematoxylin and eosin staining of liver samples from C57B1 / 6 mice injected with the indicated vectors. Magnification: 20x. Arrows indicate areas of lymphocytic infiltrate. [Figure 12E] Figures 12A, 12B, 12C, 12D, 12E, and 12F show hematoxylin and eosin staining of liver samples from C57B1 / 6 mice injected with the indicated vectors. Magnification: 20x. Arrows indicate areas of lymphocytic infiltrate. [Figure 12F] Figures 12A, 12B, 12C, 12D, 12E, and 12F show hematoxylin and eosin staining of liver samples from C57B1 / 6 mice injected with the indicated vectors. Magnification: 20x. Arrows indicate areas of lymphocytic infiltrate. [Figure 13A] Figures 13A and 13B show markers of vaccine-induced CD8+ T cell activation / exhaustion in the livers of C57B1 / 6 mice injected with the indicated vectors. **p-values ​​between 0.001 and 0.01, ***p-values ​​between 0.0001 and 0.001 by one-way ANOVA. [Figure 13B] Figures 13A and 13B show markers of vaccine-induced CD8+ T cell activation / exhaustion in the livers of C57B1 / 6 mice injected with the indicated vectors. **p-values ​​between 0.001 and 0.01, ***p-values ​​between 0.0001 and 0.001 by one-way ANOVA. [Figure 14A]Figures 14A and 14B show HBV viral kinetics in C57B1 / 6 mice injected with an exemplary AdC6-gDPolN vector. Median HBV DNA VL / ml at week 4 is -7.3 log10 cps / mL. n=7, one mouse was excluded due to missing data. [Figure 14B] Figures 14A and 14B show HBV viral kinetics in C57B1 / 6 mice injected with an exemplary AdC6-gDPolN vector. Median HBV DNA VL / ml at week 4 was -7.3 log10 cps / mL. n=7, with one mouse excluded due to missing data. [Figure 15A] Figures 15A and 15B show the effect of AAV-induced HBV on CD8+ T cell responses in C57B1 / 6 mice initially injected with 10 or 10 vg of AAV-1.3HBV and boosted 4 weeks later with 10 vp of an exemplary AdC6-gDPolN vector. In Figure 15B, each slice represents a distinct epitope, with size representing a percentage of the total; only responses greater than 0.1% were included. The slice represents epitopes recognized only in mice infected with AAV8-1.3HBV. [Figure 15B] Figures 15A and 15B show the effect of AAV-induced HBV on CD8+ T cell responses in C57B1 / 6 mice initially injected with 10 or 10 vg of AAV-1.3HBV and boosted 4 weeks later with 10 vp of an exemplary AdC6-gDPolN vector. In Figure 15B, each slice represents a distinct epitope, with size representing a percentage of the total; only responses greater than 0.1% were included. The slice represents epitopes recognized only in mice infected with AAV8-1.3HBV. [Figure 16] Figure 16 shows the frequency of IFN-γ-producing CD8+ T cells in individual C57B1 / 6 mice that were intravenously injected with 10 vg of AAV8-1.3HBV vector, vaccinated 4 weeks later with 5 x 10 vp of AdC6-gDPolN vector, and boosted 2 months later with the same dose of AdC7-gDPolN vaccine. Control mice received the vaccine only. Untreated mice served as additional controls. [Figure 17A] Figures 17A and 17B show A) the percentage of CD8+ T cells within lymphoid infiltrates in the livers of individual mice, and B) the frequency of PolN-tetramer+ CD8+ T cells within the same infiltrates. C57B1 / 6 mice were intravenously injected with 10 or 10 vg of the AAV8-1.3HBV vector and vaccinated 4 weeks later with 5 x 10 vp of the AdC6-gDPolN vector, followed by a booster vaccination with the same dose of AdC7-gDPolN vaccine 2 months later. Control mice received the vaccine alone. Untreated mice served as additional controls. [Figure 17B] Figures 17A and 17B show A) the percentage of CD8+ T cells within lymphoid infiltrates in the livers of individual mice, and B) the frequency of PolN-tetramer+ CD8+ T cells within the same infiltrates. C57B1 / 6 mice were intravenously injected with 10 or 10 vg of the AAV8-1.3HBV vector and vaccinated 4 weeks later with 5 x 10 vp of the AdC6-gDPolN vector, followed by a booster vaccination with the same dose of AdC7-gDPolN vaccine 2 months later. Control mice received the vaccine alone. Untreated mice served as additional controls. [Figure 18A] Figures 18A, 18B, 18C, 18D, 18E, and 18F show the phenotype of infiltrating tetramer+ CD8+ T cells compared to naive (i.e., tetramer-CD44-CD8+) T cells analyzed by mean fluorescence intensity (MFI) of the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001. [Figure 18B] Figures 18A, 18B, 18C, 18D, 18E, and 18F show the phenotype of infiltrating tetramer+ CD8+ T cells compared to naive (i.e., tetramer-CD44-CD8+) T cells analyzed by mean fluorescence intensity (MFI) of the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001. [Figure 18C] Figures 18A, 18B, 18C, 18D, 18E, and 18F show the phenotype of infiltrating tetramer+ CD8+ T cells compared to naive (i.e., tetramer-CD44-CD8+) T cells analyzed by mean fluorescence intensity (MFI) of the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001. [Figure 18D] Figures 18A, 18B, 18C, 18D, 18E, and 18F show the phenotype of infiltrating tetramer+ CD8+ T cells compared to naive (i.e., tetramer-CD44-CD8+) T cells analyzed by mean fluorescence intensity (MFI) of the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001. [Figure 18E] Figures 18A, 18B, 18C, 18D, 18E, and 18F show the phenotype of infiltrating tetramer+ CD8+ T cells compared to naive (i.e., tetramer-CD44-CD8+) T cells analyzed by mean fluorescence intensity (MFI) of the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001. [Figure 18F] Figures 18A, 18B, 18C, 18D, 18E, and 18F show the phenotype of infiltrating tetramer+ CD8+ T cells compared to naive (i.e., tetramer-CD44-CD8+) T cells analyzed by mean fluorescence intensity (MFI) of the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001. [Figure 19A]Figures 19A, 19B, 19C, 19D, 19E, and 19F show the percentage of Tet+ or naive CD8+ T cells positive for the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p≦0.05-0.01, (**) p≦0.01-0.001, (***) p≦0.001-0.0001, (****) p≦0.0001. [Figure 19B] Figures 19A, 19B, 19C, 19D, 19E, and 19F show the percentage of Tet+ or naive CD8+ T cells positive for the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p≦0.05-0.01, (**) p≦0.01-0.001, (***) p≦0.001-0.0001, (****) p≦0.0001. [Figure 19C] Figures 19A, 19B, 19C, 19D, 19E, and 19F show the percentage of Tet+ or naive CD8+ T cells positive for the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p≦0.05-0.01, (**) p≦0.01-0.001, (***) p≦0.001-0.0001, (****) p≦0.0001. [Figure 19D] Figures 19A, 19B, 19C, 19D, 19E, and 19F show the percentage of Tet+ or naive CD8+ T cells positive for the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p≦0.05-0.01, (**) p≦0.01-0.001, (***) p≦0.001-0.0001, (****) p≦0.0001. [Figure 19E] Figures 19A, 19B, 19C, 19D, 19E, and 19F show the percentage of Tet+ or naive CD8+ T cells positive for the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p≦0.05-0.01, (**) p≦0.01-0.001, (***) p≦0.001-0.0001, (****) p≦0.0001. [Figure 19F]Figures 19A, 19B, 19C, 19D, 19E, and 19F show the percentage of Tet+ or naive CD8+ T cells positive for the indicated markers. Lines with stars above indicate significant differences by multiple t-test. (*) p≦0.05-0.01, (**) p≦0.01-0.001, (***) p≦0.001-0.0001, (****) p≦0.0001. [Figure 20A] Figures 20A, 20B, 20C, 20D, 20E, and 20F show CD8+ T cell responses to individual peptides spanning the PolN sequence. Pooled—response to a mixture of all PolN peptides; Naive—response of naive mice to a mixture of all PolN peptides. Figures 20A and 20D show CD8+ T cell responses from mice that received only the AdC6-gDPolN vaccine. Figures 20B and 20E show CD8+ T cell responses from mice that were injected with 1010 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20C and 20F show CD8+ T cell responses from mice that were injected with 1011 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20A, 20B and 20C can be used to calculate the breadth of immune responses due to individual epitopes using the peptide pool shown in Figure 7D and the individual peptide sequences recognized using Figure 7E. [Figure 20B]Figures 20A, 20B, 20C, 20D, 20E, and 20F show CD8+ T cell responses to individual peptides spanning the PolN sequence. Pooled—response to a mixture of all PolN peptides; Naive—response of naive mice to a mixture of all PolN peptides. Figures 20A and 20D show CD8+ T cell responses from mice that received only the AdC6-gDPolN vaccine. Figures 20B and 20E show CD8+ T cell responses from mice that were injected with 1010 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20C and 20F show CD8+ T cell responses from mice that were injected with 1011 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20A, 20B and 20C can be used to calculate the breadth of immune responses due to individual epitopes using the peptide pool shown in Figure 7D and the individual peptide sequences recognized using Figure 7E. [Figure 20C] Figures 20A, 20B, 20C, 20D, 20E, and 20F show CD8+ T cell responses to individual peptides spanning the PolN sequence. Pooled—response to a mixture of all PolN peptides; Naive—response of naive mice to a mixture of all PolN peptides. Figures 20A and 20D show CD8+ T cell responses from mice that received only the AdC6-gDPolN vaccine. Figures 20B and 20E show CD8+ T cell responses from mice that were injected with 1010 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20C and 20F show CD8+ T cell responses from mice that were injected with 1011 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20A, 20B and 20C can be used to calculate the breadth of immune responses due to individual epitopes using the peptide pool shown in Figure 7D and the individual peptide sequences recognized using Figure 7E. [Figure 20D]Figures 20A, 20B, 20C, 20D, 20E, and 20F show CD8+ T cell responses to individual peptides spanning the PolN sequence. Pooled—response to a mixture of all PolN peptides; Naive—response of naive mice to a mixture of all PolN peptides. Figures 20A and 20D show CD8+ T cell responses from mice that received only the AdC6-gDPolN vaccine. Figures 20B and 20E show CD8+ T cell responses from mice that were injected with 1010 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20C and 20F show CD8+ T cell responses from mice that were injected with 1011 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20A, 20B and 20C can be used to calculate the breadth of immune responses due to individual epitopes using the peptide pool shown in Figure 7D and the individual peptide sequences recognized using Figure 7E. [Figure 20E] Figures 20A, 20B, 20C, 20D, 20E, and 20F show CD8+ T cell responses to individual peptides spanning the PolN sequence. Pooled—response to a mixture of all PolN peptides; Naive—response of naive mice to a mixture of all PolN peptides. Figures 20A and 20D show CD8+ T cell responses from mice that received only the AdC6-gDPolN vaccine. Figures 20B and 20E show CD8+ T cell responses from mice that were injected with 1010 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20C and 20F show CD8+ T cell responses from mice that were injected with 1011 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20A, 20B and 20C can be used to calculate the breadth of immune responses due to individual epitopes using the peptide pool shown in Figure 7D and the individual peptide sequences recognized using Figure 7E. [Figure 20F]Figures 20A, 20B, 20C, 20D, 20E, and 20F show CD8+ T cell responses to individual peptides spanning the PolN sequence. Pooled—response to a mixture of all PolN peptides; Naive—response of naive mice to a mixture of all PolN peptides. Figures 20A and 20D show CD8+ T cell responses from mice that received only the AdC6-gDPolN vaccine. Figures 20B and 20E show CD8+ T cell responses from mice that were injected with 1010 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20C and 20F show CD8+ T cell responses from mice that were injected with 1011 vg of AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. Figures 20A, 20B and 20C can be used to calculate the breadth of immune responses due to individual epitopes using the peptide pool shown in Figure 7D and the individual peptide sequences recognized using Figure 7E. [Figure 21A] Figures 21A and 21B show PolN-specific CD8+ T cells in mouse spleens or livers. The left panel of Figure 21A shows the CD8+ T cell responses in the spleens of mice injected with AAV8-1.3HBV followed by a prime-boost schedule of vaccines, with or without 5x1010 vp of AdC6-gDPolN. The middle panel of Figure 21A shows the CD8+ T cell frequencies in the livers of mice treated with different doses of AAV8-1.3HBV and vaccinated in a prime-boost schedule. The right panel of Figure 21A shows the Tox-1 expression levels of PolN-specific CD8+ T cells or naive CD8+ T cells from the same experiment. Figure 21B shows the percentage of IFN-γ+ CD8+ T cells. [Figure 21B]Figures 21A and 21B show PolN-specific CD8+ T cells in mouse spleens or livers. The left panel of Figure 21A shows the CD8+ T cell responses in the spleens of mice injected with AAV8-1.3HBV followed by a prime-boost schedule of vaccines, with or without 5x1010 vp of AdC6-gDPolN. The middle panel of Figure 21A shows the CD8+ T cell frequencies in the livers of mice treated with different doses of AAV8-1.3HBV and vaccinated in a prime-boost schedule. The right panel of Figure 21A shows the Tox-1 expression levels of PolN-specific CD8+ T cells or naive CD8+ T cells from the same experiment. Figure 21B shows the percentage of IFN-γ+ CD8+ T cells. [Figure 22A] Figures 22A and 22B show A) the frequency of CD8+ T cells in the blood of mice injected with the indicated AdC6 vectors, and B) the frequency of tetramer+ CD8+ T cells. [Figure 22B] Figures 22A and 22B show A) the frequency of CD8+ T cells in the blood of mice injected with the indicated AdC6 vectors, and B) the frequency of tetramer+ CD8+ T cells. [Figure 23] FIG. 23 shows CD8+ T cell frequencies in the blood of mice injected with the indicated AdC7 vectors. [Figure 24A] Figures 24A, 24B, 24C, 24D, 24E, and 24F show the frequencies of CD8+ (Figures 24A-C) and CD4+ (Figures 24D-F) T cells in response to gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vectors ("prime") and boosted with the corresponding AdC6 vectors ("booster"). Graphs show the frequencies of IFN-γ-producing T cells, TNF-α-producing T cells, and the sum of the frequencies of T cells producing either cytokine. [Figure 24B]Figures 24A, 24B, 24C, 24D, 24E, and 24F show the frequencies of CD8+ (Figures 24A-C) and CD4+ (Figures 24D-F) T cells in response to gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vectors ("prime") and boosted with the corresponding AdC6 vectors ("booster"). Graphs show the frequencies of IFN-γ-producing T cells, TNF-α-producing T cells, and the sum of the frequencies of T cells producing either cytokine. [Figure 24C] Figures 24A, 24B, 24C, 24D, 24E, and 24F show the frequencies of CD8+ (Figures 24A-C) and CD4+ (Figures 24D-F) T cells in response to gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vectors ("prime") and boosted with the corresponding AdC6 vectors ("booster"). Graphs show the frequencies of IFN-γ-producing T cells, TNF-α-producing T cells, and the sum of the frequencies of T cells producing either cytokine. [Figure 24D] Figures 24A, 24B, 24C, 24D, 24E, and 24F show the frequencies of CD8+ (Figures 24A-C) and CD4+ (Figures 24D-F) T cells in response to gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vectors ("prime") and boosted with the corresponding AdC6 vectors ("booster"). Graphs show the frequencies of IFN-γ-producing T cells, TNF-α-producing T cells, and the sum of the frequencies of T cells producing either cytokine. [Figure 24E]Figures 24A, 24B, 24C, 24D, 24E, and 24F show the frequencies of CD8+ (Figures 24A-C) and CD4+ (Figures 24D-F) T cells in response to gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vectors ("prime") and boosted with the corresponding AdC6 vectors ("booster"). Graphs show the frequencies of IFN-γ-producing T cells, TNF-α-producing T cells, and the sum of the frequencies of T cells producing either cytokine. [Figure 24F] Figures 24A, 24B, 24C, 24D, 24E, and 24F show the frequencies of CD8+ (Figures 24A-C) and CD4+ (Figures 24D-F) T cells in response to gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vectors ("prime") and boosted with the corresponding AdC6 vectors ("booster"). Graphs show the frequencies of IFN-γ-producing T cells, TNF-α-producing T cells, and the sum of the frequencies of T cells producing either cytokine. [Figure 25A] Figures 25A and 25B show HBV DNA viral titers in C57B1 / 6 mice challenged with 1 x 10 vg of AAV8-1.3HBV and vaccinated 4 weeks later with 1 x 10 vp of AdC6-gDPolN ("gDPolN"), AdC6-gDHBV2 ("gDHBV2"), AdC6-gDHBV3 ("gDHBV3"), or AdC6-HBV2 without gD ("HBV2"). Animals infected with AAV but not vaccinated ("Naive") and animals not infected with AAV but not vaccinated (data not shown) served as controls. Figure 25A shows viral titers for each group at weeks 4 and 8 after AAV challenge, and Figure 25B shows results for individual mice at weeks 4 and 8 after AAV challenge. [Figure 25B]Figures 25A and 25B show HBV DNA viral titers in C57B1 / 6 mice challenged with 1 x 10 vg of AAV8-1.3HBV and vaccinated 4 weeks later with 1 x 10 vp of AdC6-gDPolN ("gDPolN"), AdC6-gDHBV2 ("gDHBV2"), AdC6-gDHBV3 ("gDHBV3"), or AdC6-HBV2 without gD ("HBV2"). Animals infected with AAV but not vaccinated ("Naive") and animals not infected with AAV but not vaccinated (data not shown) served as controls. Figure 25A shows viral titers for each group at weeks 4 and 8 after AAV challenge, and Figure 25B shows results for individual mice at weeks 4 and 8 after AAV challenge. [Figure 26A] Figures 26A, 26B, 26C, and 26D show the percentages (as mean) of parental IFN-γ and / or TNF-α-producing CD8+ T cells (Figure 26A), CD44+ CD8+ T cells (Figure 26B), CD4+ T cells (Figure 26C), or CD44+ CD4+ T cells (Figure 26D) at 2 and 8 weeks after the primary vaccination and at 2 and 4 weeks after the booster vaccination with the indicated constructs. [Figure 26B] Figures 26A, 26B, 26C, and 26D show the percentages (as mean) of parental IFN-γ and / or TNF-α-producing CD8+ T cells (Figure 26A), CD44+ CD8+ T cells (Figure 26B), CD4+ T cells (Figure 26C), or CD44+ CD4+ T cells (Figure 26D) at 2 and 8 weeks after the primary vaccination and at 2 and 4 weeks after the booster vaccination with the indicated constructs. [Figure 26C] Figures 26A, 26B, 26C, and 26D show the percentages (as mean) of parental IFN-γ and / or TNF-α-producing CD8+ T cells (Figure 26A), CD44+ CD8+ T cells (Figure 26B), CD4+ T cells (Figure 26C), or CD44+ CD4+ T cells (Figure 26D) at 2 and 8 weeks after the primary vaccination and at 2 and 4 weeks after the booster vaccination with the indicated constructs. [Figure 26D]Figures 26A, 26B, 26C, and 26D show the percentages (as mean) of parental IFN-γ and / or TNF-α-producing CD8+ T cells (Figure 26A), CD44+ CD8+ T cells (Figure 26B), CD4+ T cells (Figure 26C), or CD44+ CD4+ T cells (Figure 26D) at 2 and 8 weeks after the primary vaccination and at 2 and 4 weeks after the booster vaccination with the indicated constructs. [Figure 27A] Figures 27A, 27B, and 27C show CD8+ T cells at multiple time points: 4 weeks after the primary vaccination (Figure 27A); 2 weeks after the booster vaccination (Figure 27B); and 4 weeks after the booster vaccination (Figure 27C) with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3). Graphs show the overall frequency of IFN-γ+-producing CD8+ T cells as assessed by ICS. [Figure 27B] Figures 27A, 27B, and 27C show CD8+ T cells at multiple time points: 4 weeks after the primary vaccination (Figure 27A); 2 weeks after the booster vaccination (Figure 27B); and 4 weeks after the booster vaccination (Figure 27C) with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3). Graphs show the overall frequency of IFN-γ+-producing CD8+ T cells as assessed by ICS. [Figure 27C] Figures 27A, 27B, and 27C show CD8+ T cells at multiple time points: 4 weeks after the primary vaccination (Figure 27A); 2 weeks after the booster vaccination (Figure 27B); and 4 weeks after the booster vaccination (Figure 27C) with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3). Graphs show the overall frequency of IFN-γ+-producing CD8+ T cells as assessed by ICS. [Figure 28A] Figures 28A, 28B, and 28C show cytokine-producing CD4+ T cells assessed by ICS at multiple time points: 4 weeks after the primary vaccination (Figure 28A); 2 weeks after the booster vaccination (Figure 28B); and 4 weeks after the booster vaccination (Figure 28C) with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3). The dashed line indicates the cutoff for a positive response based on results from naive mice. [Figure 28B] Figures 28A, 28B, and 28C show cytokine-producing CD4+ T cells assessed by ICS at multiple time points: 4 weeks after the primary vaccination (Figure 28A); 2 weeks after the booster vaccination (Figure 28B); and 4 weeks after the booster vaccination (Figure 28C) with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3). The dashed line indicates the cutoff for a positive response based on results from naive mice. [Figure 28C] Figures 28A, 28B, and 28C show cytokine-producing CD4+ T cells assessed by ICS at multiple time points: 4 weeks after the primary vaccination (Figure 28A); 2 weeks after the booster vaccination (Figure 28B); and 4 weeks after the booster vaccination (Figure 28C) with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3). The dashed line indicates the cutoff for a positive response based on results from naive mice. [Figure 29A] Figures 29A and 29B show tetramer staining results gated on CD8+ T cells (Figure 29A) or CD44+CD8+ T cells (Figure 29B) 4 weeks after primary inoculation with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2). [Figure 29B] Figures 29A and 29B show tetramer staining results gated on CD8+ T cells (Figure 29A) or CD44+CD8+ T cells (Figure 29B) 4 weeks after primary inoculation with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2). [Figure 30A] Figures 30A, 30B, 30C, 30D, 30E, and 30F show the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of the dye conjugated to the indicated antibody: Figure 30A - anti-PD1 antibody conjugated with BV605, Figure 30B - anti-LAG3 antibody conjugated with BV650, Figure 30C - anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D - anti-CTLA4 antibody conjugated with PE-A; Figure 30E - anti-EOMES antibody conjugated with AF488; and Figure 30F - anti-T-bet antibody conjugated with BV786. [Figure 30B]Figures 30A, 30B, 30C, 30D, 30E, and 30F show the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of the dye conjugated to the indicated antibody: Figure 30A - anti-PD1 antibody conjugated with BV605, Figure 30B - anti-LAG3 antibody conjugated with BV650, Figure 30C - anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D - anti-CTLA4 antibody conjugated with PE-A; Figure 30E - anti-EOMES antibody conjugated with AF488; and Figure 30F - anti-T-bet antibody conjugated with BV786. [Figure 30C] Figures 30A, 30B, 30C, 30D, 30E, and 30F show the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of the dye conjugated to the indicated antibody: Figure 30A - anti-PD1 antibody conjugated with BV605, Figure 30B - anti-LAG3 antibody conjugated with BV650, Figure 30C - anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D - anti-CTLA4 antibody conjugated with PE-A; Figure 30E - anti-EOMES antibody conjugated with AF488; and Figure 30F - anti-T-bet antibody conjugated with BV786. [Figure 30D] Figures 30A, 30B, 30C, 30D, 30E, and 30F show the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of the dye conjugated to the indicated antibody: Figure 30A - anti-PD1 antibody conjugated with BV605, Figure 30B - anti-LAG3 antibody conjugated with BV650, Figure 30C - anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D - anti-CTLA4 antibody conjugated with PE-A; Figure 30E - anti-EOMES antibody conjugated with AF488; and Figure 30F - anti-T-bet antibody conjugated with BV786. [Figure 30E] Figures 30A, 30B, 30C, 30D, 30E, and 30F show the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of the dye conjugated to the indicated antibody: Figure 30A - anti-PD1 antibody conjugated with BV605, Figure 30B - anti-LAG3 antibody conjugated with BV650, Figure 30C - anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D - anti-CTLA4 antibody conjugated with PE-A; Figure 30E - anti-EOMES antibody conjugated with AF488; and Figure 30F - anti-T-bet antibody conjugated with BV786. [Figure 30F]Figures 30A, 30B, 30C, 30D, 30E, and 30F show the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of the dye conjugated to the indicated antibody: Figure 30A - anti-PD1 antibody conjugated with BV605, Figure 30B - anti-LAG3 antibody conjugated with BV650, Figure 30C - anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D - anti-CTLA4 antibody conjugated with PE-A; Figure 30E - anti-EOMES antibody conjugated with AF488; and Figure 30F - anti-T-bet antibody conjugated with BV786. [Figure 31] Figure 31 shows CD8+ T cell responses after a primary vaccination with 5x1010 vp AdC7-gDHBV2 followed two months later by vaccination with 5x1010 vp AdC6-gDHBV2. Numbers on the X-axis correspond to the SEQ ID NOs provided herein. [Figure 32] Figure 32 shows CD8+ T cell responses after a primary vaccination with 5x109 vp AdC7-gDHBV2 followed two months later by vaccination with 5x109 vp AdC6-gDHBV2. Numbers on the x-axis correspond to the SEQ ID NOs provided herein. [Figure 33] Figure 33 shows immunogenicity after a primary vaccination with 5x1010 vp AdC7-gDHBV3 followed two months later by vaccination with 5x1010 vp AdC6-gDHBV3. Numbers on the X-axis correspond to the SEQ ID NOs provided herein. [Figure 34] Figure 34 shows the immunogenicity of AdC6-gDHBV2 and AdC7-gDHBV2 vaccines, corresponding to the SEQ ID NOs (X-axis) provided herein. The Core, PolC, and PolN regions of both HBV2 constructs were immunogenic. [Figure 35] Figure 35 shows the immunogenicity of AdC6-gDHBV3 and AdC7-gDHBV3 vaccines, corresponding to the SEQ ID NOs (X-axis) provided herein. The Core, PolC, and PolN regions of both HBV3 constructs were immunogenic. DETAILED DESCRIPTION OF THE INVENTION

[0015] The disclosed proteins, vaccines, and methods may be readily understood by reference to the following detailed description considered in connection with the accompanying drawings, which form a part of this disclosure: It is to be understood that the disclosed proteins, vaccines, and methods are not limited to the specific proteins, vaccines, and methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to be limiting to the claimed proteins, vaccines, and methods.

[0016] Unless otherwise specifically stated, any description of a possible mechanism or mode of operation or reason for improvement is intended to be exemplary only, and the disclosed proteins, vaccines, and methods should not be constrained by the precision or imprecision of any such proposed mechanism or mode of operation or reason for improvement.

[0017] Throughout this text, the description refers to proteins and methods of using the proteins. Where this disclosure describes or claims features or embodiments relating to proteins, such features or embodiments may equally apply to methods of using the proteins. Similarly, where this disclosure describes or claims features or embodiments relating to methods of using the proteins, such features or embodiments may equally apply to the proteins.

[0018] When a numerical range is described or set forth in this specification, the range includes its endpoints and all individual integers and rational numbers within the range, and also includes each of the smaller ranges formed by all the various possible combinations of those endpoints and internal integers and rational numbers to form subgroups of values ​​within the larger grouping within the stated range, to the same extent, as if each of these smaller ranges were explicitly stated. When a numerical range is defined herein as being greater than a stated value, the range is also finite and bounded on the upper end by values ​​usable within the context of the invention described herein. When a numerical range is defined herein as being less than a stated value, the range is also bounded on the lower end by a non-zero value. When defining a range, it is not intended that the scope of the invention be limited to the specific values ​​set forth. All ranges are inclusive and combinable.

[0019] When values ​​are expressed as approximations, by use of the antecedent word "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise.

[0020] It should be understood that certain features of the disclosed proteins, vaccines, and methods that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed proteins, vaccines, and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0021] As used herein, the singular forms "a", "an" and "the" include the plural forms.

[0022] Various terms relating to the described embodiments are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art unless otherwise specified. Other specifically defined terms should be interpreted consistent with the definitions provided herein.

[0023] As used herein, "immunogenic fragment thereof" refers to a portion of the disclosed HBV core (Core), HBV polymerase N-terminal domain (PolN), or HBV polymerase C-terminal domain (PolC) that is capable of eliciting an immune response in a subject.

[0024] As used herein, "providing to a subject" and like terms refer to a method of delivering a fusion protein, nucleic acid molecule, vector, or vaccine to a subject such that a target cell, tissue, or area of ​​the subject's body comes into contact with the fusion protein, nucleic acid molecule, vector, or vaccine. "Providing to a subject" includes parenteral and oral routes of administration.

[0025] The term "biosimilar" (of an approved reference product / biological product, i.e., reference-listed drug) refers to a biological material that is so similar to the reference product that, despite minor variations in clinically inactive components, there are no clinically meaningful differences between the biosimilar and the reference product with respect to safety, purity, and potency, based on data from (a) analytical studies demonstrating that the biological material is highly similar to the reference product despite minor variations in clinically inactive components, (b) animal studies (including toxicology evaluation), and / or (c) one or more clinical trials (immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and potency under one or more appropriate conditions of use for which the reference product is approved and intended to be used and for which licensing is sought. A biosimilar may be an interchangeable product that may be substituted for the reference product at the pharmacy without the intervention of a prescribing healthcare professional. To meet additional criteria for interchangeability, a biosimilar should be expected to produce the same clinical results as the reference product in any given patient, and if the biosimilar is administered more than once to an individual, the risks of reduced safety or effectiveness from alternating or switching between the use of the biosimilar and the reference product are no greater than the risks of using the reference product without such alternation or switching. The biosimilar utilizes the same mechanism of action for the proposed conditions of use to the extent that the mechanism is known for the reference product. One or more conditions of use prescribed, recommended, or suggested in the proposed labeling for the biosimilar have previously been approved for the reference product. The route of administration, dosage form, and / or strength of the biosimilar are identical to those of the reference product, and the biosimilar is manufactured, processed, packaged, and held in facilities meeting standards designed to ensure that the biosimilar remains safe, pure, and potent. Biosimilars may contain minor amino acid sequence modifications, such as N- or C-terminal deletions, that are not expected to alter the performance of the biosimilar when compared to the reference product. Biosimilars of the disclosed proteins and fusion proteins are included within the scope of this disclosure.

[0026] As used herein, the term "subject" is intended to refer to any animal, particularly a mammal. While the induction of an immune response in mice is exemplified herein, the disclosed methods can be used to treat any type of mammal. Thus, the methods are preferably used in mice and humans, most preferably humans, but are applicable to human and non-human animals.

[0027] The term "comprising" is intended to include examples encompassed by the terms "consisting essentially of" and "consisting of," and similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of."

[0028] The following abbreviations are used herein: hepatitis B virus (HBV); adenovirus (Ad); herpes simplex virus (HSV); glycoprotein (gD), and viral genome (vg).

[0029] Provided herein are non-naturally occurring variants of Hepatitis B virus (HBV) core protein. The disclosed HBV core protein can comprise the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof. Exemplary immunogenic fragments of SEQ ID NO: 6 include SEQ ID NOs: 20-54, provided in Table 3 below. In some embodiments, the immunogenic fragment of HBV core protein comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, the immunogenic fragment of HBV core protein comprises the amino acid sequence of SEQ ID NO: 183.

[0030] Nucleic acid molecules encoding HBV core protein or immunogenic fragments thereof are also provided. The nucleic acid molecule can encode an HBV core protein comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:7. The nucleic acid molecule can encode a core fragment provided in Table 3. In some embodiments, the nucleic acid molecule can encode the amino acid sequence of SEQ ID NO:180. In some embodiments, the nucleic acid molecule can encode the amino acid sequence of SEQ ID NO:183.

[0031] Also provided is a vector comprising a nucleic acid molecule encoding HBV core protein or its immunogenic fragment. Suitable vectors include viral vectors such as lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus replicons, herpesvirus vectors, poxvirus vectors, and rhabdovirus vectors. In some embodiments, the viral vector is an adenoviral vector. The adenoviral vector can be a chimpanzee-derived adenoviral vector. In some embodiments, the vector is the AdC68 vector described in Farina SF, Gao GP, Xiang ZQ, Rux JJ, Burnett RM, Alvira MR, Marsh J, Ertl HC, Wilson JM. "Replication-defective vector based on a chimpanzee adenovirus." J Virol. 2001 Dec; 75(23):11603-13. In some embodiments, the vector is the AdC7 vector described in Reyes-Sandoval A, Fitzgerald JC, Grant R, Roy S, Xiang ZQ, Li Y, Gao GP, Wilson JM, Ertl HC. "Human immunodeficiency virus type 1-specific immune responses in primates upon sequential immunization with adenoviral vaccine carriers of human and simian serotypes" J Virol. 2004 Jul; 78(14):7392-9.In some embodiments, the vector is the AdC6 vector described in Pinto AR, Fitzgerald JC, Giles-Davis W, Gao GP, Wilson JM, Ertl HC. "Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers" J Immunol. 2003 Dec 15; 171(12):6774-9.

[0032] In some embodiments, the vector comprises a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the vector is an AdC6 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the vector is an AdC7 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7.

[0033] In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 180. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 183. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector.

[0034] Vaccines comprising a vector comprising a nucleic acid molecule encoding an HBV core protein or an immunogenic fragment thereof are also disclosed. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the vaccine comprises an AdC6 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the vaccine comprises an AdC7 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the vaccine comprises an AdC6 vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 180. In some embodiments, the vaccine comprises an AdC7 vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 180. In some embodiments, the vaccine comprises an AdC6 vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 183. In some embodiments, the vaccine comprises an AdC7 vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 183.

[0035] The vaccine may further comprise a pharmaceutically acceptable carrier or excipient. As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance that, when combined with the disclosed fusion protein, nucleic acid, or vector, allows the fusion protein, nucleic acid, or vector to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions such as oil / water emulsions, and any of various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

[0036] Also disclosed herein are non-naturally occurring variants of the HBV polymerase N-terminal domain (PolN) and the HBV polymerase C-terminal domain (PolC). The disclosed HBV polymerase N-terminal domain can comprise the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof. Exemplary immunogenic fragments of SEQ ID NO:8 include SEQ ID NOs:55-113, as provided in Table 4 below. In some embodiments, an immunogenic fragment of HBV PolN comprises the amino acid sequence of SEQ ID NO:178. In some embodiments, an immunogenic fragment of HBV PolN comprises the amino acid sequence of SEQ ID NO:181. The disclosed HBV polymerase C-terminal domain can comprise the amino acid sequence of SEQ ID NO:10 or an immunogenic fragment thereof. Exemplary immunogenic fragments of SEQ ID NO:10 include SEQ ID NOs:114-172, as provided in Table 5 below. In some embodiments, an immunogenic fragment of HBV PolC comprises the amino acid sequence of SEQ ID NO:179. In some embodiments, an immunogenic fragment of HBV PolC comprises the amino acid sequence of SEQ ID NO:182.

[0037] Nucleic acid molecules encoding the HBV polymerase N-terminal domain or an immunogenic fragment thereof, or the HBV polymerase C-terminal domain or an immunogenic fragment thereof are also provided. The nucleic acid molecule can encode the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the nucleic acid molecule encoding the HBV polymerase N-terminal domain comprises the nucleic acid sequence of SEQ ID NO:9. The nucleic acid molecule can encode the HBV polymerase N-terminal domain fragment provided in Table 4. The nucleic acid molecule can encode the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the nucleic acid molecule encoding the HBV polymerase C-terminal domain comprises the nucleic acid sequence of SEQ ID NO:11. The nucleic acid molecule can encode the HBV polymerase C-terminal domain fragment provided in Table 5. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:178. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:181. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:179. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:182.

[0038] Also provided are vectors comprising a nucleic acid molecule encoding the HBV polymerase N-terminal domain or an immunogenic fragment thereof, or the C-terminal domain or an immunogenic fragment thereof. Suitable vectors include those described above. In some embodiments, the vector comprises a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the vector comprises a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:11. In some aspects, the vector is an adenoviral vector. Suitable adenoviral vectors include, for example, an AdC6 vector or an AdC7 vector. In some embodiments, the vector is an AdC6 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the vector is an AdC7 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the vector is an AdC6 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the vector is an AdC7 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:178. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 181. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 179. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 182. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector.

[0039] Vaccines comprising a vector comprising a nucleic acid molecule encoding the HBV polymerase N-terminal domain or an immunogenic fragment thereof, or the HBV polymerase C-terminal domain or an immunogenic fragment thereof are also disclosed. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:9. The vaccine can comprise an AdC6 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:9. The vaccine can comprise an AdC7 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:11. The vaccine can comprise an AdC6 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:11. The vaccine can comprise an AdC7 vector comprising a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:11. The vaccine can further comprise a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient as disclosed above. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:178. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:181. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 179. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector. In some embodiments, the vaccine comprises a vector comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 182. In some embodiments, the vector is an AdC6 vector. In some embodiments, the vector is an AdC7 vector.

[0040] Also provided herein are fusion proteins comprising a combination of the disclosed HBV core protein or immunogenic fragment thereof, the HBV polymerase N-terminal domain or immunogenic fragment thereof, and / or the HBV polymerase C-terminal domain or immunogenic fragment thereof. For example, the fusion protein may comprise: (1) an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6 and an HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 8; (2) One or more immunogenic fragments of the HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 and one or more immunogenic fragments of the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8. For example, one or more immunogenic fragments of SEQ ID NO: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) provided in Table 3 and one or more immunogenic fragments of SEQ ID NO: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) provided in Table 4; (3) an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6 and an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 10; (4) One or more immunogenic fragments of the HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 and one or more immunogenic fragments of the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more immunogenic fragments of SEQ ID NO: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) provided in Table 3 and one or more immunogenic fragments of SEQ ID NO: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) provided in Table 5; (5) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; (6) One or more immunogenic fragments of the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 and one or more immunogenic fragments of the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more immunogenic fragments of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) provided in Table 4 and one or more immunogenic fragments of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) provided in Table 5; (7) HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6, HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 8, and HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 10; (8) One or more immunogenic fragments of the HBV core protein comprising the amino acid sequence of SEQ ID NO: 6, one or more immunogenic fragments of the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8, one or more immunogenic fragments of the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more immunogenic fragments of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) provided in Table 3, one or more immunogenic fragments of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) provided in Table 4, and one or more immunogenic fragments of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) provided in Table 5, (9) An HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 178, an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 179, and an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 180, or (10) An HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 181, an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 182, and an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 183. may include:

[0041] The fusion protein can comprise an HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 178, an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 179, and an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 180. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 174.

[0042] The fusion protein can comprise an HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 181, an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 182, or an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 183. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 175.

[0043] Also provided herein are fusion proteins comprising a herpes simplex virus (HSV) glycoprotein (gD) sequence and the disclosed HBV core protein, HBV polymerase N-terminal domain, HBV polymerase C-terminal domain, or various combinations thereof.

[0044] HSV gD is the receptor-binding glycoprotein of HSV. The gD ectodomain is organized into two structurally and functionally distinct regions: an amino-terminus containing the signal sequence and receptor-binding site, and a carboxy-terminus containing the pro-fusion domain and transmembrane domain. gD interacts with the herpesvirus entry mediator (HVEM) receptor and nectin receptor. Interaction of gD with these receptors reduces the binding of the HVEM receptor to BTLA or CD160, immunosuppressive molecules expressed on T cells. In some embodiments, the disclosed fusion proteins comprising gD and the disclosed HBV core protein, HBV polymerase N-terminal domain, HBV polymerase C-terminal domain (referred to as "gDCore," "gDPolN," or "gDPolC," respectively), or combinations thereof, are expected to enhance a subject's immune response to HBV to a greater extent than HBV core and / or polymerase antigens alone (i.e., without gD).

[0045] HSV gD proteins suitable for use in the disclosed fusion proteins include wild-type or mutant gD that retain the ability to 1) increase stimulation of CD8+ T cell responses to antigens and / or 2) interfere with HVEM-BTLA pathway activation.

[0046] The fusion protein can comprise an HBV core protein or an immunogenic fragment thereof disclosed herein, an HBV polymerase N-terminal domain or an immunogenic fragment thereof, an HBV polymerase C-terminal domain or an immunogenic fragment thereof, or any combination thereof, an N-terminal HSV gD protein sequence, and a C-terminal HSV gD protein sequence. The HBV core protein, HBV polymerase N-terminal domain, and HBV polymerase C-terminal domain can be those provided in Table 9 or immunogenic fragments provided in Tables 3-5. The HBV core protein, HBV polymerase N-terminal domain, HBV polymerase C-terminal domain, or immunogenic fragments thereof can be inserted between the N-terminal HSV gD protein sequence and the C-terminal HSV gD protein sequence. In some embodiments, the N-terminal HSV gD protein sequence comprises the amino acid sequence of SEQ ID NO: 12, and the C-terminal HSV gD protein sequence comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the N-terminal HSV gD protein sequence comprises amino acid residues 26-269 of SEQ ID NO: 12.

[0047] The fusion protein is N-terminal HSV gD sequence or a variant thereof, HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6, and C-terminal HSV gD sequence or variants thereof may include:

[0048] The immunogenic fragment of HBV core protein may comprise any one of SEQ ID NOs: 20-54, 180, or 183.

[0049] The fusion protein is N-terminal HSV gD sequence or a variant thereof, HBV core protein comprising the amino acid sequence of SEQ ID NO: 180 or SEQ ID NO: 183, and C-terminal HSV gD sequence or variants thereof may include:

[0050] The fusion protein is N-terminal HSV gD sequence or a variant thereof, HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and C-terminal HSV gD protein sequence or variants thereof may include:

[0051] An immunogenic fragment of the HBV polymerase N-terminal domain can comprise any one of SEQ ID NOs: 55-113, 178, or 181.

[0052] The fusion protein is N-terminal HSV gD sequence or a variant thereof, an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178 or SEQ ID NO: 181; and C-terminal HSV gD protein sequence or variants thereof may include:

[0053] The fusion protein is N-terminal HSV gD sequence or a variant thereof, HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof, and C-terminal HSV gD protein sequence or variants thereof may include:

[0054] An immunogenic fragment of the HBV polymerase C-terminal domain can comprise any one of SEQ ID NOs: 114-172, 179, or 182.

[0055] The fusion protein is N-terminal HSV gD sequence or a variant thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179 or SEQ ID NO: 182; and C-terminal HSV gD protein sequence or variants thereof may include:

[0056] The fusion protein is N-terminal HSV gD sequence or a variant thereof, (1) an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6 and an HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 8; (2) One or more immunogenic fragments of the HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 and one or more immunogenic fragments of the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8. For example, one or more immunogenic fragments of SEQ ID NO: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) provided in Table 3 and one or more immunogenic fragments of SEQ ID NO: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) provided in Table 4; (3) an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6 and an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 10; (4) One or more immunogenic fragments of HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 and the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) provided in Table 3 and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) provided in Table 5; (5) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; (6) One or more immunogenic fragments of the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 and one or more immunogenic fragments of the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more immunogenic fragments of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) provided in Table 4 and one or more immunogenic fragments of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) provided in Table 5; (7) An HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6, an HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 8, and an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 10, or (8) One or more immunogenic fragments of the HBV core protein comprising the amino acid sequence of SEQ ID NO: 6, one or more immunogenic fragments of the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8, one or more immunogenic fragments of the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more immunogenic fragments of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) provided in Table 3, one or more immunogenic fragments of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) provided in Table 4, one or more immunogenic fragments of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) provided in Table 5, (9) An HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 178, an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 179, and an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 180, or (10) An HBV polymerase N-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 181, an HBV polymerase C-terminal domain or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 182, and an HBV core protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 183; an HBV sequence comprising: C-terminal HSVgD protein sequence or variants thereof may include:

[0057] In some embodiments, the N-terminal HSV gD sequence can include at least amino acids 1-269 of HSV gD. The N-terminal HSV gD sequence can include, for example, the amino acid sequence of SEQ ID NO: 12. In some embodiments, the N-terminal HSV gD sequence includes amino acid residues 26-269 of SEQ ID NO: 12.

[0058] In some embodiments, the C-terminal HSV gD sequence comprises the transmembrane domain of HSV gD. The C-terminal HSV gD sequence can comprise, for example, the amino acid sequence of SEQ ID NO:13.

[0059] The fusion protein can comprise the amino acid sequence of SEQ ID NO: 14 (corresponding to gDCore) or an immunogenic fragment thereof. The fusion protein can comprise the amino acid sequence of SEQ ID NO: 16 (corresponding to gDPolN) or an immunogenic fragment thereof. The fusion protein can comprise the amino acid sequence of SEQ ID NO: 18 (corresponding to gDPolC) or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18 or an immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide.

[0060] The fusion protein can comprise the amino acid sequence of SEQ ID NO: 185 (gDHBV2). The fusion protein can comprise the amino acid sequence of SEQ ID NO: 187 (gDHBV3).

[0061] Nucleic acid molecules encoding any of the disclosed fusion proteins are also provided. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 15 (corresponding to gDCore). In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 17 (corresponding to gDPolN). In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 19 (corresponding to gDPolC).

[0062] The nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). The nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3).

[0063] Also disclosed are vectors comprising a nucleic acid molecule encoding the fusion protein. Suitable vectors include those described above, for example, adenoviral vectors. In some embodiments, the adenoviral vector is an AdC6 vector. In some embodiments, the adenoviral vector is an AdC7 vector. The vector can comprise the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). In some embodiments, the vector is an AdC6 vector comprising the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). In some embodiments, the vector is an AdC7 vector comprising the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). The vector can comprise the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3). In some embodiments, the vector is an AdC6 vector comprising the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3). In some embodiments, the vector is an AdC7 vector comprising the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3).

[0064] Vaccines comprising any of the disclosed vectors are also provided. The vaccine may further comprise a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient as described above. The vaccine may comprise a vector comprising the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). In some embodiments, the vaccine comprises an AdC6 vector comprising the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). In some embodiments, the vaccine comprises an AdC7 vector comprising the nucleic acid sequence of SEQ ID NO: 184 (gDHBV2). The vaccine may comprise a vector comprising the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3). In some embodiments, the vaccine comprises an AdC6 vector comprising the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3). In some embodiments, the vaccine comprises an AdC7 vector comprising the nucleic acid sequence of SEQ ID NO: 186 (gDHBV3).

[0065] Provided herein are methods for inducing an immune response against HBV in a subject, the method comprising providing to the subject an effective amount of any of the disclosed fusion proteins, any of the disclosed nucleic acid molecules, any of the disclosed vectors, or any of the disclosed vaccines, thereby inducing an immune response against HBV. In some embodiments, the method comprises providing to the subject an effective amount of any of the disclosed fusion proteins, thereby inducing an immune response against HBV. In some embodiments, the method comprises providing to the subject an effective amount of any of the disclosed nucleic acid molecules, thereby inducing an immune response against HBV. In some embodiments, the method comprises providing to the subject an effective amount of any of the disclosed vectors, thereby inducing an immune response against HBV. In some embodiments, the method comprises providing to the subject an effective amount of any of the disclosed vaccines, thereby inducing an immune response against HBV.

[0066] The method includes providing to the subject an effective amount of a vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18. In some embodiments, the method further includes providing to the subject, after providing the vaccine comprising the AdC6 vector, a vaccine comprising an AdC7 vector comprising a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18. Such a prime-boost method comprises: The method can include providing the subject with a vaccine comprising an AdC6 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 14, and subsequently providing the subject with a vaccine comprising an AdC7 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the amino acid sequence of SEQ ID NO: 14 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide. The method can include providing the subject with a vaccine comprising an AdC6 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 16, and subsequently providing the subject with a vaccine comprising an AdC7 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the amino acid sequence of SEQ ID NO: 16 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide. The method can include providing the subject with a vaccine comprising an AdC6 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 18, and subsequently providing the subject with a vaccine comprising an AdC7 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the amino acid sequence of SEQ ID NO: 18 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide.

[0067] The method includes providing to the subject an effective amount of a vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18. In some embodiments, the method further includes providing to the subject, after providing the vaccine comprising the AdC7 vector, a vaccine comprising an AdC6 vector comprising a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18. Such prime-boost methods include: The method can include providing the subject with a vaccine comprising an AdC7 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 14, and subsequently providing the subject with a vaccine comprising an AdC6 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the amino acid sequence of SEQ ID NO: 14 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide. The method can include providing the subject with a vaccine comprising an AdC7 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 16, and subsequently providing the subject with a vaccine comprising an AdC6 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the amino acid sequence of SEQ ID NO: 16 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide. The method can include providing the subject with a vaccine comprising an AdC7 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 18, and subsequently providing the subject with a vaccine comprising an AdC6 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the amino acid sequence of SEQ ID NO: 18 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide.

[0068] The method can include providing to the subject an effective amount of a vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185 or 187. In some embodiments, the method can further include providing to the subject, after providing the vaccine comprising the AdC6 vector, a vaccine comprising an AdC7 vector comprising a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185 or 187. Such a prime-boost method can include: The method can include providing the subject with a vaccine comprising an AdC6 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185, and subsequently providing the subject with a vaccine comprising an AdC7 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185. In some embodiments, the amino acid sequence of SEQ ID NO: 185 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide, or The method can include providing the subject with a vaccine comprising an AdC6 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 187, and subsequently providing the subject with a vaccine comprising an AdC7 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 187. In some embodiments, the amino acid sequence of SEQ ID NO: 187 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide.

[0069] The method can include providing to the subject an effective amount of a vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185 or 187. In some embodiments, the method can further include providing to the subject, after providing the vaccine comprising the AdC7 vector, a vaccine comprising an AdC6 vector comprising a fusion protein or an immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185 or 187. Such a prime-boost method can include: The method can include providing the subject with a vaccine comprising an AdC7 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185, and subsequently providing the subject with a vaccine comprising an AdC6 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 185. In some embodiments, the amino acid sequence of SEQ ID NO: 185 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide, or The method can include providing the subject with a vaccine comprising an AdC7 vector comprising a fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 187, and subsequently providing the subject with a vaccine comprising an AdC6 vector comprising the fusion protein or immunogenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 187. In some embodiments, the amino acid sequence of SEQ ID NO: 187 or immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide.

[0070] The immune response induced by the disclosed methods includes, but is not limited to, a T cell response, a B cell response, or both (i.e., a cellular and / or humoral immune response). The immune response can be a primary immune response or a secondary immune response. The disclosed methods can induce a subject's immune response against HBV to a greater extent than HBV core and / or polymerase antigen alone (i.e., without gD).

[0071] The disclosed methods can be used for both therapeutic and prophylactic or preventative treatment, reducing the severity and / or frequency of symptoms, eliminating symptoms and / or the underlying causes of symptoms, reducing the frequency or likelihood of symptoms and / or their underlying causes, and ameliorating or treating damage caused directly or indirectly by HBV. Treatment also includes prolonging survival compared to the expected survival of a subject not receiving treatment. Subjects to be treated include those with HBV as well as those prone to have HBV or in whom HBV is to be prevented.

[0072] Thus, the amount of the disclosed fusion proteins, nucleic acid molecules, vectors, or vaccines required to induce an immune response against HBV (e.g., an "effective amount") may vary depending on factors such as the stage of disease, age, sex, and weight of the subject, and the ability of the fusion protein, nucleic acid molecule, vector, or vaccine to elicit a desired response in the subject. Exemplary indicators of an effective amount include, for example, improved well-being in the subject, and reduction, elimination, or prevention of HBV symptoms.

[0073] Also provided is the use of any of the disclosed fusion proteins, nucleic acid molecules, vectors, or vaccines in the manufacture of a medicament for inducing an immune response against HBV in a subject.

[0074] Also provided is the disclosed fusion protein, nucleic acid molecule, vector, or vaccine for use in inducing an immune response against HBV in a subject. [Example]

[0075] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, but not to limit, the disclosed embodiments.

[0076] Generation of epitope-optimized Core sequences Hepatitis B virus (HBV) can be classified into several genotypes based on phylogenetic clustering. To aid in the development of antigen inserts for a multi-genotype HBV vaccine for patients with chronic infection, a preliminary bioinformatics evaluation of the genes encoding HBV core and HBV polymerase across genotypes A, B, C, and D was performed.

[0077] Core amino acid sequences from the four major HBV clades were downloaded as aligned ClustalW sequences from the Hepatitis B Virus Database (HBVdb) (release version 45.0, last updated August 2, 2018). These amino acid sequences represent thousands of HBV genomes entered by users across Europe, as summarized in the table below. [Table 1]

[0078] A "consensus" Core sequence was first identified for each genotype using the Shannon Entropy tool provided by Los Alamos National Laboratory (www.hiv.lanl.gov / content / sequence / ENTROPY / entropy), which calculates the variation and frequency at each amino acid position. These calculations were repeated for each genotype, generating four "consensus" Core sequences, one for each genotype analyzed (SEQ ID NOS: 1-4). Genotype A consensus sequence (SEQ ID NO: 1) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLeDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC- Genotype B consensus sequence (SEQ ID NO: 2) MDID p YKEFGAS v ELLSFLPSDFFPS i RDLLDTA s ALYREALESPEHCSPHHTALRQAI l CWGELMNLATWVGSNL e DPASRELVV s YVNVNMGLK i RQLLWFHISCLTFGRETVLEYLVSFGVWIRTP p AYRP p NAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRE s QC- Genotype C consensus sequence (SEQ ID NO: 3) MDID p YKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVV s YVNVNMGLK i RQlLWFHISCLTFGRETVLEYLVSFGVWIRTP p AYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRSQSRESQC- Genotype D consensus sequence (SEQ ID NO: 4) MDIDPYKEFGA t VELLSFLP s DFFPSVRDLLDTASALYR e ALESPEHCSPHHTALRQAILCWG e LMtLATWVG g NLEDP aSRDLVVSYVNTN m GLKFRQLLWFHISCLTFGR e TV i EYLVSFGVWIRTP p AYRPPNAPILSTLPETTV v RRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC- (Bold underlined residues represent amino acids with a frequency of less than 90%).

[0079] The above "consensus" Core sequences were combined to generate epitope-optimized Core sequences. Conserved amino acids were identified at each amino acid residue in the Core proteins of each genotype (A, B, C, and D), and their frequency and mutations within a given sample of genotype genomes were determined. To select amino acids at mutation sites, each mutation was examined using an epitope prediction algorithm across multiple HLA types, and the sequence with the highest immunogenicity was selected. Specifically, (1) Each residue that was identical across the four genotypes was kept. To align these sequences for mutations, genome-weighted frequencies were also calculated to characterize variability, adding spacers, where applicable. (2) Residues that were not identical across the four genotypes were identified and the amino acid mutations were recorded (see Table 2). Residues that were not identical across the four genotypes were designated X1 to X2. 11 The initial core sequence (SEQ ID NO: 5) is provided below, with residues labeled as * and residues with a frequency less than 90% in bold and underlined font. MDID P YKEFGAX1VELLSFLPSDFFPSX2DLLDTASALYREALESPEHCSPHHTALRQAILCWGELMX3LATWVGX4NLeDPASRX5LVV X 6YVNX7NMGLKX8RQLLWFHISCLTFGRETVX9EYLVSFGVWIRTP P AYRP P NAPILSTLPETTVVRRRX 10 X 11 GRSPRRRTPSPRRRRSQSPRRRRSQSRESQC [Table 2] (3) To determine the final amino acids at these positions, an epitope prediction algorithm was used to select appropriate amino acids. For amino acids that showed variation between genotypes, either the amino acid present in three of these genotypes was selected, or MHC class I epitope prediction software was used to select the most immunogenic amino acid. This approach maximizes potential immunogenicity across the greatest number of HLA types. The epitope-optimized Core sequence across and within all genotypes is shown below (SEQ ID NO: 6). didpykefgatvellsflpsdffpsirdlldtasalyrealespehcsphhtalrqailcwgelmtlatwvgsnledpasrelvvsyvnvnmglkirqllwfhiscltfgretvieylvsfgvwirtppayrppnapilstlpettvvrrrdrgrsprrrtpsprrrrsqsprrrrsqsresqc

[0080] The average variation at each site across all genomes, weighted by the number of clade-specific genomes analyzed, was calculated, and large residues with high area and conservation were shown (Figure 1).

[0081] Generation of epitope-optimized polymerase sequences Epitope-optimized polymerase sequences were generated from the four major HBV clades as discussed above for the Core sequences. Because the polymerases are long, two fragments were generated: an N-terminal fragment (from which segments with high variability between genotypes were removed) and a C-terminal fragment. Both fragments are approximately 300 amino acids in length. The epitope-optimized polymerase amino acid sequences are shown below and in Table 9. Epitope-optimized HBV polymerase N-terminal amino acid sequence (SEQ ID NO: 8) plsyqhfrklllldeeagpleeelprladeglnrrvaedlnlgnlnvsipwthkvgnftglysstvpvfnpewqtpsfpkihlqedivdrckqfvgpltvnekrrlklimparfypnvtkylpldkgikpyypehavnhyfqtrhylhtlwk agilykrettrsasfcgspysweqelqhgscwwlqfrnskpcseyclthlvnlledwgpcdehgehhiriprtparvtggvflvdknphntaesrlvvdfsqfsrgitrvswpkfavpnlqsltnllssnlswlsldvsaafyhiplhpaamp Epitope-optimized HBV polymerase C-terminal amino acid sequence (SEQ ID NO: 10) hllvgssglsryvarlssnsriinhqhgtmqnlhdscsrnlyvsllllyktfgrklhlyshpiilktkrwgyslnfmgyvigswgslpqdhiiqkikecfrklpvnrpidwkvcqrivgllgfaapftqcgypalmplyaciqskqaftfs ptykaflskqylnlypvarqrpglcqvfadatptgwglamghqrmrgtfvaplpihtaellaacfarsrsgakilgtdnsvvlsrkytsfpwllgcaanwilrgtsfvyvpsalnpaddpsrgrlglsrpllrlpfrpttgrtslyavspsv

[0082] Generation of AdC6 and AdC7 vectors expressing epitope-optimized Core and polymerase sequences Genes encoding the epitope-optimized Core and polymerase amino acid sequences were cloned into a transfer vector containing the herpes simplex virus (HSV) glycoprotein D (gD) sequence under the control of a CMV promoter. This gene was then cloned into an E1-deleted, E3 ORF3, 4, 5, 6, and 7-deleted replication-deficient adenoviral vector (described in PCT / US2017 / 043315) to generate the following vectors: AdC6 containing the epitope-optimized Core sequence fused to gD (AdC6-gDCore), AdC6 containing an epitope-optimized polymerase N-terminal sequence fused to gD (AdC6-gDPolN), AdC6 containing an epitope-optimized polymerase C-terminal sequence fused to gD (AdC6-gDPolC), AdC7 containing the epitope-optimized Core sequence fused to gD (AdC7-gDCore), AdC7 containing the epitope-optimized polymerase N-terminal sequence fused to gD (AdC7-gDPolN), and · AdC7 containing an epitope-optimized polymerase C-terminal sequence fused to gD (AdC7-gDPolC).

[0083] Correct clones were identified by restriction enzyme digestion, and the cloning site was sequenced. The vector was rescued, propagated in HEK 293 cells, purified by cesium chloride (CsCl) gradient centrifugation, and vector concentration (vp) was measured spectrophotometrically. The vector was titrated for infectious units when propagated in serial dilutions in HEK 293 cells, after which RNA was isolated, reverse transcribed, and subjected to nested hexon-specific PCR. Genetic integrity of the vector was determined by restriction enzyme digestion followed by gel electrophoresis of purified viral DNA. Protein expression was measured by Western blotting using a gD-specific antigen. Genetic stability was determined by serial passage of the vector in HEK 293 cells (12–15 times) followed by restriction enzyme digestion and gel electrophoresis of purified viral DNA.

[0084] Immunogenicity testing of the vaccine in mice C57Bl / 6, BALB / c, and HLA-A2 tg mice (n = 5 per group) were injected with various concentrations of each of the above vectors. Untreated mice served as controls. Mice were bled at various times postinjection, and the frequencies of insert-specific CD8+ and CD4+ T cells were measured by intracellular cytokine staining (ICS) for IFN-γ. Two months after the first injection, AdC6-immunized mice were boosted with a heterologous vector (AdC7) expressing the same insert. The frequencies of HBV-specific T cells were again tested. Results after the first inoculation are shown in Figures 2A-2F and 3A (C57Bl / 6 mice), 3B (BALB / c mice), and 3C (HLA-A2 mice). Results after the booster inoculation are shown in Figures 4A-4C and 5A-5B.

[0085] C57Bl / 6 mice showed very robust CD8+ T cell responses to the epitope-optimized polymerase N-terminal sequence and poor responses to the epitope-optimized polymerase C-terminal sequence and epitope-optimized Core sequence, whereas CD4+ responses were favorable to the epitope-optimized Core sequence and epitope-optimized polymerase C-terminal sequence (Figures 2A-F). Epitope mapping in C57Bl / 6 mice demonstrated a higher and broader response to PolN than to PolC (Figure 3A). A total of 14 peptides within PolN were recognized by CD8+ T cells, whereas only two adjacent peptides within PolC were recognized, most likely representing a single epitope. CD4+ T cells did not respond to PolN or PolC. This pattern was largely mirrored in BALB / c mice, where CD8+ T cell responses were highest to PolN, recognizing 12 peptides, followed by PolC, recognizing 4 peptides (Figure 3B). Responses to Core were low but surprisingly broad, recognizing 10 peptides (Figure 3B). BALB / c CD4+ T cells responded best to Core, recognizing 15 peptides, with lower recognition of PolC (4 peptides) or PolN (2 peptides). CD8+ T cell responses were also tested in HLA-A2tg mice, where PolN again elicited the highest response, involving 12 peptides (Figure 3C). Responses to PolC were low but broad (16 peptides), whereas only one peptide from Core was detected (Figure 3C). The sequences of the peptides tested in the priming experiments are provided in Table 3 (Core peptides), Table 4 (PolN peptides), and Table 5 (PolC peptides). The peptide composition of the peptide pools from the priming experiments is provided in Tables 6-8. Overall these data indicate that in many cases the inserts that elicited detectable T cell responses were directed against multiple epitopes within each sequence. [Table 3] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 6] [Table 7] [Table 8]

[0086] After booster inoculations tested in C57Bl / 6, BALB / c, and HLA-A2 tg mice, the increase in response was observed against inserts at vector doses that induced suboptimal responses at the initial inoculation, i.e., 1 × 10 9 This was primarily seen against Core at the vp vector dose tested (Figures 4A-4C). Although booster immunizations with higher vector doses failed to increase responses to PolN or PolC, booster immunizations nonetheless broadened T cell responses (Figures 5A-5C).

[0087] Immunogenicity Summary From the above results, The vaccine is immunogenic, with PolN>PolC>Core for CD8+ T cells and Core>PolC>PolN for CD4+ T cell responses. The immune response can be boosted by heterologous vaccine carriers. The immune response is broad, and After booster vaccination, the breadth of T cell responses increases, This shows that:

[0088] Effect of low-dose AAV-1.3 vaccination on HBV titers during HBV exposure 1 x 10 in groups of 3 mice 10 , 1×10 11 or 1.5 x 10 11 1.3HBV (vg) and 8 weeks later, they were vaccinated with AdC6-gDPolN. Viral titers were measured 8 weeks after vaccination and compared with pre-vaccination titers. Figure 6 shows the change in viral load from baseline for each treatment group.

[0089] Epitope changes CD8+ T cells directed against HBV antigens were exhausted during chronic HBV infection. The progression to CD8+ T cell exhaustion was more rapid and pronounced for dominant epitopes than for subdominant epitopes. The underlying reason is that exhaustion is driven by overwhelming antigen-driven stimulation through the T cell receptor, and dominant epitopes are present at higher levels on MHC class I antigens expressed by antigen-presenting cells than subdominant epitopes, which have lower avidity for restriction molecules. Typical vaccine strategies primarily induce immune responses against significant epitopes. Therapeutic vaccines should take into account the decline in T cells directed against dominant epitopes during chronic viral infection and be designed to favor the expansion of CD8+ T cells directed against subdominant epitopes, which is more likely to resist disease-driven exhaustion and translate into better disease control.

[0090] The epitope profile of naive mice immunized with an adenoviral vector containing a nucleic acid sequence encoding the HBV polymerase N-terminal domain (PolN) fused to herpes simplex virus glycoprotein D ("AdC6-gDPolN"; the amino acid sequence of gDPolN is SEQ ID NO: 16) was determined. The responses of mice not pre-treated with the AAV8-1.3HBV vector were compared with those obtained from mice infected with an AAV8 vector expressing the 1.3HBV genome before vaccination with AdC6-gDPolN. The AAV8-1.3HBV vector induced high serum titers of HBV, potentially driving CD8+ T cell exhaustion.

[0091] In the first series of experiments, a peptide pool matrix was used to identify epitopes in mice vaccinated with the AdC6-gDPolN vector but not exposed to the AAV-1.3HBV vector. The number of regions that elicited a protein response in these naive mice was identified (e.g., greater than 1% of IFN-γ-producing CD8+CD44+ T cells). Figures 7A and 8A. In the second experiment, mice were administered 1 x 10 10 Mice were challenged with the AAV-1.3 HBV vector containing the viral genome (vg) and vaccinated 4 weeks later with an AdC6 vector expressing the same HBV polymerase sequence (gDPolN) as unchallenged mice in the first experiment. Ten weeks later, HBV PolN-specific CD8+ T cell epitope profiles were determined using a peptide pool matrix on splenocytes from unchallenged mice prior to vaccination (Figures 7B and 8B). Mice were challenged with 1.5 x 10 11 The experiment was repeated using stringent conditions by challenging mice with a 1000mg dose of AAV8-1.3HBV vector. Four weeks later, mice were vaccinated again, and CD8+ T cell responses to the peptide pool matrix were tested approximately 10 weeks post-vaccination (Figures 7C and 8C). In both experiments, a shift in the epitope profile of mice infected with AAV8-1.3HBV was observed compared to results obtained from unvaccinated mice, with a 1000mg dose per ml serum at the time of vaccination. 7 ~10 9 The mice exposed to the high-dose AAV8-1.3HBV vector had a high viral load of 1.3vg. This effect was more pronounced in mice exposed to the high-dose AAV8-1.3HBV vector. In both experiments, a reduced response was observed. Moreover, particularly in mice exposed to the high-dose AAV8-1.3HBV vector, the results showed a decrease in CD8+ T cells against many of the epitopes that were immunodominant in uninfected, vaccinated mice (e.g., within the region represented by peptides 50–59; Figure 8), with good preservation of subdominant epitopes (such as those within the region represented by peptides 2–8) as well as novel epitopes, such as those within the region represented by peptides 10–29. These data confirm a shift from dominant to subdominant epitope recognition.

[0092] Based on these data, a novel HBV polymerase N-terminal domain insert (HBV PolN v2) was generated (SEQ ID NO: 173). HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT This insert induced CD8+ T cell responses primarily against subdominant epitopes while leaving responses intact in mice with high HBV viral loads.

[0093] Immunogenicity and efficacy of gDCore, gDPolN, and gDPolC vaccines The immunogenicity and efficacy of AdC6-gDCore, AdC6-gDPolN, AdC6-gDPolC, AdC7-gDCore, AdC7-gDPolN, and AdC7-gDPolC vaccines in the AAV8-HBV mouse model were analyzed.

[0094] Methods - Immunogenicity C57Bl / 6 mice (n = 5 per group) were injected with various doses of AdC6-gDCore (gDCore nucleic acid sequence corresponding to SEQ ID NO: 15); AdC6-gDPolN (gDPolN nucleic acid sequence corresponding to SEQ ID NO: 17); or AdC6-gDPolC (gDPolC nucleic acid sequence corresponding to SEQ ID NO: 19). Two months after the first injection, mice immunized with the AdC6 vector were boosted with an AdC7 vector containing the same insert (e.g., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC). Mice were bled 14 and 56 days after injection, and the frequency of T cells against various HBV inserts was analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when cells were stimulated with overlapping peptides representing HBV sequences. Control cells were cultured without peptide. The frequency and phenotype of CD8+ T cells against a single immunodominant epitope within PolN were examined by staining with MHCI tetramer. The breadth and specificity of the CD8+ T cell response to individual peptides within the target sequence was performed by epitope mapping of splenocytes (IFN-γ of CD8+ T cells was tested by ICS).

[0095] To assess CD8+ T cells in the liver, C57Bl / 6 mice (n = 8 per group) were injected with 1 × 10 10 Intravenous administration of AAV8-1.3HBV viral genome (vg), 1 × 10 11 4 weeks after intravenous administration of 5 × 10 9 Mice received a single IM injection of 10 viral particles (vp) of AdC6-gDPolN. Eight weeks after IM injection, mice were sacrificed, livers were removed, and lymphocytes were isolated and stained with T cell markers and tetramers that recognize T cell receptors against immunodominant epitopes present in the PolN sequence.

[0096] In another experiment, three groups of C57Bl / 6 mice (n = 4 per group) were treated with 5 × 10 9 vp (-) or 4 weeks later received a single IM injection of 5 × 10 AdC6-gDPolN9 1 × 10 via the tail vein with or without vp AdC6-gDPolN 11 Mice received intravenous administration of AAV8-1.3HBV, a viral genome (vg). Approximately two months after administration of AAV8-1.3HBV, mice were sacrificed, livers removed, and liver sections from each of the three groups were prepared, stained with hematoxylin and eosin, and evaluated for lymphocytic infiltrates. From the same experiment, cells were stained with specific tetramer- and fluorochrome-conjugated antibodies against T-bet (clone 4B10, BV785), or antibodies against PD-1 (clone 29F.1A12, BF605), TIM-3 (clone RMT3-23, Pe / Cy7), CTLA-4 (clone UC10-4B9, PE), or LAG-3 (clone C9B7W, BV650). Cells were analyzed by flow cytometry and gated on CD44+CD8 tetramer-positive cells and markers. The percentage of marker-positive cells was identified by comparison of the histograms to untreated T cells.

[0097] Methods - Effectiveness AAV8-1.3 HBV vector study—To evaluate the effect of AdC6-gDPolN on chronic HBV viral exposure, C57Bl / 6 mice (n = 8 per group) were injected with 1 × 10 10 vg of AAV8-1.3HBV intravenously via the tail vein, and 4 weeks later, 5 × 10 9 Patients were immunized with a single IM injection of vp AdC6-gDPolN. HBV DNA viral titers were assessed by qPCR, and the change (log ) from baseline before and after vaccine exposure was evaluated. 10 The viral genome copy numbers were reported as HBV copies / mL. Viral genome copy numbers were assessed 4, 6, 8, 10, and 12 weeks after AAV8 challenge. Viral kinetics was assessed by PCR over time, and the log10 change in HBV copies per mL was assessed. The number of mice showing a 1-, 2-, or 3-log reduction at different time points after treatment was assessed.

[0098] Effect of chronic HBV exposure on CD8+ T cell antigen recognition over time - The effect of AAV8-1.3HBV on vaccine-induced liver CD8+ T cells was evaluated. 5 × 10 9 The epitope profile of splenocytes from naive mice immunized with a single IM injection of vp AdC6-gDPolN was determined 4 weeks after vaccination. 10 and 1.5 x 10 11 vg of AAV8-1.3HBV, followed 4 weeks later by 5 × 10 9 Mice vaccinated with vp AdC6-gDPolN had CD8+ T cell epitope profiles in splenocytes obtained 10 weeks after vaccination (14 weeks after AAV injection). Epitope profiles of AAV-naive and AAV-treated vaccinated animals were compared. PolN-specific CD8+ T cells from the liver were analyzed for different markers.

[0099] result Immunogenicity—Vaccination induced robust and sustained CD8+ T cell responses to PolN (median frequency across all circulating CD8+ T cells: 6.0%) and lower responses to PolC and core (median frequencies: 1.0% and 0.4%, respectively; Figures 9A and 9B). After a booster vaccination at 8 weeks, a significant shift was observed for core (p=0.007), with increased responses in all regions (Figure 9C). Figures 9A-9C show CD8+ T cell changes across all CD8+ T cells for individual mice, with medians indicated by lines. Vaccination induced broader epitope recognition by CD8+ T cells, which was further enhanced after a booster vaccination (27% to 34%, Figure 10).

[0100] Twelve weeks after AdC6-gDPolN vaccination, vaccinated mice infected with AAV8-1.3HBV showed a preferential increase in liver CD8+ infiltrates (Figures 11A-11B and Figures 12A-12F), a decrease in the abundance of vaccine-induced HBV-specific CD8+ T cells (Figures 11A and 11B), and a slight decrease in T-bet levels (suggesting loss of effector function) (Figures 13A-13B). Figure 11A shows the CD8+ T cell changes across all recovered lymphocytes from individual livers. Figure 11B shows the tetramer-positive CD8+ T cell changes across all recovered lymphocytes. + Although cellular changes were identified in histograms compared to naive T cells, no clear pattern of cellular markers suggesting differentiation of T cells toward an exhausted phenotype was observed between vaccinated AAV1.3HBV-infected and uninfected mice (Figures 13A-13B).

[0101] Efficacy—After a single IM injection of the AdC6-gDPolN vector, mice infected with AAV8-1.3HBV exhibited a multi-log reduction in serum HBV DNA that persisted for 8 weeks after vaccination (Figure 14). The median reduction in serum HBV DNA viral load levels at 4 and 8 weeks after vaccination was 0.86 and 2.69 log, respectively. 10 At 8 weeks, all animals had >1 log 10 6 / 7 (86%) had a reduction from baseline in cps / mL >2 log 10 had a decrease from baseline in cps / mL, and 2 / 7 (29%) had a >3 log 10 There was a decrease from baseline in cps / mL (Figure 14B).

[0102] After a single AdC6-gDPolN vector injection, distinct CD8+ T cell recognition patterns against PolN peptides were observed in splenocytes when comparing AAV-HBV-infected and untreated mice. Figures 15A and 15B show that mice were first infected with AAV-1.3HBV and then infected with PolN peptides 4 weeks later. 10After booster vaccination with the vp AdC6-gDPolN vector, splenocytes were harvested 8 weeks post-immunization, and IFN-γ was assessed by ICS upon brief ex vivo stimulation with a series of individual peptides spanning PolN. Background frequencies obtained without peptide were subtracted. Figure 15A shows the peptide recognition profile of mice initially injected with the indicated doses of AAV8-1.3HBV vector and subsequently receiving only the AdC6-gDPolN vaccine. The pie chart in Figure 15B shows the total CD44 + CD8 + Responses corresponding to peptides that reached a threshold of 0.1% of cells are shown (data corresponding to those in Figure 15A). Each size / color represents the frequency of responses for that individual peptide, with the size representing a percentage of the total; only responses greater than 0.1% were included. The subtraction shows epitopes recognized only in mice infected with AAV8-1.3HBV. Pretreatment with AAV significantly increased the number of epitopes recognized after a single IM stimulation and CD8 + We found that AAV pretreatment reduced both the magnitude of the immune response as a function of the total number of IFN-γ-producing CD8+ T cells in the T cell pool and the magnitude of the immune response as a function of the total number of IFN-γ-producing CD8+ T cells in the T cell pool. + These responses represented approximately one-third of the T cell responses. The percentage of functional HBV-specific CD8+ T cell responses was highest in untreated mice (4.4%, Figure 15B), but was reduced with low and high doses of AAV8-1.3HBV (2.0% and 0.6%, respectively, Figure 15B). Animals not infected with AAV8-1.3HBV exhibited strong CD8+ T cell responses to many epitopes, which were reduced or altered in animals infected with AAV-HBV, including T cell recognition of novel epitopes.

[0103] Consideration To create a therapeutic HBV vaccine that targets early CD8+ T cell activation using gD as a genetically encoded checkpoint inhibitor. Induce potent and durable CD8+ T cell responses against key HBV antigens (Figure 9). stimulate a very broad CD8+ T cell response (Figure 10), including subdominant epitope recognition (Figure 15), and Functional CD8+ T cells were preferentially trafficked to the liver (Figures 11 and 12) and achieved a sustained multi-log reduction in HBV DNA viral load in an AAV mouse model (Figure 14).

[0104] In the disclosed AAV study, AAV-induced HBV infection resulted in a decrease in CD8+ T cell recognition of the dominant epitope of PolN after vaccination with AdC6-gDPolN (Figure 15). Without intending to be bound by theory, it is believed that the breadth of CD8+ T cells induced by gD and their ability to recognize subdominant epitopes leads to a sustained immune response and multi-log suppression of HBV.

[0105] Immunogenicity of AdC6 / 7-gDPolN in blood and liver after vaccination in AAV-induced HBV-infected animals CD8 responses to AdC6-gDPolN vaccine in the blood, spleen, and liver of animals in the presence of pre-existing AAV-induced HBV infection + To assess T cell responses, the following tests were performed.

[0106] Experiment #1 CD8 of AAV8-1.3HBV-infected mice + T cell response: Response dynamics in the blood Objective: CD8 against gDPolN antigen expressed in AdC6 vector + To evaluate the effect of persistent titers of HBV antigen on T-cell responses.

[0107] Methods: C57Bl / 6 mice were cultured in 10 10 Four weeks later, they received 5 × 10 9 The mice were vaccinated with the AdC6-gDPolN vector at 1000 ng / vp. Control mice received only the AdC6-gDPolN vector. Untreated mice served as additional controls. Mice were boosted two months later with the same dose of AdC7-gDPolN vaccine. Blood was collected at various times after the primary and booster vaccinations to measure IFN-γ-producing CD8+ cells. +PBMCs for T cells were examined.

[0108] Results—As shown in Figure 16, mice expressed active PolN-specific CD8 + CD8 T cell responses were initiated, which gradually declined by week 8 and rose again after a booster vaccination. + T cell responses were more stable after the booster inoculation than after the primary inoculation, and at most time points, responses in the tested mice injected with the AAV8-1.3HBV vector were lower than those in controls that were not injected with the AAV vector.

[0109] Experiment #2 CD8 of AAV8-1.3HBV-infected mice + T cell responses: Responses in the liver Objectives: To investigate the expression of CD8 containing markers indicative of T cell exhaustion in the livers of vaccinated mice infected with AAV8-1.3HBV. + To assess T cell responses.

[0110] Methods: C57Bl / 6 mice were cultured in 10 10 or 10 11 vg of AAV8-1.3HBV vector was injected intravenously. Four weeks later, these were 9 Mice were vaccinated with the AdC6-gDPolN vector at 100 ng / vp. Control mice received only the AdC6-gDPolN vector. Untreated mice served as additional controls. Mice were boosted two months later with the same dose of AdC7-gDPolN vaccine.

[0111] To obtain liver lymphocytes, livers were cut into small pieces and treated with 2 mg / ml collagenase P, 1 mg / ml DNase I (all from Roche, Basel, Switzerland) and 2% FBS (Tissue Culture Biologicals, Tulare, CA) in L15 for 1 hour under stirring. Liver fragments were homogenized and filtered through a 70 μm filter, and lymphocytes were purified by Percoll gradient centrifugation and washed with DMEM supplemented with 10% FBS. Lymphocytes were stained at +4°C in the dark for 30 min with blue-violet live / dead dye (Thermo Fisher Scientific), anti-CD8-APC (clone 53-6.7, BioLegend), anti-CD44-Alexa Flour 700 (clone IM7, BioLegend), anti-EOMES-Alexa Fluor 488 (clone Dan11mag, eBioscience), anti-PD1-BV605 (clone 29F.1A12, BioLegend), anti-LAG3-BV650 (clone C9B7W, BioLegend), anti-T-bet-BV786 (clone 4B10, BioLegend), anti-CTLA-4-PE-A (clone UC10-4B9, BioLegend), and anti-TIM-3-Pe-Cy7-A (clone RMT3-23, BioLegend). Cells were stained with an APC-labeled MHC class I tetramer (NIH Tetramer Facility, Emory University, Atlanta, GA) corresponding to amino acids 396-404 of HBV polymerase (BioLegend) and FAVPNLQSL (SEQ ID NO: 188) (peptide 55). Cells were washed and analyzed using BD FACS Celesta (BD Biosciences, San Jose, CA) and DiVa software. Post-acquisition analysis was performed with FlowJo (TreeStar, Ashland, OR).

[0112] Results: CD8 in lymphocytic liver infiltrates + The frequency of T cells was analyzed. CD8 in lymphocytic liver infiltrates +The frequency of T cells was elevated in vaccinated mice compared with naive mice, and was further elevated in mice injected with the AAV8-1.3HBV vector prior to vaccination (Fig. 17A). PolN-specific CD8 T cells were identified by staining with a tetramer specific for an epitope present in the PolN insert. + The frequency of T cells was reduced in mice injected with AAV-1.3HBV (Fig. 17B).

[0113] Untreated (i.e., tetramer - CD44 - CD8 + Infiltrating tetramers compared to T cells + CD8 + T cell phenotypes were assessed by measuring the mean fluorescence intensity of dyes conjugated to certain antibodies (Figures 18A-18F) and CD8+ cells positive for the indicated markers. + This was assessed by assessing the proportion of T cells (FIGS. 19A to 19F).

[0114] CD8 + T-bet, which regulates the number of T cell functions, increased liver CD8 cells from mice injected with AAV8-1.3HBV before vaccination compared to the vaccine-only group. + The group pretreated with AAV8-1.3HBV did not show an increase in exhaustion markers, and PolN-specific CD8 + The observed decline in T cells is due to the classical CD8 + This suggests that this is unlikely to be caused by T cell exhaustion (Figures 18A-18F and 19A-19F).

[0115] Experiment #3: Breadth of PolN-specific CD8+ T cell responses in mice infected with AAV8-1.3HBV Objective: The presence of HBV stimulates CD8 responses to PolN expressed in gD by AdC vaccine. + To assess whether it affects the breadth of T cell responses.

[0116] Method - 10 mice 10 or 10 11Mice were intravenously injected with 1000 mg of the AAV8-1.3 HBV vector and boosted 2 months later with the corresponding AdC7 vector. Control mice received only the AdC6-gDPolN vector. Ten weeks later, mice were euthanized, and pooled splenocytes were tested against a pool of peptides from non-AAV-infected animals. Results are shown in Figures 20A-C.

[0117] In the second experiment, mice were given 10 10 or 10 11 vg of AAV8-1.3HBV vector was injected intravenously. Four weeks later, these were 10 The mice were vaccinated with the AdC6-gDPolN vector (vp). Control mice received only the AdC6-gDPolN vector. Untreated mice served as additional controls. Six weeks later, IFN-γ-producing CD8+ cells were detected in response to individual peptides spanning the PolN sequence. + Splenocytes were analyzed for T cells, and the results are presented in Figures 20D-20F.

[0118] Results: Presence of HBV, especially 10 11 High HBV titers, such as those after injection of a 2000mg dose of AAV8-HBV1.3, were observed in total CD8 cells against the PolN sequence presented by the AdC6-gDPolN vaccine. + Not only did it reduce T cell responses, but it also caused a shift in epitope recognition profiles.

[0119] Experiment #4 - Function of hepatic PolN-specific CD8+ T cells in mice infected with AAV8-1.3HBV Objective: Liver-infiltrating PolN-specific CD8 in mice infected with AAV8-1.3HBV + To assess whether T cells remain functional.

[0120] Methods—In the first experiment, C57BL / 6 mice were inoculated with 3 × 10 11 One group received 5 × 10 vg of AAV8-1.3HBV intravenously. After 8 weeks, one group received 5 × 10 10The other group was vaccinated with the AdC6-gDPolN vector vp. After 4.5 months, mice were euthanized and analyzed for CD8 responses to the PolN peptide pool. + Splenocytes were examined for the frequency of T cell-producing IFN-γ.

[0121] In the second experiment, mice were inoculated with graded concentrations of AAV8-1.3HBV (1 × 10 10 ,4×10 10 , or 1×10 11 ) after 4 weeks. After 4 weeks, all mice were injected with 5 × 10 10 Mice were vaccinated with 1000 mg of AdC6-gDPolN vector (vp). Two months later, the mice were boosted with the same dose of AdC7-gDPolN vector. Two months later, the mice were euthanized, and lymphocytes were isolated from the liver and analyzed for CD8 responses to the PolN peptide pool. + T cells were examined for IFN-γ production. Cells were also stained with an antibody against Tox, a transcription factor that is increased in exhausted T cells.

[0122] Results—As shown in Figure 21, vaccine-induced CD8 + T cells remained functional in mice injected with the AAV8-1.3HBV vector.

[0123] Experiment #5 - Effect of vaccination of mice infected with AAV8-1.3HBV on liver tissue diagnosis Objective—To evaluate whether AdC6 / 7-gDPolN vaccination of AAV.8-1.3HBV-vaccinated mice results in persistent liver injury.

[0124] Methods: Mice were given 10 mg of 10 ... 10 vg of AAV8-1.3HPV. One month later, these 9Mice were vaccinated with the AdC6-gDPolN vector at 200 ng / vp. Two months later, the mice were boosted with the same dose of AdC7-gDPolN vector given at the same dose. Two months later, the mice were euthanized. Liver sections were collected and fixed in 10% formaldehyde. Sections (up to 3 μm thick) were prepared and stained with hematoxylin and eosin (H&E). These were observed under a light microscope at 20x magnification.

[0125] Results—One of 33 sections from mice that received both the AAV vector and vaccine showed a small lymphocytic infiltrate in the peripheral portion of the liver section.

[0126] As shown in Figure 21B, after a single gDPolN vaccination of HLA-A2-tg mice, IFN-γ-producing liver CD8 + The frequency of T cells was reduced in mice that received AAV compared with those that were simply vaccinated. conclusion CD8 against PolN + T cell responses were reduced in mice infected with AAV8-1.3HBV, yet they remained detectable. Animals pretreated with AAV8-1.3HBV did not elevate exhaustion markers, and PolN-specific CD8 + The observed reduction in T cells is due to the classical CD8 + This suggests that it is unlikely to be caused by T cell exhaustion. AAV-induced HBV infection inhibits CD8 expression of PolN + The epitope recognition profile of the T cell response was altered. Vaccine-induced CD8 in mice previously infected with the AAV8-1.3 HBV vector + T cells remained functional. The vaccine used in the prime-boost regimen did not cause overt liver damage in HBV-positive mice.

[0127] Generation of HBV PolN-PolC-Core construct Two multi-antigen inserts (second-generation PolN-PolC-Core and third-generation PolN-PolC-Core) were generated, the sequences of which are shown below. Second Generation HBV Vaccine Insert ("HBV2") (Pol N (italics)-Pol C (underlined)-Core) (SEQ ID NO: 174) YLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDV QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR Third Generation HBV Vaccine Insert ("HBV3") (Pol N (italics)-Pol C (underlined)-Core) (SEQ ID NO: 175) HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR

[0128] The second-generation HBV ("HBV2") insert contains immunodominant PolN epitopes identified in mice not infected with the AAV8-1.3HBV vector prior to vaccination. Many of these epitopes were found to be diminished in a mouse model of chronic HBV infection induced by prior administration of the AAV8-1.3HBV vector (defined above as "epitope shift"). The third-generation HBV ("HBV3") insert was selected for its proximity to PolN, which is preferentially recognized by mice with high HBV burden (see above). Regions of Core and PolC were chosen for both constructs using the following general formula: the regions with the highest immune response to either the primary (Figure 3) or booster (Figure 5) regions in C57Bl / 6, BALBc, and HLA-A2tg mice, with the goal of selecting unique epitopes and selecting large proximity regions instead of inserting spacer sequences between them.

[0129] Genetic integrity and stability of second- and third-generation HBV inserts (HBV2 and HBV3) Western Blot—The ability of purified recombinant viral vector preparations (AdC6-gDHBV2, AdC6-gDHBV3, AdC7-gDHBV2, and AdC7-gDHBV3) to induce transgene product expression in vitro was evaluated. To this end, Western blot assays were performed to assess gD protein expression in cell lysates following cell culture infection with the vector of interest. Adherent HEK293 cell monolayers were infected with known amounts of purified vector, harvested 48 h postinfection, resuspended in lysis and extraction buffer containing protease inhibitors, and lysed by sonication. Total protein extracts were denatured using dithiothreitol as a redox agent and subjected to electrophoresis in a 12% Bis-Tris polyacrylamide gel (PAGE). After protein separation by SDS-PAGE, samples were transferred onto activated polyvinylidene fluoride membranes by wet electrophoretic transfer. The membrane was immunostained for 1 hour at room temperature to detect gD protein using a primary antibody against gD (clone PA1-30233, Invitrogen, Carlsbad, CA) diluted 1:1000 in saline. The membrane was washed with 1X TBS-T before incubating with HRP-conjugated goat anti-rabbit secondary IgG (ab6721, Abcam, Cambridge UK) for 1 hour at room temperature. This was followed by the addition of a luminol-based chemiluminescent substrate. The stained membrane was exposed to autoradiography film and processed in an automatic film developer to assess signal emission. After describing gD protein expression in infected HEK293 cell lysates, the membrane was stripped and the presence of β-actin was again probed in total protein extract samples. This staining step was utilized to assess the consistency of the PAGE sample loading step and thus better support semiquantitative analysis of in vitro stimulation of gD protein expression by recombinant viral vectors.

[0130] Stability—To ensure the genetic integrity of the viral constructs, the genetic stability of each recombinant viral vector lot was assessed by serial viral passage in adherent HEK293 cell cultures. Recombinant viral pools resulting from each transfection were cultured under standard growth conditions for a total of 12 passages. At the final passage, viral pools were expanded, and crude harvests were purified through a cesium chloride (CsCl) gradient. After vector purification, viral DNA was isolated using the QIAGEN DNeasy Blood & Tissue Kit and assessed by restriction enzyme digestion with two restriction enzymes, Ase I and Bgl II, which cleave the DNA template into distinct, construct-specific, predefined banding patterns. After digestion, samples were electrophoresed in a 1% agarose gel containing ethidium bromide to allow visualization of the digested bands, and the results were subsequently documented using a digital gel imaging system. Virus preparations exhibiting banding patterns identical to those of the early-passage virus were considered to maintain the original molecular clonal structure and, therefore, to be stable through the 12 viral passages.

[0131] Results—The banding pattern of the viral vector DNA remained stable after 12 passages compared with that after 5 passages, indicating that the vector genome was stable (data not shown).

[0132] Immunogenicity of second- and third-generation HBV inserts (HBV2 and HBV3) expressed by AdC6 or AdC7 vectors Objective: CD8 against HBV2 and HBV3 inserts expressed by AdC6 or AdC7 vectors + To assess T cell responses.

[0133] Methods—Groups of C57Bl / 6 mice were cultured at 5 × 10 9 or 5 x 10 10Mice were injected with 1000 bp of AdC6-gDHBV2 or AdC6-gDHBV3 vectors. Mice injected with the same dose of AdC6-gDPolN vector served as a positive control, and untreated mice served as a negative control. 14 days later, mice were bled, and PBMCs were isolated from CD8+ cells that produce IFN-γ in response to a peptide pool corresponding to the HBV insert. + T cell frequencies were assessed. Four weeks later (6 weeks post-vaccination), mice were bled again and challenged with PolN-specific tetramers. Mice immunized with AdC6-gDHBV3 were excluded because this insert lacks the epitope corresponding to the tetramer.

[0134] 5 × 10 groups of C57Bl / 6 mice 9 Or 5 x 10 10 vp AdC7-gDHBV2 or 5 × 10 10 vp AdC7-gDHBV3 vector. Untreated mice served as negative controls. 14 days later, mice were bled, and PBMCs were isolated from CD8+ cells that produce IFN-γ in response to a peptide pool corresponding to the HBV insert. + The frequency of T cells was examined.

[0135] Immunogenicity of AdC7 prime / AdC6 booster Mice were bled 4 weeks later and PBMCs were isolated from CD8+ cells that produce IFN-γ and / or TNF-α in response to peptides related to the insert. + T cells were again examined by ICS. Two months after the primary inoculation, mice were boosted with the same dose of heterologous vector expressing the same insert. Two months later, PBMCs were examined by ICS, and CD8 + and CD4 + T cell responses were compared. After the first inoculation, the AdC7-gDHBV2 vector elicited robust frequencies of CD8 T cells producing IFN-γ and / or TNF-α. + The frequency of T cells increased after the AdC6-gDHBV2 booster vaccination, which was associated with a low dose of vector and IFN-γ-producing CD8 +This was particularly evident for T cells. Although the AdC7-gDHBV3 vector was poorly immunogenic, it significantly increased CD8 T cells after the AdC6-gDHBV3 booster vaccination. + T cell responses became positive. Similarly, CD4+ T cell responses were small after the primary vaccination but increased after the booster vaccination. There was no significant difference in CD4 responses to HBV2 or HBV3 inserts. conclusion Both AdC6-gDHBV2 and AdC7-gDHBV2 vectors were highly immunogenic (Figures 22A, 22B, and 23), but responses were increased after booster vaccination with a heterologous AdC vector expressing the same insert (Figure 24). The AdC7-gDHBV2 and AdC7-gDHBV3 vectors exhibited borderline immunogenicity, consistent with their design, as they lack epitopes corresponding to the tetramers used (Figures 22A and 23). Boosting AdC7-gDHBV2 with AdC6-gDHBV2 increased CD8 + T cell responses are enhanced.

[0136] Comparison of HBV DNA viral titers in mice infected with AdC6-gDPolN, AdC6-gDHBV2, AdC6-gDHBV3, or AdC6-HBV2 AAV method 1 × 10 to five groups of C57Bl / 6 mice 9 vg AAV8-1.3HBV, and 4 weeks later 1 x 10 10 Animals vaccinated with either AdC6-gDPolN (n = 10), AdC6-gDHBV2 (n = 10), AdC6-gDHBV3 (n = 10), or AdC6-HBV2 without gD (n = 10) were infected with AAV but not vaccinated ("naive") (n = 10), and unvaccinated animals not infected with AAV (n = 2-5) served as controls. Viral titers were determined 4 weeks after AAV injection (prevaccination) and compared with levels 4 weeks after vaccination (8 weeks after AAV injection).

[0137] result At week 8, the median HBV viral titer was 0.98 log in untreated mice and 0.98 log in untreated mice, respectively. 10 cps / mL increased and remained unchanged in mice vaccinated with AdC6-HBV2, and −0.04, −1.09, and −2.13 log in animals vaccinated with AdC6-gDHBV3, AdC6-gDPolN, and AdC6-gDHBV2. 10 The results for individual mice are shown in Figure 25B, with all AdC6-gDPolN and AdC6-gDHBV2-vaccinated animals showing a 1 log decrease in cps / mL (Figure 25A). 10 copies / mL, greater than 2 log 10 copies / mL at week 8, whereas untreated, AdC6-HBV2- or AdC6-gDHBV3-vaccinated animals showed a 1 log 10 The number of copies / mL decreased by more than 1.

[0138] Immunogenicity studies of gDHBV2 and gDHBV3 CD8 responses to HBV core and polymerase segments contained in either gDHBV2 or gDHBV3 after a single priming injection or a priming vaccination followed by a booster vaccination with a heterologous vector containing the same insert. + The T cell response and its breadth were assessed.

[0139] Experiment 1 Objective—IFN-γ after primary and booster vaccinations with gD-HBV2 and gD-HBV3 expressed by heterologous chimpanzee adenoviral vectors (AdC6 and AdC7) in C57Bl / 6 mice + CD8 + T cell responses are assessed.

[0140] Methods: Four groups of five C57Bl / 6 mice were treated with: (a) 5 × 10 10 vp AdC7-gDHBV2, 5 × 10 after 2 months 10 vp AdC6-gDHBV2; (b) 5 × 10 9 vp AdC7-gDHBV2, 5 × 10 after 2 months9 vp AdC6-gDHBV2; (c) 5 × 10 10 vp AdC7-gDHBV3, 5 × 10 after 2 months 10 (d) unvaccinated, immunized by intramuscular injection. Blood IFN-γ levels were measured 2 and 6 weeks after the primary vaccination, before the booster vaccination, and 2 and 4 weeks after the booster vaccination. + CD8 + T cell responses were assessed by ICS.

[0141] Results: Each vaccine construct produced IFN-γ at all time points examined. + CD8 + Figure 26 shows parental IFN-γ and / or TNF-α producing CD8 T cells. + The percentages of T cells (Figure 26A), CD44+CD8+ T cells (Figure 26B), CD4+ T cells (Figure 26C), or CD44+CD4+ T cells (Figure 26D) are shown. The mean immune responses assessed by ICS from PBMCs of individual mice are shown 2 and 8 weeks after the primary vaccination and 2 and 4 weeks after the booster vaccination.

[0142] Experiment 2 Objective—To evaluate IFN-γ after primary and booster vaccinations with different doses of gD-HBV2 and gD-HBV3 using heterologous chimpanzee adenovirus vectors (AdC6 and AdC7) in C57Bl / 6 mice. + CD8 + T cell responses are compared to those with gD-PolN.

[0143] Methods: Groups of C57Bl / 6 mice (n=5 mice / group) were treated as follows: gDPolN group (a) 5 × 10 9 vp AdC6-gDPolN, 5 × 10 after 3 months 9 vp AdC7-gDPolN, and (b) 5 × 10 10 vp AdC6-gDPolN, 5 × 10 after 3 months 10 vp AdC7-gDPolN gDHBV2 group (c) 5 × 10 9 vp AdC6-gDHBV2, 3 months later 5 × 10 9 vp AdC7-gDHBV2, and (d) 5 × 10 10 vp AdC6-gDHBV2, 3 months later 5 × 10 10 vp AdC7-gDHBV2 gDHBV3 group (e) 5 × 10 9 vp AdC6-gDHBV3, 5 × 10 after 3 months 9 vp AdC7-gDHBV3, and (f) 5 × 10 10 vp AdC6-gDHBV3, 5 × 10 after 3 months 10 vp AdC7-gDHBV3 No treatment served as a control. were immunized as follows.

[0144] For all treatment groups, immunogenic CD8 + T cell responses were measured using IFN-γ at 2 and 6 weeks after the primary vaccination, and at 2 and 6 weeks before and after the booster vaccination. + Immunogenicity was also assessed 4 weeks after the first vaccination by tetramer staining using APC-labeled MHC class I tetramers (NIH tetramer Facility, Emory University, Atlanta, GA) corresponding to amino acids 396-404 FAVPNLQSL (peptide 55) of the HBV polymerase. HBV3 does not contain the FAVPNLQSL peptide.

[0145] Results: At all time points, each vaccine tested produced IFN-γ + CD8 + The results obtained with the gDHBV2 vaccine were similar to those obtained with the gDPolN vaccine, and the gDHBV3 vaccine was less immunogenic. +T cell frequencies were comparable between the two vaccines, and many activation markers tended to be more highly expressed on tetramer+CD8+ T cells from the gDHBV2-immunized group. Figure 27 shows CD8 T cells at multiple time points: 4 weeks after the primary vaccination (Figure 27A), 2 weeks after the booster vaccination (Figure 27B), and 4 weeks after the booster vaccination (Figure 27C). + The graph shows T cells. IFN-γ as assessed by ICS. + CD8 producing + The overall frequency of T cells is shown.

[0146] Figure 28 shows cytokine-producing CD4+ T cells assessed by ICS at multiple time points: 4 weeks after the primary vaccination (Figure 28A), 2 weeks after the booster vaccination (Figure 28B), and 4 weeks after the booster vaccination (Figure 28C). The dashed line indicates the cutoff for a positive response based on results from naive mice.

[0147] Figure 29 shows the results of tetramer staining gated on either CD8+ T cells (Figure 29A) or CD44+CD8+ T cells (Figure 29B) 4 weeks after the first vaccination.

[0148] Figure 30 shows the phenotype of tetramer+ CD8+ T cells, shown as the mean fluorescence intensity of dye bound to the indicated antibodies: Figure 30A anti-PD1 antibody bound to BV605; Figure 30B anti-LAG3 antibody bound to BV650; Figure 30C anti-TIM3 antibody bound to Pe-Cy7-A; Figure 30D anti-CTLA4 antibody bound to PE-A; Figure 30E anti-EOMES antibody bound to AF488; and Figure 30F anti-T-bet antibody bound to BV786.

[0149] Experiment 3 The breadth of responses was assessed from pooled splenocytes from vaccinated C57BL / 6 mice and was tested by ICS against individual peptides present in the HBV vaccine insert.

[0150] Methods: Four groups of five C57Bl / 6 mice were treated with: (a) 5 × 10 10vp AdC7-gDHBV2, 5 × 10 after 2 months 10 vp AdC6-gDHBV2; (b) 5 × 10 9 vp AdC7-gDHBV2, 5 × 10 after 2 months 9 vp AdC6-gDHBV2; (c) 5 × 10 10 vp AdC7-gDHBV3, 5 × 10 after 2 months 10 (3) No vaccine was administered intramuscularly. Eight weeks after the booster vaccination, the animals were sacrificed and pooled splenocytes were cultured to detect IFN-γ against individual HBV2 or HBV3 peptides. + CD8 + T cell responses were assessed by ICS (cutoff for positive responses was set at 0.1%).

[0151] Results: Independent of dose, the prime-boost regimen with gDHBV2 vaccine induced responses to several epitopes within the core and polymerase. Figure 31 shows that 5x10 10 vp AdC7-gDHBV2 first vaccination and 5 × 10 10 CD8 after vaccination with vpAdC6-gDHBV2 + Figure 32 shows T cell responses. The numbers on the X axis correspond to the SEQ ID NOs provided herein. Figure 32 shows the T cell responses of 5 x 10 9 vp AdC7-gDHBV2 first vaccination and 5 × 10 9 Figure 33 shows the CD8+ T cell response after vaccination with vp AdC6-gDHBV2. The numbers on the X-axis correspond to the SEQ ID NOs. provided herein. Figure 33 shows the CD8+ T cell response after vaccination with 5x10 10 vp AdC7-gDHBV3 first vaccination and 5 × 10 10 1 shows immunogenicity after vaccination with vp AdC6-gDHBV3. Numbers on the X-axis correspond to the SEQ ID NOs provided herein.

[0152] Experiment 4 The breadth of the response was assessed from pooled splenocytes of vaccinated BALB / c mice and was tested by ICS against individual peptides present in the HBV insert.

[0153] Methods: Five groups of five BALB / c mice were cultured as follows: (a) 5 × 10 10 vp AdC6-gDHBV2; (b) 5 × 10 10 vp AdC6-gDHBV3; (c) 5 × 10 10 vp AdC7-gDHBV2; (d) 5 × 10 10 (e) or no vaccine, by intramuscular injection. Twelve weeks after vaccination, the animals were sacrificed, the spleens were harvested, and pooled splenocytes were incubated with IFN-γ against individual HBV2 or HBV3 peptides. + CD8 + T cell responses were assessed by ICS (cutoff for positive responses was set at 0.1%).

[0154] Results: At week 12, each vaccine construct was found to be immunogenic across multiple regions of the core and polymerase genes delivered by the vaccine. Figure 34 shows the immunogenicity of AdC6-gDHBV2 and AdC7-gDHBV2 vaccines, corresponding to the SEQ ID NOs (X-axis) provided herein. The Core, PolC, and PolN regions of both HBV2 constructs were immunogenic. Figure 35 shows the immunogenicity of AdC6-gDHBV3 and AdC7-gDHBV3 vaccines, corresponding to the SEQ ID NOs (X-axis) provided herein. The Core, PolC, and PolN regions of both HBV3 constructs were immunogenic.

[0155] Experiment 5 Methods: Five groups of C57Bl / 6 mice were treated with 1 × 10 9 vg of AAV8-1.3HBV, and 4 weeks later, 1 x 10 10Animals vaccinated with either AdC6-gDPolN (n = 10), AdC6-gDHBV2 (n = 10), AdC6-gDHBV3 (n = 10), or AdC6-HBV2 without gD (n = 10) and infected with AAV but not vaccinated (n = 10) served as controls. Animals not infected with AAV but not vaccinated (n = 2–5) served as controls. Mice were bled at various times postinjection, and the frequencies of insert-specific CD8+ and CD4+ T cells were determined by intracellular cytokine staining (ICS) for IFN-γ. PCR was performed at weeks 2, 6, and 8 after the primary vaccination, and T cell assays were performed at week 4 after the primary vaccination.

[0156] Eight weeks after the primary vaccination, mice were boosted with AdC7 vectors containing the same antigen insert used in the primary vaccination ("booster vaccination"), and blood and serum CD8+ / CD4+ T cells were examined at various times after vaccination as previously described. PCR was performed at weeks 2, 6, and 10 after the booster vaccination, and T cell assays were performed at weeks 4 and 12 after the booster vaccination.

[0157] Those skilled in the art will appreciate that numerical changes and modifications can be made to the preferred embodiments of the present invention and that such changes and modifications can be made without departing from the spirit of the present invention. It is therefore intended that the appended claims cover all equivalent variations as fall within the true spirit and scope of the present invention.

[0158] The disclosures of each patent, patent application, and patent publication cited or described in this document are hereby incorporated by reference in their entirety. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11]

[0159] Embodiment The following list of embodiments is intended to supplement rather than replace or supersede the previous descriptions. Embodiment 1. A Hepatitis B virus (HBV) core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof. Embodiment 2. The HBV core protein of embodiment 1, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 20-54. Embodiment 3. A Hepatitis B virus (HBV) core protein comprising the amino acid sequence of SEQ ID NO: 180 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO: 183 or an immunogenic fragment thereof. Embodiment 4. A nucleic acid molecule encoding the HBV core protein according to any one of embodiments 1 to 3. Embodiment 5. The nucleic acid molecule of embodiment 4, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:7. Embodiment 6. A vector comprising the nucleic acid molecule of embodiment 4 or 5. Embodiment 7. The vector of embodiment 6, wherein the vector is an adenoviral vector. Embodiment 8. The vector of embodiment 7, wherein the adenoviral vector is an AdC6 vector or an AdC7 vector. Embodiment 9. A vaccine comprising the vector according to any one of embodiments 6 to 8. Embodiment 10. An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof. Embodiment 11. The HBV polymerase N-terminal domain of embodiment 10, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 55 to 113. Embodiment 12. An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof. Embodiment 13. An HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof. Embodiment 14. The HBV polymerase C-terminal domain of embodiment 13, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 114-172. Embodiment 15. An HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof. Embodiment 16. A nucleic acid molecule encoding the HBV polymerase according to any one of embodiments 10 to 15. Embodiment 17. The nucleic acid molecule of embodiment 16, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:9. Embodiment 18. The nucleic acid molecule of embodiment 16, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 11. Embodiment 19. A vector comprising the nucleic acid molecule of any one of embodiments 16 to 18. Embodiment 20. The vector of embodiment 19, wherein the vector is an adenovirus vector. Embodiment 21. The vector of embodiment 20, wherein the adenoviral vector is an AdC6 vector or an AdC7 vector. Embodiment 22. A vaccine comprising the vector of any one of embodiments 19 to 21. Embodiment 23. A fusion protein comprising one or more of an HBV core protein having the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV polymerase N-terminal domain having the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV polymerase C-terminal domain having the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof. Embodiment 24. (1) an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof; (2) one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8); (3) an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; (4) one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10); (5) an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; (6) one or more of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10); (7) an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; or (8) one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6), one or more of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8), and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10) 24. The fusion protein of embodiment 23, comprising: Embodiment 25. A fusion protein comprising an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment thereof, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 180 or an immunogenic fragment thereof. Embodiment 26. The fusion protein of embodiment 25, comprising the amino acid sequence of SEQ ID NO: 174. Embodiment 27. A fusion protein comprising an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 183 or an immunogenic fragment thereof. Embodiment 28. The fusion protein of embodiment 27, comprising the amino acid sequence of SEQ ID NO: 175. Embodiment 29. An N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or a variant thereof. HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, and A fusion protein comprising a C-terminal HSV gD sequence or a variant thereof. Embodiment 30. The fusion protein of embodiment 29, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 20-54. Embodiment 31. An N-terminal HSV gD sequence or a variant thereof. an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof; and A fusion protein comprising a C-terminal HSV gD protein sequence or a variant thereof. Embodiment 32. The fusion protein of embodiment 31, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 55-113. Embodiment 33. An N-terminal HSV gD sequence or a variant thereof. an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; and C-terminal HSV gD protein sequence or variants thereof A fusion protein comprising: Embodiment 34. The fusion protein of embodiment 33, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 114-172. Embodiment 35. An N-terminal HSV gD sequence or a variant thereof. (1) an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof; (2) one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8); (3) an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; (4) one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10); (5) an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; (6) one or more of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10); (7) an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof; or (8) HBV sequences comprising one or more of SEQ ID NOs: 20 to 54 (immunogenic fragments of SEQ ID NO: 6), one or more of SEQ ID NOs: 55 to 113 (immunogenic fragments of SEQ ID NO: 8), and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragments of SEQ ID NO: 10), and C-terminal HSV gD protein sequence or variants thereof A fusion protein comprising: Embodiment 36. An N-terminal HSV gD sequence or a variant thereof. HBV core protein comprising the amino acid sequence of SEQ ID NO: 180 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO: 183 or an immunogenic fragment thereof, and A fusion protein comprising a C-terminal HSV gD sequence or a variant thereof. Embodiment 37. An N-terminal HSV gD sequence or a variant thereof. an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof; and A fusion protein comprising a C-terminal HSV gD protein sequence or a variant thereof. Embodiment 38. An N-terminal HSV gD sequence or a variant thereof. an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof; and A fusion protein comprising a C-terminal HSV gD protein sequence or a variant thereof. Embodiment 39. N-terminal HSV gD sequence or a variant thereof, (1) an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment thereof, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 180 or an immunogenic fragment thereof; or (2) an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 183 or an immunogenic fragment thereof; an HBV sequence comprising: A fusion protein comprising a C-terminal HSV gD protein sequence or a variant thereof. Embodiment 40. The fusion protein of embodiment 39, wherein the HBV sequence comprises an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179 or an immunogenic fragment thereof, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 180 or an immunogenic fragment thereof. Embodiment 41. The fusion protein of embodiment 40, wherein the HBV sequence comprises the amino acid sequence of SEQ ID NO: 174. Embodiment 42. The fusion protein of embodiment 39, wherein the HBV sequence comprises an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 183 or an immunogenic fragment thereof. Embodiment 43. The fusion protein of embodiment 42, wherein the HBV sequence comprises the amino acid sequence of SEQ ID NO: 175. Embodiment 44. The fusion protein of any one of embodiments 29 to 43, wherein the N-terminal HSV gD sequence comprises the amino acid sequence of SEQ ID NO: 12. Embodiment 45. The fusion protein of any one of embodiments 29-43, wherein the N-terminal HSV gD sequence comprises amino acid residues 26-269 of SEQ ID NO:12. Embodiment 46. The fusion protein of any one of embodiments 29 to 45, wherein the C-terminal HSV gD sequence comprises the transmembrane domain of HSV gD. Embodiment 47. The fusion protein of any one of embodiments 29 to 46, wherein the C-terminal HSV gD sequence comprises the amino acid sequence of SEQ ID NO: 13. Embodiment 48. A fusion protein according to any one of embodiments 29 to 47, wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 14 or an immunogenic fragment thereof, SEQ ID NO: 16 or an immunogenic fragment thereof, or SEQ ID NO: 18 or an immunogenic fragment thereof. Embodiment 49. The fusion protein of any one of embodiments 39 to 47, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 185. Embodiment 50. The fusion protein of any one of embodiments 39 to 47, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 187. Embodiment 51. A nucleic acid molecule encoding the fusion protein of any one of embodiments 23 to 50. Embodiment 52. The nucleic acid molecule of embodiment 51, wherein the nucleic acid molecule comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 15, 17, or 19. Embodiment 53. The nucleic acid molecule of embodiment 51, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 176. Embodiment 54. The nucleic acid molecule of embodiment 51, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 177. Embodiment 55. The nucleic acid molecule of embodiment 51, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 184. Embodiment 56. The nucleic acid molecule of embodiment 51, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 186. Embodiment 57. A vector comprising the nucleic acid molecule of any one of embodiments 51 to 56. Embodiment 58. The vector of embodiment 57, wherein the vector is an adenovirus vector. Embodiment 59. The vector of embodiment 58, wherein the adenoviral vector is an AdC6 vector or an AdC7 vector. Embodiment 60. A vaccine comprising the vector of any one of embodiments 57 to 59. Embodiment 61. A method for inducing an immune response against HBV in a subject, the method comprising providing to the subject an effective amount of a fusion protein described in any one of embodiments 23 to 50, a nucleic acid molecule described in any one of embodiments 51 to 56, a vector described in any one of embodiments 57 to 59, or a vaccine described in embodiment 60, thereby inducing an immune response against HBV. Embodiment 62. The method of embodiment 61, wherein the vaccine comprises an AdC6 vector comprising a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic fragment thereof. Embodiment 63. The method of embodiment 62, further comprising providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic fragment thereof, subsequent to providing to the subject a vaccine comprising an AdC6 vector. Embodiment 64. The method of embodiment 61, wherein the vaccine comprises an AdC7 vector comprising a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic fragment thereof. Embodiment 65. The method of embodiment 64, further comprising providing to the subject a vaccine comprising an AdC7 vector, followed by providing to the subject a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic fragment thereof. Embodiment 66. The method of embodiment 61, wherein the vaccine comprises an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 185. Embodiment 67. The method of embodiment 66, further comprising providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 185, subsequent to providing to the subject a vaccine comprising the AdC6 vector. Embodiment 68. The method of embodiment 61, wherein the vaccine comprises an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 185. Embodiment 69. The method of embodiment 68, further comprising providing to the subject a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 185, subsequent to providing to the subject a vaccine comprising the AdC7 vector. Embodiment 70. The method of embodiment 61, wherein the vaccine comprises an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 187. Embodiment 71. The method of embodiment 70, further comprising providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 187, subsequent to providing to the subject a vaccine comprising the AdC6 vector. Embodiment 72. The method of embodiment 61, wherein the vaccine comprises an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 187. Embodiment 73. The method of embodiment 72, further comprising providing to the subject a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 187, subsequent to providing to the subject a vaccine comprising the AdC7 vector. Embodiment 74. The method of any one of embodiments 61 to 73, wherein the amino acid sequence of any one of SEQ ID NOs: 14, 16, 18, 185, or 187, or an immunogenic fragment thereof, does not include the N-terminal 25 amino acid signal peptide.

Claims

1. 1. Use of a vaccine in the manufacture of a medicament for inducing an immune response against HBV in a subject, comprising: The vaccine a nucleic acid sequence encoding the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178; A nucleic acid sequence encoding the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179; and a nucleic acid sequence encoding an HBV core protein comprising the amino acid sequence of SEQ ID NO: 180; a nucleic acid molecule comprising The use comprising providing the subject with a first dose of the vaccine, and then providing the subject with a second dose of the vaccine following the first dose.

2. The nucleic acid molecule of the first vaccine, the nucleic acid molecule of the second vaccine, or the nucleic acid molecule of the first and second vaccines further comprises: the N-terminal HSV gD sequence, C-terminal HSV gD sequence, or both, The use according to claim 1, comprising a nucleic acid sequence encoding

3. the first vaccine further comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an N-terminal HSV gD sequence and a nucleic acid sequence encoding a C-terminal HSV gD sequence; and the second vaccine further comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an N-terminal HSV gD sequence and a nucleic acid sequence encoding a C-terminal HSV gD sequence; 3. The use according to claim 2.

4. 4. The use of claim 3, wherein the N-terminal HSV gD sequence of the first vaccine, the N-terminal HSV gD sequence of the second vaccine, or both, comprises the amino acid sequence of SEQ ID NO:

12.

5. 4. The use of claim 3, wherein the N-terminal HSV gD sequence of the first vaccine, the N-terminal HSV gD sequence of the second vaccine, or both, comprises amino acid residues 26-269 of SEQ ID NO:

12.

6. 4. The use of claim 3, wherein the C-terminal HSV gD sequence of the first vaccine, the C-terminal HSV gD sequence of the second vaccine, or both, comprises the transmembrane domain of HSV gD.

7. 7. The use of claim 6, wherein the C-terminal HSV gD sequence of the first vaccine, the C-terminal HSV gD sequence of the second vaccine, or both, comprises the amino acid sequence of SEQ ID NO:

13.

8. The first vaccine comprises: an N-terminal HSV gD sequence comprising amino acid residues 26-269 of SEQ ID NO: 12; HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178; HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179; HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, and a C-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO: 13; and a nucleic acid molecule comprising a nucleic acid sequence encoding The second vaccine comprises: an N-terminal HSV gD sequence comprising amino acid residues 26-269 of SEQ ID NO: 12; HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178; HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179; HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, and a C-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO: 13; a nucleic acid molecule comprising a nucleic acid sequence encoding 4. The use according to claim 3.

9. The first vaccine comprises: an N-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO: 12; HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178; HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179; HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, and a C-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO: 13; and a nucleic acid molecule comprising a nucleic acid sequence encoding The second vaccine comprises: an N-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO: 12; HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178; HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179; HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, and a C-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO: 13; a nucleic acid molecule comprising a nucleic acid sequence encoding 9. The use according to claim 8.

10. 10. The use of any one of claims 1 to 9, wherein the first vaccine nucleic acid molecule, the second vaccine nucleic acid molecule, or both, encode an HBV fusion protein comprising the amino acid sequence of SEQ ID NO:

174.

11. The use of any one of claims 1 to 10, wherein the nucleic acid molecule of the first vaccine, the nucleic acid molecule of the second vaccine, or both, comprises the nucleic acid sequence of SEQ ID NO:

176.

12. 12. The use of any one of claims 1 to 11, wherein the first vaccine nucleic acid molecule, the second vaccine nucleic acid molecule, or both, encode a fusion protein comprising the amino acid sequence of SEQ ID NO:

185.

13. 13. The use of any one of claims 1 to 12, wherein the first vaccine nucleic acid molecule, the second vaccine nucleic acid molecule, or both, comprise the nucleic acid sequence of SEQ ID NO:

184.

14. The use according to claim 12 or 13, wherein the amino acid sequence of SEQ ID NO: 185 does not include the N-terminal 25 amino acid signal peptide.

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