Adenovirus vector encoding hepatitis B virus antigen fused with herpes simplex virus glycoprotein D and method for using the same

Abstract

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 herpes simplex virus (HSV) glycoprotein (gD) sequences, and methods of using the fusion proteins.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 958,809, filed on January 9, 2020; U.S. Provisional Patent Application No. 62 / 958,827, filed on January 9, 2020; U.S. Provisional Patent Application No. 62 / 967,242, filed on January 29, 2020; U.S. Provisional Patent Application No. 62 / 967,104, filed on January 29, 2020; U.S. Provisional Patent Application No. 63 / 064,506, filed on August 12, 2020; U.S. Provisional Patent Application No. 63 / 064,571, filed on August 12, 2020; U.S. Provisional Patent Application No. 63 / 112,202, filed on November 11, 2020; and U.S. Provisional Patent Application No. 63 / 112,219, filed on November 11, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted 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 variants of hepatitis B virus (HBV) core protein, the N - terminal domain of HBV polymerase, and the C - terminal domain of HBV polymerase of non - natural origin, as well as immunogenic fragments thereof and fusion proteins containing the same.

Background Art

[0004] The World Health Organization estimated in 2015 that 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, mainly due to cirrhosis and hepatocellular carcinoma (i.e., primary liver cancer). Assuming that women of reproductive age account for 25.3% of the world's population (United Nations data), chronically infected adults can 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 population (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) in the United States.

[0005] Despite the availability of HBV preventive vaccines, the burden of chronic HBV infection continues to be a major unaddressed global medical problem due to suboptimal treatment options and the continuing rate of new infections in most regions of the developing world.

Summary of the Invention

[0006] This specification provides 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] Disclosed is also an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof.

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

[0010] This specification also provides a fusion protein comprising a combination of an N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or a variant thereof, 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 a variant thereof.

[0011] Also 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] This specification also provides a method of inducing an immune response against HBV in a subject, the method comprising providing to the subject an effective amount of any one 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 further 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. In the drawings.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

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

[0016] Unless otherwise specifically stated, any description of possible mechanisms or modes of operation or reasons for improvement are intended only as examples, and the disclosed proteins, vaccines, and methods should not be constrained by the accuracy or inaccuracy of any such proposed mechanisms or modes of operation or reasons for improvement.

[0017] Throughout the text, the description relates to proteins and methods of using such proteins. When the disclosure describes or claims features or embodiments related to a protein, such features or embodiments are equally applicable to methods of using the protein. Similarly, when the disclosure describes or claims features or embodiments related to methods of using a protein, such features or embodiments are equally applicable to the protein.

[0018] When a numerical range is recited or set forth in the specification, that range includes its endpoints and all individual integers and rational numbers within that range, and each of these narrower ranges formed by all the various possible combinations of those endpoints and internal integers and rational numbers forms sub-groups of a larger group of values within the defined range to the same extent as if each of these narrower ranges was explicitly recited. When a numerical range is defined herein to be greater than a defined value, that range is still finite and is bounded at the upper end by the values that are usable within the context of the invention described herein. When a numerical range is defined herein to be less than a defined value, that range is still bounded at the lower end by non-zero values. When defining a range, it is not intended that the scope of the invention be limited to the specific values recited. All ranges are inclusive and combinable.

[0019] By use of the antecedent term “about” where a value is expressed as an approximation, a particular value is understood to form another embodiment. A reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

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

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

[0022] Various terms related to the mode of description are used throughout this specification and the claims. Such terms should be given their ordinary meaning in the art, unless otherwise specified. Other specifically defined terms should be construed to be consistent with the definitions provided herein.

[0023] As used herein, "its immunogenic fragment" refers to a part of the disclosed HBV Core, HBV polymerase N-terminal domain (PolN), or HBV polymerase C-terminal domain (PolC) that can elicit an immune response in a subject.

[0024] As used herein, "providing to a subject" and similar 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 region 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 administration routes.

[0025] The term "biological follow-on product" (of an approved reference product / biological product, i.e., a reference listed drug) refers to a biological substance that is very similar to the reference product and, based on (a) analytical studies demonstrating similarity to the reference product despite minor differences in clinically inactive components, (b) animal studies (including toxicity assessments), and / or (c) data from one or more clinical trials (immunogenicity and pharmacokinetics or pharmacodynamics), is shown to have safety, purity, and potency sufficient to demonstrate safety, purity, and potency under one or more appropriate conditions of use for which the reference product is approved, intended to be used, and for which approval is sought for the biological follow-on product. A biological follow-on product 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 further criteria for interchangeability, a biological follow-on product should be expected to provide the same clinical outcome as the reference product in any given patient, and if the biological follow-on product is administered to an individual more than once, the risk of decreased safety or efficacy associated with alternating or switching between the use of the biological follow-on product and the reference product should not be higher than the risk of using the reference product without such alternation or switching. A biological follow-on product utilizes the same mechanism of action for the proposed conditions of use to the extent known to the agency for the reference product. One or more conditions of use prescribed, recommended, or proposed in the labeling proposed for the biological follow-on product have been previously approved for the reference product. The route of administration, dosage form, and / or strength of the biological follow-on product are the same as those of the reference product, and the biological follow-on product is manufactured, processed, packaged, and held in a facility that meets standards designed to ensure that the biological follow-on product remains safe, pure, and potent. A biological follow-on product may include minor modifications to the amino acid sequence, such as N-terminal or C-terminal deletions, that are not expected to change the performance of the biological follow-on product when compared to the reference product. Biological follow-on products of the disclosed proteins and fusion proteins are included within the scope of the present disclosure.

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

[0027] The term "comprising" is intended to include examples subsumed by the terms "consisting essentially of" and "consisting of", and similarly the term "consisting essentially of" is intended to include examples subsumed 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 the 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 the HBV core protein comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, the immunogenic fragment of the HBV core protein comprises the amino acid sequence of SEQ ID NO: 183.

[0030] Also provided are nucleic acid molecules encoding an HBV core protein or an immunogenic fragment thereof. 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 are vectors comprising a nucleic acid molecule encoding an HBV core protein or an immunogenic fragment thereof. 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 an adenoviral vector derived from chimpanzee. In some aspects, 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 aspects, 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 an AdC6 vector as 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] Also disclosed are vaccines comprising a vector comprising a nucleic acid molecule encoding an HBV core protein or an immunogenic fragment thereof. 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 a pharmaceutically acceptable excipient. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance that, when combined with the disclosed fusion protein, nucleic acid, or vector, retains the biological activity of this fusion protein, nucleic acid, or vector and is non-reactive with the immune system of the subject. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil / water emulsions, and 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 in this specification are variants of non-natural origin of the HBV polymerase N-terminal domain (PolN) and the HBV polymerase C-terminal domain (PolC). The disclosed HBV polymerase N-terminal domain can include 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 to 113 provided in Table 4 below. In some embodiments, the immunogenic fragment of HBV PolN includes the amino acid sequence of SEQ ID NO: 178. In some embodiments, the immunogenic fragment of HBV PolN includes the amino acid sequence of SEQ ID NO: 181. The disclosed HBV polymerase C-terminal domain can include 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 to 172 provided in Table 5 below. In some embodiments, the immunogenic fragment of HBV PolC includes the amino acid sequence of SEQ ID NO: 179. In some embodiments, the immunogenic fragment of HBV PolC includes the amino acid sequence of SEQ ID NO: 182.

[0037] Also provided are 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. 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 adenovirus vector. Suitable adenovirus vectors include, for example, the AdC6 vector or the 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 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: 179. 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: 182. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

[0039] Also disclosed are vaccines comprising a vector comprising a nucleic acid molecule encoding an HBV polymerase N-terminal domain or an immunogenic fragment thereof or an HBV polymerase C-terminal domain or an immunogenic fragment thereof. 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 excipient 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 aspects, the vector is an AdC6 vector. In some aspects, 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 aspects, the vector is an AdC6 vector. In some aspects, 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 aspects, the vector is an AdC6 vector. In some aspects, 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 aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

[0040] This specification also provides fusion proteins comprising combinations of the disclosed HBV core protein or its immunogenic fragments, the HBV polymerase N-terminal domain or its immunogenic fragments, and / or the HBV polymerase C-terminal domain or its immunogenic fragments. For example, the fusion proteins may be: (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 immunogenic fragments of an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 and one or more immunogenic fragments of an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8. For example, one or more of SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ ID NOs: 55-113 provided in Table 4 (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 immunogenic fragments of an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 and one or more immunogenic fragments of an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more of SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ ID NO: 6) and one or more of SEQ ID NOs: 114-172 provided in Table 5 (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 immunogenic fragments of an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 and one or more immunogenic fragments of an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10. For example, one or more of SEQ ID NOs: 55-113 provided in Table 4 (immunogenic fragments of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172 provided in Table 5 (immunogenic fragments of SEQ ID NO: 10), (7) The HBV core protein containing the amino acid sequence of SEQ ID NO: 6 or its immunogenic fragment, the HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO: 8 or its immunogenic fragment, and the HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO: 10 or its immunogenic fragment, (8) One or more immunogenic fragments of the HBV core protein containing the amino acid sequence of SEQ ID NO: 6, one or more immunogenic fragments of the HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO: 8, and one or more immunogenic fragments of the HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO: 10. For example, one or more of SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ ID NO: 6), one or more of SEQ ID NOs: 55-113 provided in Table 4 (immunogenic fragments of SEQ ID NO: 8), and one or more of SEQ ID NOs: 114-172 provided in Table 5 (immunogenic fragments of SEQ ID NO: 10), (9) The HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO: 178 or its immunogenic fragment, the HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO: 179 or its immunogenic fragment, and the HBV core protein containing the amino acid sequence of SEQ ID NO: 180 or its immunogenic fragment, or (10) The HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO: 181 or its immunogenic fragment, the HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO: 182 or its immunogenic fragment, and the HBV core protein containing the amino acid sequence of SEQ ID NO: 183 or its immunogenic fragment can include.

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

[0042] The fusion protein can comprise 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, or an HBV core protein comprising the amino acid sequence of SEQ ID NO: 183 or an immunogenic fragment thereof. 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 a receptor-binding glycoprotein of HSV. The gD extracellular domain is organized into two structurally and functionally distinct regions, namely the amino terminus containing the signal sequence and receptor-binding site, and the carboxy terminus containing the prefusion domain and transmembrane domain. gD interacts with the herpesvirus entry mediator (HVEM) receptor and the nectin receptor. Interaction of gD with these receptors reduces the binding of the HVEM receptor to BTLA or CD160, which are 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 (collectively referred to as "gDCore", "gDPolN", or "gDPolC", respectively), or combinations thereof are expected to enhance the subject's immune response to HBV to a greater extent compared to 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 the stimulation of CD8+ T cell responses to an antigen and / or 2) interfere with HVEM-BTLA pathway activation.

[0046] The fusion protein can comprise an HBV core protein or an immunogenic fragment thereof, 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, as disclosed herein. The HBV core protein, the HBV polymerase N-terminal domain, and the HBV polymerase C-terminal domain can be those provided in Table 9 or the immunogenic fragments provided in Tables 3-5. The HBV core protein, the HBV polymerase N-terminal domain, the 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 an N-terminal HSV gD sequence or a variant thereof, an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, and a C-terminal HSV gD sequence or a variant thereof and can comprise.

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

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

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

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

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

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

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

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

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

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

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

[0059] The fusion protein can include the amino acid sequence of SEQ ID NO: 14 (corresponding to gDCore) or an immunogenic fragment thereof. The fusion protein can include the amino acid sequence of SEQ ID NO: 16 (corresponding to gDPolN) or an immunogenic fragment thereof. The fusion protein can include the amino acid sequence of SEQ ID NO: 18 (corresponding to gDPolC) or an immunogenic fragment thereof. In some embodiments, any one of the amino acid sequences of SEQ ID NO: 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 include the amino acid sequence of SEQ ID NO: 185 (gDHBV2). The fusion protein can include the amino acid sequence of SEQ ID NO: 187 (gDHBV3).

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

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

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

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

[0065] This specification provides 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, 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 comprises providing to the subject an effective amount of a vaccine comprising an AdC6 vector, the AdC6 vector comprising a fusion protein comprising any one of the amino acid sequences of SEQ ID NO: 14, 16, or 18 or an immunogenic fragment thereof. In some embodiments, the method further comprises providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising any one of the amino acid sequences of SEQ ID NO: 14, 16, or 18 or an immunogenic fragment thereof after providing to the subject a vaccine comprising an AdC6 vector. Such a prime-boost method can comprise providing to the subject a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 14 or an immunogenic fragment thereof, and subsequently providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 14 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO: 14 or an immunogenic fragment thereof does not comprise the N-terminal 25 amino acid signal peptide. Providing a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 16 or an immunogenic fragment thereof to the subject, and subsequently providing a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 16 or an immunogenic fragment thereof to the subject can be included. In some embodiments, the amino acid sequence of SEQ ID NO: 16 or an immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide. Providing a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 18 or an immunogenic fragment thereof to the subject, and subsequently providing a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 18 or an immunogenic fragment thereof to the subject can be included. In some embodiments, the amino acid sequence of SEQ ID NO: 18 or an immunogenic fragment thereof does not include the N-terminal 25 amino acid signal peptide.

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

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

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

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

[0071] The disclosed methods can be used for both therapeutic treatment and prophylactic or preventative treatment, to reduce the severity and / or frequency of symptoms, to eliminate symptoms and / or the causes underlying the symptoms, to reduce the frequency or likelihood of symptoms and / or the causes underlying them, and to ameliorate or treat the damage caused directly or indirectly by HBV. The treatment also includes extending the survival time compared to the expected survival time of untreated subjects. Subjects to be treated include those having HBV, those having a tendency to have HBV, or those in whom HBV should be prevented.

[0072] The amount (e.g., "effective amount") of the disclosed fusion protein, nucleic acid molecule, vector, or vaccine required to induce an immune response against HBV can vary depending on factors such as the subject's disease stage, age, gender, and weight, and the ability of the fusion protein, nucleic acid molecule, vector, or vaccine to elicit the desired response in the subject. Exemplary indicators of an effective amount include, for example, improved well-being of the subject, 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.

Examples

[0075] The following examples are provided to further illustrate some of the embodiments disclosed in this specification. The examples are intended to be illustrative and not intended 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 assist in the development of antigen inserts for multiple-genotype HBV vaccines for patients with chronic infections, 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 four major HBV clades were downloaded as ClustalW-aligned sequences from the Hepatitis B Virus Database (HBVdb) (release version 45.0, last update August 2, 2018). These amino acid sequences represent thousands of HBV genomes entered by users across Europe, as summarized in the following table.

Table 1

[0078] The Shannon Entropy tool provided by the Los Alamos National Laboratory (www.hiv.lanl.gov / content / sequence / ENTROPY / entropy), which calculates mutations and frequencies at each amino acid position, was used to first identify a "common" Core sequence for each genotype. These calculations were repeated for each genotype, generating four "common" Core sequences (SEQ ID NOs: 1-4) that are those of each analyzed genotype. Genotype A Common Sequence (SEQ ID NO: 1) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLeDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC- Genotype B common 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 common sequence (SEQ ID NO: 3) MDID p YKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVV s YVNVNMGLK i RQlLWFHISCLTFGRETVLEYLVSFGVWIRTP p AYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRSQSRESQC- Genotype D common 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- (The residues underlined in bold represent amino acids with a frequency of less than 90%).

[0079] The above "common" Core sequences were combined to generate epitope-optimized Core sequences. The conserved amino acids were identified with each amino acid residue of the Core proteins of each genotype (A, B, C, and D), and the frequency and variation within a given sample of the genotype genome were determined. To select the amino acids at the mutation sites, each mutation was examined using an epitope prediction algorithm among multiple HLA types, and the sequence with the highest immunogenicity was selected. Specifically, (1) Each residue that was identical across the four genotypes was maintained. To align these sequences for mutations, genomic weighted frequencies were also calculated to characterize the variability with spacers added where applicable. (2) Residues that were not identical across the four genotypes were identified, and amino acid mutations were recorded (see Table 2). Residues that were not identical across the four genotypes were labeled as X1 to X 11 and the initial core sequence (SEQ ID NO: 5) with residues having a frequency of less than 90% represented in bold underlined font is provided below. MDID P YKEFGAX1VELLSFLPSDFFPSX2DLLDTASALYREALESPEHCSPHHTALRQAILCWGELMX3LATWVGX4NLeDPASRX5LVV X 6YVNX7NMGLKX8RQLLWFHISCLTFGRETVX9EYLVSFGVWIRTP P AYRP P NAPILSTLPETTVVRRRX 10 X 11 GRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

Table 2

[0080] The average variation at each site across all genomes, weighted by the number of clade-specific genomes analyzed, was calculated, showing residues with high area and conservation. 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 sequence. Since the polymerase is 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) plsyqhfrklllldeeagpleeelprladeglnrrvaedlnlgnlnvsipwthkvgnftglysstvpvfnpewqtpsfpkihlqedivdrckqfvgpltvnekrrlklimparfypnvtkylpldkgikpyypehavnhyfqtrhylhtlwkagilykrettrsasfcgspysweqelqhgscwwlqfrnskpcseyclthlvnlledwgpcdehgehhiriprtparvtggvflvdknphntaesrlvvdfsqfsrgitrvswpkfavpnlqsltnllssnlswlsldvsaafyhiplhpaamp Epitope-optimized HBV polymerase C-terminal amino acid sequence (SEQ ID NO: 10) hllvgssglsryvarlssnsriinhqhgtmqnlhdscsrnlyvsllllyktfgrklhlyshpiilktkrwgyslnfmgyvigswgslpqdhiiqkikecfrklpvnrpidwkvcqrivgllgfaapftqcgypalmplyaciqskqaftfsptykaflskqylnlypvarqrpglcqvfadatptgwglamghqrmrgtfvaplpihtaellaacfarsrsgakilgtdnsvvlsrkytsfpwllgcaanwilrgtsfvyvpsalnpaddpsrgrlglsrpllrlpfrpttgrtslyavspsv

[0082] Generation of AdC6 and AdC7 vectors expressing epitope-optimized Core and polymerase sequences Genes encoding 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 the CMV promoter. This gene was then cloned into an E1-deleted, E3 ORF3, 4, 5, 6, and 7-deleted replication-deficient adenovirus vector (described in PCT / US2017 / 043315) to generate the following vectors. · AdC6 (AdC6-gDCore) containing an epitope-optimized Core sequence fused with gD, · AdC6 (AdC6-gDPolN) containing an epitope-optimized polymerase N-terminal sequence fused with gD, · AdC6 (AdC6-gDPolC) containing an epitope-optimized polymerase C-terminal sequence fused with gD, · AdC7 (AdC7-gDCore) containing an epitope-optimized Core sequence fused with gD, · AdC7 (AdC7-gDPolN) containing an epitope-optimized polymerase N-terminal sequence fused with gD, and · AdC7 (AdC7-gDPolC) containing an epitope-optimized polymerase C-terminal sequence fused with gD.

[0083] Appropriate clones were identified by restriction enzyme digestion, and the cloning sites were sequenced. The vector was rescued, propagated in HEK 293 cells, purified by cesium chloride (CsCl) gradient centrifugation, and the vector concentration (vp) was measured by spectrophotometry. When the vector was propagated in serial dilutions in HEK 293 cells, it was titrated against infectious units, and then RNA was isolated and reverse transcribed, and a nested hexon-specific PCR reaction was performed. The genetic integrity of the vector was determined by restriction enzyme digestion and subsequent gel electrophoresis of the purified viral DNA. Protein expression was measured by Western blotting using a gD-specific antigen. Genetic stability was determined by serial passage (12 - 15 times) of the vector in HEK 293 cells and subsequent restriction enzyme digestion and gel electrophoresis of the purified viral DNA.

[0084] Immunogenicity test of the vaccine in mice C57Bl / 6, BALB / c, and HLA-A2 tg mice (n = 5 per group) were injected with each of the above vectors at various concentrations. Untreated mice served as controls. Mice were bled at various times after injection, 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 boostered with a heterologous vector (AdC7) expressing the same insert. The frequencies of HBV-specific T cells were tested again. The results after the primary inoculation are shown in Figures 2A - 2F and 3A (C57Bl / 6 mice), 3B (BALB / c mice), and 3C (HLA-A2 mice). The results after the booster inoculation are shown in Figures 4A - 4C and 5A - 5B.

[0085] C57Bl / 6 mice showed a very robust CD8+ T cell response to the epitope-optimized polymerase N-terminal sequence, a low response to the epitope-optimized polymerase C-terminal sequence and the epitope-optimized Core sequence, while the CD4+ response was good to the epitope-optimized core sequence and the epitope-optimized polymerase C-terminal sequence (Figs. 2A-2F). Epitope mapping in C57Bl / 6 mice showed a higher and broader response to PolN than to PolC (Fig. 3A). A total of 14 peptides within PolN were recognized by CD8+ T cells, while within PolC, only 2 adjacent peptides, which appear to show one epitope the most, were recognized. CD4+ T cells did not respond to PolN or PolC. This pattern was mostly replicated in BALB / c mice, where the CD8+ T cell response recognized 12 peptides and was highest to PolN, then 4 peptides and was high to PolC (Fig. 3B). The response to the core was low but surprisingly broad, recognizing 10 peptides (Fig. 3B). BALB / c CD4+ T cells recognized 15 peptides and responded best to the core, with low recognition of PolC (4 peptides) or PolN (2 peptides). The CD8+ T cell response was also tested in HLA-A2tg mice, and PolN again triggered the highest response, involving 12 peptides (Fig. 3C). The response to PolC was low but broad (16 peptides), while only 1 peptide of the core was detected (Fig. 3C). The sequences of the peptides tested in the primary stimulation experiments are provided in Tables 3 (Core peptides), 4 (PolN peptides), and 5 (PolC peptides). The peptide compositions of the peptide pools in the primary inoculation experiments are provided in Tables 6-8. Collectively, these data indicate that the inserts that induce a detectable T cell response in many cases are directed to 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 vaccination in C57Bl / 6, BALB / c, and HLA-A2 tg mice, the increased response was mainly seen against the inserted fragment at vector doses that induced suboptimal responses at the time of the primary vaccination, i.e., 1×10 9 vp vector dose tested against Core (Figures 4A - 4C). Even when injecting the vector at high doses, booster immunization could not increase the response against PolN or PolC, but nevertheless, the booster vaccination broadened the T cell response (Figures 5A - 5C).

[0087] Summary of immunogenicity 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 a heterologous vaccine carrier. · The immune response is broad, and · After booster vaccination, the breadth of the T cell response expands, as shown.

[0088] Effect of vaccination on HBV titer at low-dose AAV-1.3HBV exposure Groups of three mice were administered 1×10 10 、1×10 11 or 1.5×10 11 vg of AAV-1.3HBV and vaccinated with AdC6-gDPolN 8 weeks later. The viral titers were examined 8 weeks after vaccination and compared with the titers before vaccination. Figure 6 shows the change in the amount of virus from the baseline for each treatment group.

[0089] Epitope change CD8+ T cells against HBV antigens were depleted during chronic HBV infection. The progression to CD8+ T cell depletion was more rapid and apparent for dominant epitopes compared to subdominant epitopes. The underlying reason is that depletion is driven by stimulation by overwhelming antigens through the T cell receptor, and dominant epitopes are present at high levels on MHC class I antigens expressed by antigen-presenting cells compared to subdominant epitopes with low binding activity to restricting molecules. Typical vaccination methods mainly induce an immune response against significant epitopes. Therapeutic vaccines should take into account the decrease in T cells against dominant epitopes during chronic viral infections and should be designed to favor the proliferation of CD8+ T cells against subdominant epitopes, which may then resist depletion driven by the disease and are likely to translate into excellent disease management.

[0090] The epitope profile of untreated mice immunized with an adenovirus vector (「AdC6-gDPolN」, the amino acid sequence of gDPolN is SEQ ID NO: 16) containing a nucleic acid sequence encoding the HBV polymerase N-terminal domain (PolN) fused to herpes simplex virus glycoprotein D was determined. The response of mice not pre-treated with the AAV8-1.3HBV vector was compared with that obtained from mice infected with an AAV8 vector expressing the 1.3HBV genome before vaccination with AdC6-gDPolN. The AAV8-1.3HBV vector may induce high titers of HBV in the serum and drive the depletion of CD8+ T cells.

[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 induced a protein response in these untreated mice was identified (e.g., CD8+CD44+ T cells producing more than 1% IFN-γ). Figures 7A and 8A. In the second experiment, mice were exposed to the AAV-1.3HBV vector at 1 × 10 10 viral genomes (vg), vaccinated 4 weeks later with an AdC6 vector expressing the same HBV polymerase sequence (gDPolN) as the unexposed mice in the first experiment, and then 10 weeks later, the peptide pool matrix on splenocytes from mice exposed prior to vaccination was used to determine the HBV PolN-specific CD8+ T cell epitope profile. Figures 7B and 8B. The experiment was repeated using stringent conditions by exposing mice to a 1.5 × 10 11 vg dose of the AAV8-1.3HBV vector. Four weeks later the mice were vaccinated again, and the CD8+ T cell response to the peptide pool matrix was tested approximately 10 weeks after vaccination. Figures 7C and 8C. In both experiments, a change in the epitope profile of mice infected with AAV8-1.3HBV was observed compared to the results obtained from unvaccinated mice, with a high viral load of 10 7 ~10 9 vg per ml of serum at the time of vaccination. This effect was more pronounced in mice exposed to the high-dose AAV8-1.3HBV vector. In both experiments, a decrease in the response was observed. Moreover, particularly in mice exposed to the high-dose AAV8-1.3HBV, a decrease in CD8+ T cells against many of the epitopes that showed immunodominance in un-infected vaccinated mice (e.g., within the region represented by peptide 50 - 59, Figure 8), subdominant epitopes (such as those within the region represented by peptide 2 - 8), and good conservation of novel epitopes such as those within the region represented by peptide 10 - 29 were shown. From these data, a change from the recognition of dominant epitopes to the recognition of subdominant epitopes was confirmed.

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

[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 were analyzed in the AAV8-HBV mouse model.

[0094] Method - 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 boostered with an AdC7 vector containing the same insert (e.g., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC). Mice were bled on days 14 and 56 after injection, and the frequency of T cells specific for various HBV inserts was analyzed by intracellular cytokine staining (ICS) for interferon (IFN)γ when the cells were stimulated with overlapping peptides representing the HBV sequences. Control cells were cultured without peptides. The frequency and phenotype of CD8+ T cells specific for one immunodominant epitope within PolN were tested by staining with MHC I tetramers. The breadth and specificity of the CD8+ T cell response to individual peptides within the target sequence were performed by epitope mapping of splenocytes (IFN-γ of CD8+ T cells was tested by ICS).

[0095] To evaluate CD8+ T cells in the liver, C57Bl / 6 mice (n = 8 per group) were injected intravenously via the tail vein with 1×10 10 viral genomes (vg) of AAV8-1.3HBV, 1×10 11 vg of AAV8-1.3HBV by intravenous administration, or not receiving intravenous administration. Four weeks later, they received a single IM injection of 5×10 9 individual viral particles (vp) of AdC6-gDPolN. Eight weeks after the IM injection, the mice were sacrificed, the livers were removed, lymphocytes were isolated, and stained with tetramers that recognize the T cell receptor for the T cell marker and the immunodominant epitope present in the PolN sequence.

[0096] In another experiment, three groups of C57Bl / 6 mice (n = 4 per group) received a single IM injection of 5×10 9 vp of AdC6-gDPolN at 4 weeks (-) or 5×10 four weeks later9 Either with or without AdC6-gDPolN of vp, 1×10 was injected via the tail vein 11 The mice received intravenous administration of the viral genome (vg) of AAV8-1.3HBV. Approximately 2 months after the administration of AAV8-1.3HBV, the mice were sacrificed, the livers were removed, and liver sections were prepared from each of the 3 groups, stained with hematoxylin and eosin, and evaluated for lymphocyte infiltration. From the same experiment, the cells were stained with fluorescently labeled antibodies against specific tetramers and T-bet (clone 4B10, BV785 staining) or antibodies against PD-1 (clone 29F.1A12, BF605 staining), TIM-3 (clone RMT3-23, Pe / Cy7 staining), CTLA-4 (clone UC10-4B9, PE staining), or LAG-3 (clone C9B7W, BV650 staining). The cells were analyzed by flow cytometry, gated on CD44+CD8 tetramer-positive cells, and gated on the markers. The percentage of marker-positive cells was identified from a comparison of histograms to untreated T cells.

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

[0098] Time-course effects of chronic HBV virus exposure on CD8+ T cell antigen recognition - Evaluated the effect of AAV8-1.3HBV on vaccine-induced liver CD8+ T cells. Untreated mice immunized with a single IM injection of 5×10 9 vp of AdC6-gDPolN had their epitope profiles of splenocytes determined 4 weeks after vaccination. 1×10 10 and 1.5×10 11 vg of AAV8-1.3HBV, and then 4 weeks later were vaccinated with 5×10 9 vp of AdC6-gDPolN. Mice vaccinated had CD8+ T cell epitope profiles in splenocytes performed 10 weeks after vaccination (14 weeks after AAV injection). The epitope profiles of AAV-untreated and AAV-treated vaccinated animals were compared. PolN-specific CD8+ T cells from the liver were analyzed against different markers.

[0099] Results Immunogenicity - Vaccination induced a robust and persistent CD8+ T cell response against PolN (median frequency across all circulating CD8+ T cells: 6.0%) and low responses against PolC and core (median frequencies: 1.0% and 0.4% respectively, Figures 9A and 9B). After a booster vaccination at 8 weeks, a significant change was observed against core (p = 0.007) and responses in all regions increased (Figure 9C). Figures 9A - 9C show CD8+ T cell changes across all CD8+ T cells for individual mice with the median indicated by the line. Vaccination induced broad epitope recognition by CD8+ T cells, which was further enhanced after booster vaccination (from 27% to 34%, Figure 10).

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

[0101] Efficacy - After a single IM injection of the AdC6-gDPolN vector, mice infected with AAV8-1.3HBV had a multiple-log reduction in serum HBV DNA that persisted for 8 weeks after vaccination (Fig. 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 10 cps / mL, respectively (Fig. 14A). At 8 weeks, all animals had a >1 log 10 cps / mL reduction from baseline, 6 / 7 (86%) had a >2 log 10 cps / mL reduction from baseline, and 2 / 7 (29%) had a >3 log 10 cps / mL reduction from baseline (Fig. 14B).

[0102] When comparing AAV-HBV-infected mice and untreated mice after a single AdC6-gDPolN vector injection, different CD8+ T cell recognition patterns against the PolN peptide were observed in splenocytes. Figs. 15A and 15B show mice first infected with AAV-1.3HBV and 10 weeks after 4 10Mice were boost-inoculated with the AdC6-gDPolN vector of vp, and splenocytes were collected 8 weeks after immunization. IFN-γ during short-term in vitro stimulation with individual peptides spanning a series of PolN was examined by ICS. Background frequencies obtained without peptides were subtracted. Figure 15A shows the peptide recognition profiles of mice that were first injected with the indicated doses of the AAV8-1.3HBV vector and then received only the AdC6-gDPolN vaccine. The pie chart in Figure 15B shows the responses corresponding to peptides that reached the threshold of 0.1% of all CD44 + CD8 + cells (data corresponding to those in Figure 15A). Each size / color indicates the proportion of the whole, represents the frequency of responses of individual peptides, and includes only responses exceeding 0.1%. The extracts indicate epitopes recognized only in mice infected with AAV8-1.3HBV. Pretreatment with AAV was found to reduce both the number of epitopes recognized after single IM stimulation and the magnitude of the immune response as the total number of IFN-γ-producing CD8+ T cells in the pool of CD8 + T cells. Pretreatment with AAV changed T cell recognition to new epitopes, which represented approximately one-third of the detectable CD8 + T cell response. The proportion of functional HBV-specific CD8+ T cell responses was highest in untreated mice (4.4%, Figure 15B), but decreased with the abundance of low-dose and high-dose AAV8-1.3HBV (2.0% and 0.6% respectively, Figure 15B). Animals not infected with AAV8-1.3HBV showed strong CD8+ T cell responses to many epitopes, but decreased or changed in animals infected with AAV-HBV, including T cell recognition of new epitopes.

[0103] Discussion Develop an HBV therapeutic vaccine that targets the early activation of CD8+ T cells using gD as a genetically encoded checkpoint inhibitor, · Induce a strong and durable CD8+ T cell response against important HBV antigens (Figure 9), · Stimulate a very broad CD8+ T cell response that includes subdominant epitope recognition (Figure 15) (Figure 10), and · Functional CD8+ T cells were preferentially transported to the liver (Figs. 11 and 12), achieving a sustained multi-log reduction in HBV DNA viral load in the AAV mouse model (Fig. 14).

[0104] In the disclosed AAV tests, AAV-induced HBV infection caused a decrease in CD8+ T cell recognition of the dominant epitopes of PolN after vaccination with AdC6-gDPolN (Fig. 15). Without intending to be bound by theory, it is thought that the breadth of CD8+ T cells induced by gD and the ability of this cell to recognize subdominant epitopes lead 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 in blood, spleen and liver of animals against AdC6-gDPolN vaccine in the presence of existing AAV-induced HBV infection + To evaluate the CD8 T cell response, the following tests were performed.

[0106] Experiment #1 CD8 T cell response in AAV8-1.3HBV-infected mice + T cell response: Response kinetics in blood Objective - To evaluate the effect of the persistence titer of HBV antigen on the CD8 T cell response to the gDPolN antigen expressed within the AdC6 vector. +

[0107] Method - C57Bl / 6 mice were injected intravenously with 10 of the AAV8-1.3HBV vector. Four weeks later, they were vaccinated with 5×10 vp of the AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector. Un-treated mice served as additional controls. Two months later, the mice were boost-vaccinated with the same dose of AdC7-gDPolN vaccine. Blood was collected at various times after the first vaccination and the boost vaccination, and IFN-γ-producing CD8 10 9 +PBMC of T cells were examined.

[0108] Results - As shown in Figure 16, mice initiated an active PolN-specific CD8 + T cell response 2 weeks after vaccination, which gradually decreased until week 8 and then increased again after booster vaccination. The CD8 + T cell response was more stable after booster vaccination than after the first vaccination. At most time points, the response of the examined mice injected with the AAV8-1.3HBV vector was lower than that of the control mice not injected with the AAV vector.

[0109] Experiment #2 CD8 + T cell response in AAV8-1.3HBV-infected mice: Response in the liver Objective - To evaluate the CD8 + T cell response containing markers indicating T cell depletion in the liver of vaccinated mice infected with AAV8-1.3HBV.

[0110] Methods - C57Bl / 6 mice were intravenously injected with 10 10 or 10 11 vg of the AAV8-1.3HBV vector. Four weeks later, they were vaccinated with 5×10 9 vp of the AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector. Un-treated mice served as an additional control. The mice were booster vaccinated with the same dose of the AdC7-gDPolN vaccine 2 months later.

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

[0112] Results - Analysis of the frequency of CD8 + T cells in lymphocytic liver infiltrates. The CD8 +The frequency of T cells was increased in vaccinated mice compared to untreated mice and further increased in mice injected with the AAV8-1.3HBV vector prior to vaccination (Figure 17A). PolN-specific CD8 T cells identified by staining with tetramers specific for epitopes present in the PolN insert + The frequency of T cells was decreased in mice injected with AAV-1.3HBV (Figure 17B).

[0113] The phenotype of infiltrating tetramer - CD44 - CD8 + T cells was evaluated by measuring the mean fluorescence intensity of dyes conjugated to certain antibodies (Figure 18A - Figure 18F) and the percentage of CD8 + CD8 + T cells that were positive for the indicated markers (Figure 19A - Figure 19F). + T-bet, which controls the number of CD8

[0114] CD8 + T cell functions, was decreased on liver CD8 + T cells from mice injected with AAV8-1.3HBV prior to vaccination compared to the vaccine-only group. The exhaustion marker did not increase in the group pretreated with AAV8-1.3HBV, and the observed decrease in PolN-specific CD8 + T cells in the presence of HBV did not appear to be due to classical CD8 + T cell exhaustion (Figure 18A - Figure 18F and Figure 19A - Figure 19F).

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

[0116] Method - Mice were given 10 10 or 10 11The AAV8-1.3HBV vector of vg was injected intravenously and boosted with the corresponding AdC7 vector 2 months later. Control mice received only the AdC6-gDPolN vector. After 10 weeks, the mice were euthanized and the pooled splenocytes were examined against a pool of peptides from non-AAV-infected animal experiments. The results are provided in FIGS. 20A to 20C.

[0117] In the second experiment, mice were given 10 10 or 10 11 vg of the AAV8-1.3HBV vector was injected intravenously. Four weeks later, these were vaccinated with 5×10 10 vp of the AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector. Untreated mice served as an additional control. Six weeks later, splenocytes were analyzed for IFN-γ-producing CD8 + T cells responding to individual peptides spanning the PolN sequence. The results are provided in FIGS. 20D to 20F.

[0118] Results - The presence of HBV, particularly high HBV titers such as after injection of 10 11 vg doses of AAV8-HBV1.3, not only reduced the total CD8 + T cell response to the PolN sequence presented by the AdC6-gDPolN vaccine, but also caused a shift in the epitope recognition profile.

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

[0120] Method - In the first experiment, C57BL / 6 mice were injected intravenously with 3×10 11 vg of AAV8-1.3HBV. One group, 8 weeks later, received 5×10 10Vaccinated with the AdC6-gDPolN vector of vp. The other groups were left unvaccinated. After 4.5 months, the mice were euthanized and the spleen cells were examined for the frequency of CD8 + T cells producing IFN-γ in response to the PolN peptide pool.

[0121] In the second experiment, mice were given escalating concentrations of AAV8-1.3HBV (1×10 10 , 4×10 10 , or 1×10 11 ). Four weeks later, all mice were vaccinated with 5×10 10 vp of the AdC6-gDPolN vector. Two months later, the mice were boostered with the same dose of the AdC7-gDPolN vector. Two months later, the mice were euthanized, lymphocytes were isolated from the liver, and examined for CD8 + T cells producing IFN-γ in response to the PolN peptide pool. The cells were also stained with an antibody against Tox, a transcription factor that increases in exhausted T cells.

[0122] Results - As shown in Figure 21, the CD8 + T cells induced by the vaccine 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 vaccination of mice with AdC6 / 7-gDPolN vaccines in AAV.8-1.3HBV- causes persistent liver damage.

[0124] Method - Mice were injected with 10 10 vg of AAV8-1.3HPV intravenously. One month later, these were given 5×10 9Mice were vaccinated with the AdC6-gDPolN vector of vp. Two months later, the mice were boostered with the same dose of the AdC7-gDPolN vector that could be given at the same dose. Two months later, the mice were euthanized. Liver sections were collected and fixed in 10% formaldehyde. Sections (with a thickness of 3 μm or less) were prepared and stained with hematoxylin and eosin (H&E). These were observed under an optical microscope at a magnification of 20 times.

[0125] Results―One of the 33 sections from mice that received both the AAV vector and the vaccine showed a small lymphocyte infiltration in the peripheral part of the liver section.

[0126] As shown in Figure 21B, after a single gDPolN vaccination of HLA-A2-tg mice, the frequency of IFN-γ-producing liver CD8 + T cells decreased in mice that received AAV compared to those vaccinated only. Conclusions ·The CD8 + T cell response to PolN decreased in mice infected with AAV8-1.3HBV. Nevertheless, these remained detectable. ·In animals pre-treated with AAV8-1.3HBV, the exhaustion markers did not increase, and the observed decrease in PolN-specific CD8 + T cells in the presence of HBV was shown not to be likely caused by classical CD8 + T cell exhaustion. ·AAV-induced HBV infection changed the epitope recognition profile of the CD8 + T cell response to PolN. ·CD8 + T cells induced by the vaccine in mice previously infected with the AAV8-1.3HBV vector remained functional. ·The vaccine used in the prime-boost regimen did not cause obvious liver damage in HBV-positive mice.

[0127] Generation of the HBV PolN-PolC-Core construct Two multiple antigen insertion fragments (second-generation PolN-PolC-Core and third-generation PolN-PolC-Core) were generated. The sequences of these insertion fragments are shown below. Second-generation HBV vaccine insertion fragment ("HBV2") (Pol N (italic)-Pol C (underlined)-Core) (SEQ ID NO: 174) YLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDV QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR Third-generation HBV vaccine insertion fragment ("HBV3") (Pol N (italic)-Pol C (underlined)-Core) (SEQ ID NO: 175) HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR

[0128] The second-generation HBV ("HBV2") insert contains immunodominant PolN epitopes identified from mice not infected with the AAV8-1.3HBV vector prior to vaccination. Many of these epitopes were found to decrease in a mouse model of chronic HBV infection brought about by prior administration of the AAV8-1.3HBV vector (defined as "epitope change" above). The third-generation HBV ("HBV3") insert was selected for the region proximal to PolN that is preferentially recognized by mice with high HBV loads (see above). For both constructs, the regions of Core and PolC were chosen using the general formula of selecting the regions with the highest immune response to either the primary vaccination (Figure 3) or booster vaccination (Figure 5) regions of C57Bl / 6, BALBc, and HLA-A2tg mice, selecting the native epitopes, and instead of inserting spacer sequences between them, selecting large proximal regions for the purpose of selecting the highest immune response between these regions.

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

[0130] Stability - To ensure the genetic integrity of the viral constructs, the genetic stability of each recombinant viral vector lot was evaluated by serial viral passage in adherent HEK293 cell cultures. The recombinant viral pools resulting from each gene transfer were cultured for a total of 12 passages under standard growth conditions. At the last passage, the viral pool was expanded and the crude harvest was purified by cesium chloride (CsCl) gradient. After vector purification, viral DNA was isolated using the QIAGEN DNeasy Blood & Tissue Kit and evaluated by restriction enzyme digestion using two restriction enzymes, Ase I and Bgl II, which cut the DNA template into defined banding patterns specific to the different constructs. After digestion, the samples were subjected to electrophoresis in a 1% agarose gel containing ethidium bromide to visualize the digested bands and the results were then described using a digital gel imaging system. A viral preparation showing the same banding pattern as that of the initial passage virus was considered to maintain the original molecular clone structure and was thus considered stable up to the end of 12 viral passages.

[0131] Results - The banding pattern of the viral vector DNA remained stable after 12 passages compared to 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 - To evaluate the CD8 + T - cell responses to HBV2 and HBV3 inserts expressed by AdC6 or AdC7 vectors.

[0133] Methods - Groups of C57Bl / 6 mice were given 5×10 9 or 5×10 10Mice were injected with the AdC6-gDHBV2 or AdC6-gDHBV3 vector at vp. Mice injected with the same dose of the AdC6-gDPolN vector served as positive controls, and untreated mice served as negative controls. After 14 days, the mice were bled, and the frequency of CD8 + T cells producing IFN-γ in response to a peptide pool corresponding to the HBV insert was examined. Four weeks later (6 weeks after vaccination), the mice were bled again and examined with the PolN-specific tetramer. Mice immunized with AdC6-gDHBV3 were excluded because this insert lacks the epitope corresponding to the tetramer.

[0134] Groups of C57Bl / 6 mice were injected with 5×10 9 or 5×10 10 vp of the AdC7-gDHBV2 or 5×10 10 vp of the AdC7-gDHBV3 vector. Untreated mice served as negative controls. After 14 days, the mice were bled, and the frequency of CD8 + T cells producing IFN-γ in response to a peptide pool corresponding to the HBV insert was examined.

[0135] Immunogenicity of AdC7 prime / AdC6 booster vaccination The mice were bled 4 weeks later, and the CD8 + T cells producing IFN-γ and / or TNF-α in response to peptides related to the insert in PBMC were re-examined by ICS. Two months after the prime vaccination, the mice were boosted with the same dose of a heterologous vector expressing the same insert. Two months later, PBMC were examined by ICS, and the CD8 + and CD4 + T cell responses before and after the booster vaccination were compared. After the prime vaccination, the AdC7-gDHBV2 vector induced a robust frequency of CD8 + T cells producing IFN-γ and / or TNF-α. The frequency increased after AdC6-gDHBV2 booster vaccination, which was after a low dose of the vector and CD8 +It was particularly evident for T cells. The immunogenicity of the AdC7-gDHBV3 vector was insufficient, but the CD8 + T cell response became positive after AdC6-gDHBV3 booster vaccination. Similarly, the CD4+ T cell response after the primary vaccination was slight but increased after the booster vaccination. There was no significant difference in the CD4 response to the HBV2 or HBV3 insert fragments. Conclusion · Both AdC6-gDHBV2 and AdC7-gDHBV2 vectors were highly immunogenic (Figures 22A, 22B, and 23), but the response increased after booster vaccination with heterologous AdC vectors expressing the same insert fragment (Figure 24). · The AdC7-gDHBV2 and AdC7-gDHBV3 vectors lacked the epitopes corresponding to the tetramers used, and thus exhibited border immunogenicity consistent with these designs (Figures 22A and 23). · Boosting with AdC6-gDHBV2 after AdC7-gDHBV2 + enhanced the CD8

[0136] Comparison of HBV DNA virus titers in mice infected with AdC6-gDPolN, AdC6-gDHBV2, AdC6-gDHBV3, or AdC6-HBV2 AAV Method Five groups of C57Bl / 6 mice were exposed to 1×10 9 vg of AAV8-1.3HBV, and 4 weeks later were vaccinated with 1×10 10 vp of either AdC6-gDPolN (n = 10), AdC6-gDHBV2 (n = 10), AdC6-gDHBV3 (n = 10), or AdC6-HBV2 without gD (n = 10), infected with AAV, and animals not vaccinated ( "untreated") (n = 10) and animals not infected with AAV and not vaccinated (n = 2 - 5) served as controls. The virus titer was examined 4 weeks after AAV injection (before vaccination) and compared with the level 4 weeks after vaccination (8 weeks after AAV injection).

[0137] Result In the eighth week, the median HBV viral titer increased by 0.98 log 10 cps / mL in the untreated mice, remained unchanged in the mice vaccinated with AdC6-HBV2, and decreased by -0.04, -1.09, and -2.13 log 10 cps / mL in the animals vaccinated with AdC6-gDHBV3, AdC6-gDPolN, and AdC6-gDHBV2, respectively (Figure 25A). The results of individual mice are shown in Figure 25B, and all animals vaccinated with AdC6-gDPolN and AdC6-gDHBV2 decreased by more than 1 log 10 copies / mL and more than 2 log 10 copies / mL, respectively. In contrast, animals vaccinated with untreated, AdC6-HBV2, or AdC6-gDHBV3 decreased by more than 1 log 10 copies / mL at the eighth week.

[0138] Immunogenicity studies of gDHBV2 and gDHBV3 CD8 + T cell responses and their breadth against segments of HBV core and polymerase contained in either gDHBV2 or gDHBV3 were evaluated after a single prime injection or prime vaccination followed by booster vaccination with heterologous vectors containing the same insert.

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

[0140] Method: Four groups of 5 C57Bl / 6 mice were as follows: (a) 5×10 10 vp AdC7-gDHBV2, and 2 months later 5×10 10 vp AdC6-gDHBV2; (b) 5×10 9 vp AdC7-gDHBV2, and 2 months later 5×109 vp AdC6-gDHBV2; (c) 5×10 10 vp AdC7-gDHBV3, 2 months later 5×10 10 vp AdC6-gDHBV3; or (d) not vaccinated, immunized by intramuscular injection. Two weeks and six weeks after the first vaccination, before the booster vaccination, and two weeks and four weeks after the booster vaccination, IFN-γ in the blood + CD8 + T cell responses were evaluated by ICS.

[0141] Results: Each vaccine construct was found to induce IFN-γ + CD8 + T cells at all time points examined. Figure 26 shows the percentages 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). The means of the immune responses evaluated by ICS from PBMC of individual mice at two weeks and eight weeks after the first vaccination, and two weeks and four weeks after the booster vaccination are shown.

[0142] Experiment 2 Objective - To compare the IFN-γ + CD8 + T cell responses after the first vaccination and booster vaccination with different doses of gD-HBV2 and gD-HBV3 using heterologous chimpanzee adenovirus vectors (AdC6 and AdC7) in C57Bl / 6 mice with those with gD-PolN.

[0143] Method: Groups of C57Bl / 6 mice (n = 5 mice / group) were as follows, gDPolN group (a) 5×10 9 vp AdC6-gDPolN, 3 months later 5×10 9 vp AdC7-gDPolN, and (b) 5×10 10 vp AdC6-gDPolN, 3 months later 5×10 10 vp AdC7-gDPolN gDHBV2 group (c) 5 × 10 9 vpAdC6-gDHBV2, 3 months later 5 × 10 9 vp AdC7-gDHBV2, and (d) 5 × 10 10 vpAdC6-gDHBV2, 3 months later 5 × 10 10 vp-AdC7-gDHBV2 gDHBV3 group (e) 5 × 10 9 vpAdC6-gDHBV3, 3 months later 5 × 10 9 vp AdC7-gDHBV3, and (f) 5×10 10 vpAdC6-gDHBV3, 3 months later 5 × 10 10 vp-AdC7-gDHBV3 No treatment served as a control. immunized as follows.

[0144] For all treatment groups, immunogenic CD8 + T cell responses were measured using IFN-γ 2 and 6 weeks after the first vaccination, before the booster vaccination, and 2 and 6 weeks 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 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. Upon tetramer staining, specific CD8 +The frequency of T cells was equivalent between the two vaccines, and many activation markers tended to be more highly expressed on the 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 graphs show the overall frequency of CD8 + T cells producing IFN-γ + as evaluated by ICS.

[0146] Figure 28 shows cytokine-producing CD4+ T cells evaluated 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 cut-off for a positive response based on results from untreated 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 primary vaccination.

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

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

[0150] Method: Four groups of 5 C57Bl / 6 mice were as follows, (a) 5 × 10 10vp AdC7-gDHBV2, 2 months later 5×10 10 vp AdC6-gDHBV2; (b) 5×10 9 vp AdC7-gDHBV2, 2 months later 5×10 9 vp AdC6-gDHBV2; (c) 5×10 10 vp AdC7-gDHBV3, 2 months later 5×10 10 vp AdC6-gDHBV3; or (3) no vaccine, were immunized by intramuscular injection. Animals were sacrificed 8 weeks after booster vaccination, and IFN-γ + CD8 + T cell responses were evaluated by ICS (the cut-off for positive responses was set at 0.1%).

[0151] Results: Irrespective of dose, the prime-boost regimen with the gDHBV2 vaccine induced responses against several epitopes within the core and polymerase. Figure 31 shows the CD8 10 T cell responses after the first vaccination with 5×10 10 vp AdC7-gDHBV2 and vaccination 2 months later with 5×10 + vp AdC6-gDHBV2. The numbers on the X-axis correspond to the SEQ ID NOs provided herein. Figure 32 shows the CD8+T cell responses after the first vaccination with 5×10 9 vp AdC7-gDHBV2 and vaccination 2 months later with 5×10 9 vp AdC6-gDHBV2. The numbers on the X-axis correspond to the SEQ ID NOs provided herein. Figure 33 shows the immunogenicity after the first vaccination with 5×10 10 vp AdC7-gDHBV3 and vaccination 2 months later with 5×10 10 vp AdC6-gDHBV3. The numbers on the X-axis correspond to the SEQ ID NOs provided herein.

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

[0153] Method: Five groups of five BALB / c mice were immunized by intramuscular injection as follows: (a) 5×10 10 vp of AdC6-gDHBV2; (b) 5×10 10 vp of AdC6-gDHBV3; (c) 5×10 10 vp of AdC7-gDHBV2; (d) 5×10 10 vp of AdC7-gDHBV3; or (e) no vaccine. At 12 weeks post-vaccination, the animals were sacrificed, spleens were collected, and IFN-γ + CD8 + T cell responses to individual HBV2 or HBV3 peptides of pooled splenocytes were evaluated by ICS (the cut-off for positive responses was set at 0.1%).

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

[0155] Experiment 5 Method: Five groups of C57Bl / 6 mice were exposed to 1×10 9 vg of AAV8-1.3HBV and, 4 weeks later, 1×10 10Vaccinate with any of AdC6-gDPolN (n = 10), AdC6-gDHBV2 (n = 10), AdC6-gDHBV3 (n = 10), or AdC6-HBV2 without gD (n = 10), infect with AAV. Animals not vaccinated (n = 10) and animals not infected with AAV and not vaccinated (n = 2 - 5) served as controls. Mice were bled at various time points after injection, 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 2 weeks, 6 weeks, and 8 weeks after the first vaccination, and the T cell assay was performed at 4 weeks after the first vaccination.

[0156] At 8 weeks after the first vaccination, mice were boost-vaccinated with an AdC7 vector containing the same antigen insert used in the first vaccination ( "boost vaccination"), and CD8+ / CD4+ T cells in blood and serum were examined as previously described at various time points after vaccination. PCR was performed at 2 weeks, 6 weeks, and 10 weeks after the boost vaccination, and the T cell assay was performed at 4 weeks and 12 weeks after the boost vaccination.

[0157] Those skilled in the art will understand 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. Accordingly, the appended claims are intended to cover all equivalent changes that fall within the true spirit and scope of the present invention.

[0158] The disclosure of each patent, patent application, and patent publication cited or described in this document is hereby incorporated by reference in its entirety into this specification.

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 description. Embodiment 1. Hepatitis B virus (HBV) core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof. Embodiment 2. The immunogenic fragment comprises any one of SEQ ID NOs: 20 to 54, and the HBV core protein according to Embodiment 1. Embodiment 3. 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 according to Embodiment 4, which comprises the nucleic acid sequence of SEQ ID NO: 7. Embodiment 6. A vector comprising the nucleic acid molecule according to Embodiment 4 or 5. Embodiment 7. The vector according to Embodiment 6, wherein the vector is an adenovirus vector. Embodiment 8. The vector according to Embodiment 7, wherein the adenovirus 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. The 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 according to Embodiment 10, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 55 to 113. Embodiment 12. The 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. The 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 according to Embodiment 13, wherein the immunogenic fragment comprises any one of SEQ ID NOs: 114 to 172. Embodiment 15. The 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 according to Embodiment 16, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 9. Embodiment 18. The nucleic acid molecule according to 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 according to any one of Embodiments 16 to 18. Embodiment 20. The vector according to Embodiment 19, wherein the vector is an adenovirus vector. Embodiment 21. The adenovirus vector according to Embodiment 20, wherein the adenovirus vector is an AdC6 vector or an AdC7 vector. Vaccine comprising the vector of any one of Embodiments 19 to 21. Embodiment 23. A fusion protein comprising one or more of the HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and the HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof. Embodiment 24. (1) The HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof and the 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) The HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof and the 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) The HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof and the 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) The HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, the HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof, and the 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 fragment of SEQ ID NO: 6), one or more of SEQ ID NOs: 55 to 113 (immunogenic fragment of SEQ ID NO: 8), and one or more of SEQ ID NOs: 114 to 172 (immunogenic fragment of SEQ ID NO: 10) The fusion protein according to Embodiment 23, comprising Embodiment 25. 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, a fusion protein. Embodiment 26. The fusion protein according to Embodiment 25, comprising the amino acid sequence of SEQ ID NO: 174. Embodiment 27. 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, a fusion protein. Embodiment 28. The fusion protein according to Embodiment 27, comprising the amino acid sequence of SEQ ID NO: 175. Embodiment 29. N-terminal simple herpesvirus (HSV) glycoprotein (gD) sequence or a variant thereof, an HBV core protein comprising the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof, and a C-terminal HSV gD sequence or a variant thereof, a fusion protein. Embodiment 30. The fusion protein according to Embodiment 29, wherein this immunogenic fragment comprises any one of SEQ ID NOs: 20 to 54. Embodiment 31. 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 C-terminal HSV gD protein sequence or a variant thereof, a fusion protein. Embodiment 32. The fusion protein according to Embodiment 31, wherein this immunogenic fragment comprises any one of SEQ ID NOs: 55 to 113. Embodiment 33. An N-terminal HSV gD sequence or a variant thereof, a HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof, and a C-terminal HSV gD protein sequence or a variant thereof A fusion protein comprising the same. Embodiment 34. This immunogenic fragment comprises any one of SEQ ID NOs: 114 to 172, and is the fusion protein described in Embodiment 33. 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) One or more of SEQ ID NOs: 20-54 (immunogenic fragment of SEQ ID NO: 6), one or more of SEQ ID NOs: 55-113 (immunogenic fragment of SEQ ID NO: 8), and one or more of SEQ ID NOs: 114-172 (immunogenic fragment of SEQ ID NO: 10), an HBV sequence, and C-terminal HSV gD protein sequence or a variant thereof A fusion protein comprising the same. Embodiment 36. N-terminal HSV gD sequence or a variant thereof, An 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. 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. 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, and A fusion protein comprising a C-terminal HSV gD protein sequence or a variant thereof. Embodiment 40. This HBV sequence is the fusion protein according to Embodiment 39, which 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. This HBV sequence is the fusion protein according to Embodiment 40, which comprises the amino acid sequence of SEQ ID NO: 174. Embodiment 42. This HBV sequence is the fusion protein according to Embodiment 39, which 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 HBV sequence is the fusion protein according to Embodiment 42, which comprises the amino acid sequence of SEQ ID NO: 175. Embodiment 44. This N-terminal HSV gD sequence is the fusion protein according to any one of Embodiments 29 to 43, which comprises the amino acid sequence of SEQ ID NO: 12. Embodiment 45. The N-terminal HSV gD sequence is the fusion protein according to any one of Embodiments 29 to 43, which comprises amino acid residues 26 to 269 of SEQ ID NO: 12. Embodiment 46. The C-terminal HSV gD sequence is the fusion protein according to any one of Embodiments 29 to 45, which comprises the transmembrane domain of HSV gD. Embodiment 47. The C-terminal HSV gD sequence is the fusion protein according to any one of Embodiments 29 to 46, which contains the amino acid sequence of SEQ ID NO: 13. Embodiment 48. This fusion protein is the fusion protein according to any one of Embodiments 29 to 47, which contains the amino acid sequence of any one of SEQ ID NO: 14 or its immunogenic fragment, SEQ ID NO: 16 or its immunogenic fragment, or SEQ ID NO: 18 or its immunogenic fragment. Embodiment 49. This fusion protein is the fusion protein according to any one of Embodiments 39 to 47, which contains the amino acid sequence of SEQ ID NO: 185. Embodiment 50. This fusion protein is the fusion protein according to any one of Embodiments 39 to 47, which contains the amino acid sequence of SEQ ID NO: 187. Embodiment 51. A nucleic acid molecule encoding the fusion protein according to any one of Embodiments 23 to 50. Embodiment 52. This nucleic acid molecule is the nucleic acid molecule according to Embodiment 51, which contains the nucleic acid sequence of any one of SEQ ID NO: 15, 17, or 19. Embodiment 53. This nucleic acid molecule is the nucleic acid molecule according to Embodiment 51, which contains the nucleic acid sequence of SEQ ID NO: 176. Embodiment 54. This nucleic acid molecule is the nucleic acid molecule according to Embodiment 51, which contains the nucleic acid sequence of SEQ ID NO: 177. Embodiment 55. This nucleic acid molecule is the nucleic acid molecule according to Embodiment 51, which contains the nucleic acid sequence of SEQ ID NO: 184. Embodiment 56. This nucleic acid molecule is the nucleic acid molecule according to Embodiment 51, which contains the nucleic acid sequence of SEQ ID NO: 186. Embodiment 57. A vector containing the nucleic acid molecule according to any one of Embodiments 51 to 56. Embodiment 58. This vector is the vector according to Embodiment 57, which is an adenovirus vector. Embodiment 59. This adenovirus vector is the vector according to Embodiment 58, which is an AdC6 vector or an AdC7 vector. Embodiment 60. A vaccine containing the vector according to 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 the fusion protein according to any one of Embodiments 23 to 50, the nucleic acid molecule according to any one of Embodiments 51 to 56, the vector according to any one of Embodiments 57 to 59, or the vaccine according to Embodiment 60, thereby inducing an immune response against HBV. Embodiment 62. The method according to Embodiment 61, wherein the vaccine comprises an AdC6 vector comprising a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18 or an immunogenic fragment thereof. Embodiment 63. The method according to Embodiment 62, further comprising, subsequent to providing to the subject a vaccine comprising an AdC6 vector, providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18 or an immunogenic fragment thereof. Embodiment 64. The method according to Embodiment 61, wherein the vaccine comprises an AdC7 vector comprising a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18 or an immunogenic fragment thereof. Embodiment 65. The method according to Embodiment 64, further comprising, subsequent to providing to the subject a vaccine comprising an AdC7 vector, providing to the subject a vaccine comprising an AdC6 vector comprising a fusion protein comprising an amino acid sequence of any one of SEQ ID NOs: 14, 16, or 18 or an immunogenic fragment thereof. Embodiment 66. The method according to 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 according to Embodiment 66, further comprising, subsequent to providing to the subject a vaccine comprising the AdC6 vector, providing to the subject a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 185. Embodiment 68. The method according to 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 according to embodiment 68, further comprising, subsequent to providing the subject with a vaccine comprising the AdC7 vector, providing a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 185. Embodiment 70. The method according to 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 according to embodiment 70, further comprising, subsequent to providing the subject with a vaccine comprising the AdC6 vector, providing a vaccine comprising an AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 187. Embodiment 72. The method according to 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 according to embodiment 72, further comprising, subsequent to providing the subject with a vaccine comprising the AdC7 vector, providing a vaccine comprising an AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 187. Embodiment 74. The method according to 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. A nucleic acid sequence encoding an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178, A nucleic acid sequence encoding an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179, and An HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, A nucleic acid molecule comprising a nucleic acid sequence encoding the same.

2. The nucleic acid molecule according to claim 1, encoding a fusion protein comprising the amino acid sequence of SEQ ID NO:

174.

3. An N-terminal HSV gD sequence, A C-terminal HSV gD sequence, Or both, The nucleic acid molecule according to claim 1 or 2, further comprising a nucleic acid sequence encoding the same.

4. A nucleic acid sequence encoding an N-terminal HSV gD sequence, An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178, An HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179, and An HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, A nucleic acid sequence encoding an HBV fusion protein comprising the same, and A nucleic acid sequence encoding a C-terminal HSV gD sequence, The nucleic acid molecule according to claim 3, comprising the same.

5. The nucleic acid molecule according to claim 4, wherein the nucleic acid sequence encodes an HBV fusion protein comprising the amino acid sequence of SEQ ID NO:

174.

6. The nucleic acid molecule according to claim 5, wherein the nucleic acid sequence encodes an N-terminal HSV gD sequence comprising the amino acid sequence of SEQ ID NO:

12.

7. The nucleic acid molecule according to claim 5, wherein the nucleic acid sequence encodes an N-terminal HSV gD sequence comprising amino acid residues 26 to 269 of SEQ ID NO:

12. Claim 8 The nucleic acid sequence encodes a C-terminal HSV gD sequence containing the transmembrane domain of HSV gD, and the nucleic acid molecule according to any one of claims 4 to 7. Claim 9 The nucleic acid sequence encodes a C-terminal HSV gD sequence containing the amino acid sequence of SEQ ID NO: 13, and the nucleic acid molecule according to claim 8. Claim 10 A nucleic acid sequence encoding an N-terminal HSV gD sequence containing amino acid residues 26 to 269 of SEQ ID NO: 12 An HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO: 178, An HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO: 179, and An HBV core protein containing the amino acid sequence of SEQ ID NO: 180, A nucleic acid sequence encoding an HBV fusion protein containing A nucleic acid sequence encoding a C-terminal HSV gD sequence containing the amino acid sequence of SEQ ID NO: 13, and the nucleic acid molecule according to any one of claims 4 to 9. Claim 11 The nucleic acid sequence encodes an N-terminal HSV gD sequence containing the amino acid sequence of SEQ ID NO: 12, and the nucleic acid molecule according to claim 10. Claim 12 The nucleic acid sequence encodes an HBV fusion protein containing the amino acid sequence of SEQ ID NO: 174, and the nucleic acid molecule according to claim 10. Claim 13 The nucleic acid sequence encodes a fusion protein containing the amino acid sequence of SEQ ID NO: 185, and the nucleic acid molecule according to any one of claims 10 to 12. Claim 14 The nucleic acid molecule according to claim 12, containing the nucleic acid sequence of SEQ ID NO:

176. Claim 15 The nucleic acid molecule according to claim 13, containing the nucleic acid sequence of SEQ ID NO:

184. Claim 16 An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 180 A fusion protein comprising the same. **Claim 17** The fusion protein according to claim 16, comprising the amino acid sequence of SEQ ID NO:

174. **Claim 18** The fusion protein according to claim 16 or 17, further comprising an N-terminal HSV gD sequence, a C-terminal HSV gD protein sequence, or both. **Claim 19** An N-terminal HSV gD sequence, an HBV sequence comprising an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO: 178, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO: 179, and an HBV core protein comprising the amino acid sequence of SEQ ID NO: 180, and a C-terminal HSV gD protein sequence, The fusion protein according to claim 18. **Claim 20** The fusion protein according to claim 19, wherein the HBV sequence comprises the amino acid sequence of SEQ ID NO:

174. **Claim 21** The fusion protein according to any one of claims 18 to 20, wherein the N-terminal HSV gD sequence comprises the amino acid sequence of SEQ ID NO:

12. **Claim 22** The fusion protein according to any one of claims 18 to 20, wherein the N-terminal HSV gD sequence comprises amino acid residues 26 to 269 of SEQ ID NO:

12. **Claim 23** The fusion protein according to any one of claims 18 to 22, wherein the C-terminal HSV gD sequence comprises the transmembrane domain of HSV gD. **Claim 24** The fusion protein according to any one of claims 18 to 23, wherein the C-terminal HSV gD sequence contains the amino acid sequence of SEQ ID NO:

13.

25. The fusion protein according to any one of claims 18 to 24, wherein the fusion protein contains the amino acid sequence of SEQ ID NO:

185.

26. A virus containing the nucleic acid molecule according to any one of claims 1 to 15.

27. The virus according to claim 26, wherein the virus is an adenovirus.

28. The virus according to claim 27, wherein the adenovirus is AdC6 or AdC7.

29. A vaccine containing the virus according to any one of claims 26 to 28.

30. A pharmaceutical composition for inducing an immune response against HBV in a subject, containing the nucleic acid molecule according to any one of claims 1 to 15, the fusion protein according to any one of claims 16 to 25, the virus according to any one of claims 26 to 28, or the vaccine according to claim 29.

31. The pharmaceutical composition according to claim 30, wherein the vaccine contains an AdC6 vector having a nucleic acid molecule encoding a fusion protein containing the amino acid sequence of SEQ ID NO:

185.

32. The pharmaceutical composition according to claim 31, characterized in that after a pharmaceutical composition containing a vaccine containing the AdC6 vector is provided to the subject, a pharmaceutical composition containing a vaccine containing an AdC7 vector having a nucleic acid molecule encoding a fusion protein containing the amino acid sequence of SEQ ID NO: 185 is further provided.

33. The pharmaceutical composition according to claim 30, wherein the vaccine contains an AdC7 vector having a nucleic acid molecule encoding a fusion protein containing the amino acid sequence of SEQ ID NO:

185.

34. After the test subject is provided with a pharmaceutical composition containing a vaccine comprising the AdC7 vector, a pharmaceutical composition further containing a vaccine comprising an AdC6 vector having a nucleic acid molecule encoding a fusion protein comprising the amino acid sequence of SEQ ID NO: 185 is provided. The pharmaceutical composition according to claim 33, characterized in that.

35. The pharmaceutical composition according to any one of claims 30 to 34, wherein the amino acid sequence of SEQ ID NO: 185 does not include the 25 amino acid signal peptide at the N-terminus.

36. A nucleic acid molecule comprising a nucleic acid sequence encoding the HBV polymerase N-terminal domain, comprising the amino acid sequence of SEQ ID NO:

178.

37. The nucleic acid molecule according to claim 36, comprising the nucleic acid sequence of nucleotides 1 to 510 of the nucleic acid of SEQ ID NO:

176.

38. The HBV polymerase N-terminal domain, comprising the amino acid sequence of SEQ ID NO:

178.

39. A nucleic acid molecule comprising a nucleic acid sequence encoding the HBV polymerase C-terminal domain, comprising the amino acid sequence of SEQ ID NO:

179.

40. The nucleic acid molecule according to claim 39, comprising the nucleic acid sequence of nucleotides 511 to 885 of the nucleic acid of SEQ ID NO:

176.

41. The HBV polymerase C-terminal domain, comprising the amino acid sequence of SEQ ID NO:

179.

42. A nucleic acid molecule comprising a nucleotide sequence encoding the HBV core protein, comprising the amino acid sequence of SEQ ID NO:

180.

43. The nucleic acid molecule according to claim 42, comprising the nucleic acid sequence of nucleotides 886 to 1140 of the nucleic acid of SEQ ID NO:

176.

44. The HBV core protein, comprising the amino acid sequence of SEQ ID NO:

180.

45. A vector comprising the nucleic acid molecule according to any one of claims 36, 37, 39, 40, 42 or 43.

46. The vector according to claim 45, wherein the vector is an adenovirus vector.

47. The vector according to claim 46, wherein the adenovirus vector is an AdC6 vector or an AdC7 vector.

48. A vaccine comprising the vector according to any one of claims 45 to 47.

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