Nucleic acid constructs that utilize SNARE to induce cellular immunity against viruses

Abstract

To provide nucleic acid constructs that enhance the antigen-specific immune response to hepatitis B virus. [Solution] A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of STX7, GOSR1, and SEC22B, and a polynucleotide encoding the core protein antigen of hepatitis B virus.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid construct comprising a polynucleotide encoding a SNARE protein and a polynucleotide encoding an antigen derived from hepatitis B virus, and to the use thereof. [Background technology]

[0002] Hepatitis B virus (HBV) is an enveloped virus belonging to the Hepadnaviridae family. It is a DNA virus that forms particles with a diameter of approximately 42 nm. Chronic hepatitis B (CHB) is a viral disease caused by chronic infection with HBV. It is estimated that more than 300 million people worldwide are infected, and the annual death toll from hepatitis B is estimated to be as high as 82,000. After infection, it causes acute hepatitis, which then becomes chronic, and even after reaching a state of clinical remission, there is a certain probability of developing highly fatal liver diseases such as liver cancer and cirrhosis.

[0003] After HBV infection, covalently closed circular DNA (cccDNA) of HBV remains in the nuclei of liver cells. This viral DNA is difficult to remove, and transcription of the major proteins HBs antigen, HBc antigen, HBe antigen, and polymerase takes place from it. The HBs antigen is a surface antigen present in the envelope and is involved not only in the secretion of viral particles but also in the formation of particles on its own. The core proteins, HBc antigen and HBe antigen, are used as indicators of viral replication.

[0004] Current treatment for chronic hepatitis B primarily involves suppressing viral replication and reducing inflammation using interferon therapy and nucleoside analog drugs. While these treatments aim for clinical remission, that is, a decrease in the amount of virus in the body and negative antigen protein levels, they cannot completely eliminate the viral genome, and there is a risk of symptom relapse. Furthermore, these drugs require regular administration and are limited to patients with varying levels of symptoms. Therefore, there is a need to develop therapeutic methods that eliminate the infected cells themselves that contain viral DNA.

[0005] Vaccines are a means of preventing infection and treating diseases by administering antigens and establishing acquired immunity against them. Traditionally, vaccines have been administered mainly by subcutaneous or intramuscular injection, including live vaccines that use weakened bacteria or viruses as vaccines, inactivated vaccines in which bacteria or viruses are treated with formalin or heat to eliminate their infectivity, and toxoids, which are toxins produced by bacteria that have been isolated, purified, and inactivated with formalin. In addition to these, development is underway to improve efficacy, safety, and convenience by developing mucosal vaccines, which are administered to mucous membranes, and nucleic acid vaccines, which use nucleic acids as antigenic components. Among these, the development of nucleic acid vaccines is progressing rapidly as a new modality that can be manufactured quickly and at low cost.

[0006] Improving the immunogenicity of antigens is one of the important challenges in enhancing the effectiveness of nucleic acid vaccines, and it is necessary to guide antigens to appropriate tissues or antigen-presenting cells and efficiently induce an immune response. Patent Document 1 discloses, for example, that antigen-specific cellular immune responses were enhanced by combining allergens with specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins.

[0007] SNARE proteins are a family of proteins possessing a 20-30 kDa SNARE motif. Many SNARE proteins are anchored to lipid bilayers via a C-terminal transmembrane domain and are involved in the process of vesicle fusion with target intracellular organelles (Non-Patent Literature 1 and 2). Membrane fusion involving SNARE proteins is essential for many important life phenomena indispensable to cellular function in eukaryotic cells, including endocytosis processes such as vesicle transport, organelle membrane morphogenesis, and extracellular receptor recycling, as well as exocytosis processes such as hormone secretion and synaptic neurotransmitter release. Furthermore, the molecular mechanism of membrane fusion by SNARE proteins is thought to be conserved across all eukaryotes, from single-celled budding yeast to higher animals including humans. Each SNARE protein is thought to be localized to a specific intracellular membrane fraction (endoplasmic reticulum (ER), Golgi apparatus, endosomes, organelles such as vacuoles and lysosomes, secretory vesicles, cytoplasmic membrane, etc.) and function in the process of membrane fusion in specific intracellular transport pathways. Furthermore, SNARE proteins are known to potentially be found in exosomes. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2023-24408 [Non-patent literature]

[0009] [Non-Patent Document 1] Jahn R, Scheller RH. Nat Rev Mol Cell Biol. 2006; 7(9): 631-643. [Non-Patent Document 2] Hong W. Biochim Biophys Acta. 2005; 1744(2): 120-144. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention relates to providing nucleic acid constructs that enhance antigen-specific immune responses to hepatitis B virus and their applications. [Means for solving the problem]

[0011] The inventors have found that by using a nucleic acid construct containing a polynucleotide encoding the SNARE protein STX7, GOSR1, or SEC22B and a polynucleotide encoding the core protein antigen of the hepatitis B virus, antigen-specific cellular immune responses can be efficiently induced or enhanced compared to nucleic acid constructs containing only a polynucleotide encoding the core protein antigen of the hepatitis B virus or nucleic acid constructs containing a polynucleotide encoding a SNARE protein other than the specific SNARE proteins mentioned above and a polynucleotide encoding the core protein antigen of the hepatitis B virus.

[0012] Therefore, the present invention provides the following 1) to 4). 1) A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of STX7, GOSR1, and SEC22B, and a polynucleotide encoding the core protein antigen of the hepatitis B virus. 2) An agent for inducing or enhancing the core protein antigen-specific cellular immune response to hepatitis B virus, comprising the nucleic acid construct described in 1) as an active ingredient. 3) A prophylactic or therapeutic agent for hepatitis B containing the nucleic acid construct described in 1) as an active ingredient. 4) A nucleic acid vaccine containing the nucleic acid construct described in 1) as the active ingredient. [Effects of the Invention]

[0013] The nucleic acid construct of the present invention can efficiently induce or enhance an antigen-specific cellular immune response against hepatitis B virus. Such a nucleic acid construct is useful as a nucleic acid vaccine. [Brief explanation of the drawing]

[0014] [Figure 1] Evaluation of immunogenicity of mRNA encoding SNARE protein-HBc antigen fusion polypeptide. (A) IFNγ production is shown by the number of spot-forming cells (SFC). NT represents the control without antigen restimulation, and HBc represents the result with HBc antigen restimulation. (B) Average number of IFNγ-producing cells. (c) IL4 production is shown by the number of spot-forming cells (SFC). NT represents the control without antigen restimulation, and HBc represents the result with HBc antigen restimulation. (D) Average number of IL4-producing cells. Antigens, VAMP7, VAMP8, GOSR1, STX7, STX10, and SEC22B represent mRNA encoding HBc antigen alone, VAMP7-pc-HBc, VAMP8-pc-HBc, GOSR1-pc-HBc, STX7-pc-HBc, STX10-pc-HBc, and SEC22B-pc-HBc, respectively. [Figure 2] Immune response to administration of mRNA encoding a SNARE protein-HBc antigen fusion polypeptide to mice. (A) Percentage of CD8-positive cells possessing HBc antigen-specific T cell receptors (TCRs). (B) Average number of CD8-positive cells. Antigen, VAMP7, VAMP8, GOSR1, STX7, STX10, and SEC22B represent mRNA encoding HBc antigen alone, VAMP7-pc-HBc, VAMP8-pc-HBc, GOSR1-pc-HBc, STX7-pc-HBc, STX10-pc-HBc, and SEC22B-pc-HBc, respectively. [Figure 3] Antibody titers when mice were administered mRNA encoding a SNARE protein-HBc antigen fusion polypeptide. (A) Represents the IgG antibody titer of each group. (B) Average value of the IgG antibody titer. Antigen, VAMP7, VAMP8, GOSR1, STX7, STX10, and SEC22B represent mRNA encoding HBc antigen alone, VAMP7-pc-HBc, VAMP8-pc-HBc, GOSR1-pc-HBc, STX7-pc-HBc, STX10-pc-HBc, and SEC22B-pc-HBc, respectively.

Mode for Carrying Out the Invention

[0015] In this specification, the terms "nucleic acid", "nucleotide", "oligonucleotide" or "polynucleotide" are used interchangeably and mean DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and RNA includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA. Among them, mRNA is synthesized by in vitro transcription reaction, and then 5' Cap (methylated guanosine) is added by a Cap enzyme and poly A is added by Poly(A) Polymerase for reasons such as initiation of translation reaction in vivo, stabilization of mRNA, and suppression of degradation. The poly A sequence may be incorporated into the template DNA used in the in vitro transcription reaction. At this time, it also includes mRNA with a Cap structure and poly A added, and RNA in which some bases are modified (for example, uridine is replaced with pseudouridine or 1-methylpseudouridine).

[0016] [[ID=...]] In this specification, the "gene" includes double-stranded DNA containing genomic DNA, single-stranded DNA (sense strand) containing cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the sense strand, and fragments thereof, and means those in which some biological information is contained in the sequence information of the bases constituting the DNA. In addition, the "gene" includes not only the "gene" represented by a specific nucleotide sequence, but also nucleic acids encoding homologs (that is, homologs or orthologs) thereof, variants such as gene polymorphisms, and derivatives. The names and Gene IDs of the genes disclosed in this specification follow the Official Symbols and Gene IDs described in NCBI ([www.ncbi.nlm.nih.gov / ]).

[0017] In this specification, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably.

[0018] In this specification, "amino acid residue" means the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0019] In this specification, the identity of nucleotide or amino acid sequences is calculated using the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the homology analysis (Search homology) using the Genetyx-Win genetic information processing software with a Unit size to compare (ktup) of 2.

[0020] In this specification, "at least 80% identity" with respect to a nucleotide sequence or amino acid sequence means identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more.

[0021] In this specification, unless otherwise defined, “one or several” as used with respect to the deletion, substitution, addition or insertion of nucleotides in a nucleotide sequence may preferably mean 1 to 15, more preferably 1 to 9, and even more preferably 1 to 6. Similarly, in this specification, unless otherwise defined, “one or several” as used with respect to the deletion, substitution, addition or insertion of amino acid residues in an amino acid sequence may preferably mean 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2. In this specification, “addition” of a nucleotide or amino acid residue includes the addition of a nucleotide or amino acid residue to one end and both ends of a sequence.

[0022] In this specification, "stringent conditions" refer to the conditions for the Southern hybridization method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001), for example, conditions in which a solution containing 6×SSC (composition of 1×SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhart, and 100 mg / mL herring sperm DNA is incubated with the probe at 42°C for 8 to 16 hours to hybridize.

[0023] In this specification, "fragment" of a polynucleotide means a partial polynucleotide of the polynucleotide. The length of a partial polynucleotide is not particularly limited, as long as it encodes a polypeptide having the same function as the polypeptide encoded by the full-length polynucleotide. For example, a partial polynucleotide may mean a polynucleotide consisting of consecutive nucleotides that are preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, and preferably 90% or less, more preferably 80% or less, even more preferably 70% or less, and even more preferably 60% or less in length relative to the full-length polynucleotide. Also in this specification, "fragment" of a polypeptide means a partial polypeptide of the polypeptide. The length of a partial polypeptide is not particularly limited. For example, a partial polypeptide may mean a polypeptide consisting of consecutive amino acid residues that are preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, and preferably 90% or less, more preferably 80% or less, even more preferably 70% or less, and even more preferably 60% or less in length relative to the full-length polypeptide.

[0024] In this specification, a “regulatory region” is a region that has the function of controlling the expression of a gene located downstream of it (for example, a protein-coding region). More specifically, a “regulatory region” can be defined as a region located upstream of the coding region of a gene that interacts with RNA polymerase to control the transcription of the coding region. A regulatory region includes a transcription initiation regulatory region and / or a translation initiation regulatory region, or the region from the transcription initiation regulatory region to the translation initiation regulatory region. The transcription initiation regulatory region is the region containing the promoter and the transcription start site, and the translation initiation regulatory region is the region corresponding to the Kozak sequence necessary for translation initiation, which is recognized by the ribosome along with the start codon.

[0025] In this specification, "operable linkage" between a regulatory region and a polynucleotide of a gene (e.g., a polynucleotide encoding a protein) means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0026] In this specification, "expressible linkage" between the polynucleotide of a first gene (e.g., a protein-coding polynucleotide) and the polynucleotide of a second gene (e.g., a protein-coding polynucleotide) means that the first gene and the second gene are linked such that, when inserted into a suitable expression vector and introduced into a suitable cell, the protein encoded by the first gene and the protein encoded by the second gene are produced as a fusion protein. Here, "linkage" includes cases where the first gene and the second gene are directly linked, as well as cases where they are linked via other nucleotide sequences. The procedure for "expressible linkage" between the first gene and the second gene is well known to those skilled in the art.

[0027] In this specification, "upstream" and "downstream" with respect to a gene or its nucleotide sequence refer to the upstream and downstream directions of the gene's transcription. For example, the "upstream sequence" and "downstream sequence" of a gene refer to the sequences located on the 5' and 3' ends of the gene in the DNA sense strand, respectively.

[0028] In this specification, "antigen" means a molecule that triggers an immune response in a living organism, such as antibody production or cellular immunity. In this specification, "immunogenicity" means the property of an antigen to induce antibody production or cellular immunity.

[0029] In this specification, "nucleic acid vaccine" refers to a vaccine that induces immunity by administering a polynucleotide (DNA or RNA) encoding an antigen to a living organism. Nucleic acid vaccines include DNA vaccines, mRNA vaccines, and viral vector vaccines, all of which are thought to induce both humoral and cellular immunity. DNA vaccines contain plasmids encoding antigens. Plasmids administered to living organisms are taken up by cells, transcribed into mRNA in the nucleus, and then translated into antigen proteins in the cytoplasm, inducing an antigen-specific immune response. mRNA vaccines contain mRNA encoding antigens. mRNA administered to living organisms is taken up by cells, translated into antigen proteins in the cytoplasm, inducing an antigen-specific immune response. Viral vector vaccines contain non-pathogenic or attenuated viral vectors incorporating polynucleotides encoding antigens. Viruses administered to living organisms invade cells, causing cells to synthesize antigen proteins, inducing an antigen-specific immune response.

[0030] In this specification, "cellular immunity" refers to the acquired immune response that works to eliminate foreign substances such as pathogens, virus-infected cells, and cancer cells that have entered the body, and which uses cytotoxic T cells, macrophages, NK cells, etc. as effectors. In this specification, "humoral immunity" refers to the acquired immune response that uses antibodies as effectors.

[0031] In this specification, "Th1-type immune response" refers to the immune response promoted by Th1 cells, a subset of helper T cells. Th1 cells primarily produce IFNγ as a cytokine and target M1 macrophages, cytotoxic T cells, and NK cells, mainly inducing cellular immunity. Th1 cells are known to activate B cells and induce IgG2 production. In this specification, "Th2-type immune response" refers to the immune response promoted by Th2 cells, a subset of helper T cells. Th2 cells primarily produce IL-4, IL-5, and IL-13 as cytokines and target mast cells, M2 macrophages, eosinophils, and basophils, mainly inducing humoral immunity. Th2 cells are known to activate B cells and induce IgG1 production.

[0032] The present invention provides a nucleic acid construct comprising a polynucleotide encoding a SNARE protein selected from the group consisting of STX7, GOSR1, and SEC22B, and a polynucleotide encoding the core protein antigen of hepatitis B virus (HBV).

[0033] In the present invention, the SNARE protein is a protein belonging to the protein family having a SNARE motif, and specifically, it is one of the proteins selected from the group consisting of STX7, GOSR1, and SEC22B (hereinafter sometimes simply referred to as the SNARE protein). From the viewpoint of immune induction or enhancement, the SNARE protein is preferably one of the proteins selected from the group consisting of STX7 and GOSR1, and more preferably STX7. The SNARE protein has a transmembrane domain and is localized in intracellular vesicles.

[0034] STX7 (syntaxin 7) is a type of SNARE protein that has a transmembrane domain and is expected to localize to early and late endosomes. In a preferred example, STX7 is mammalian STX7. In a more preferred example, STX7 is human STX7, which is a protein consisting of the amino acid sequence of SEQ ID NO: 10, encoded by a gene (Gene ID: 8417) consisting of the nucleotide sequence of SEQ ID NO: 4. The STX7 used in this invention includes STX7 and polypeptides having equivalent function.

[0035] GOSR1 (golgi SNAP receptor complex member 1) is a type of SNARE protein that has a transmembrane domain and is expected to be localized to the Golgi apparatus and the trans-Golgi network. In a preferred example, GOSR1 is mammalian GOSR1. In a more preferred example, GOSR1 is human GOSR1, which is a protein consisting of the amino acid sequence of SEQ ID NO: 9, encoded by a gene (Gene ID: 9527) consisting of the nucleotide sequence of SEQ ID NO: 3. The GOSR1 used in this invention includes GOSR1 and polypeptides having equivalent function.

[0036] SEC22B (SEC22 homolog B, vesicle trafficking protein) is a type of SNARE protein that has a transmembrane domain and is expected to localize to the endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate, the Golgi apparatus, the cis-Golgi network, and the trans-Golgi network. In a preferred example, SEC22B is mammalian SEC22B. In a more preferred example, SEC22B is human SEC22B, a protein consisting of the amino acid sequence of SEQ ID NO: 12, encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 6 (Gene ID: 9554). SEC22B as used in this invention includes SEC22B and polypeptides having equivalent function.

[0037] In the present invention, a polypeptide having equivalent function to a SNARE protein refers to a polypeptide having equivalent biological activity to the SNARE protein. Examples of such polypeptides include polypeptides having a transmembrane domain and capable of localizing to intracellular vesicles (preferably intracellular vesicles where the corresponding SNARE protein is localized) or exosomes. Specific examples include the polypeptides (b) to (e) below, or the polypeptides (b), (c), and (e) below.

[0038] Specifically, the following polypeptides are examples of STX7: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 10; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 10, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 10, and which has a transmembrane domain and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes. Splicing variants of the STX7 gene consisting of the nucleotide sequence of Sequence ID No. 4 include variants that are registered in NCBI's RefSeq as NM_001326578.2, NM_001326579.2, or NM_001326580.2, and that encode proteins registered as NP_001313507.1, NP_001313508.1, or NP_001313509.1, respectively.

[0039] GOSR1 specifically includes the following polypeptides: (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 9; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 9, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 9, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (d) A polypeptide comprising an amino acid sequence encoded by a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (d) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or the trans-Golgi network) or exosomes. Splicing variants of the GOSR1 gene consisting of the nucleotide sequence of Sequence ID No. 3 include variants that encode proteins registered in NCBI's RefSeq as NM_001007025.2 or NM_001007024.2, and registered as NP_001007026.1 or NP_001007025.1, respectively.

[0040] Specifically, the following polypeptides are examples of SEC22B. (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 12; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 12, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 12, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (e) A polypeptide that is a fragment of any of the polypeptides described in (a) to (c) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes.

[0041] The polynucleotides encoding SNARE proteins used in this invention include polynucleotides encoding SNARE proteins and polynucleotides encoding polypeptides having equivalent functionality to SNARE proteins. Specific examples of polynucleotides encoding polypeptides having equivalent functionality to SNARE proteins include the following (g) to (k) and (m) to (o), or the following (g), (h), (j), (k), and (m) to (o).

[0042] The following polynucleotides are examples of polynucleotides that encode STX7. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 4; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 4, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 4, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (i) A polynucleotide encoding a polypeptide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 4, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 10, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 10, and capable of localizing to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (l) to (n) above, has a transmembrane domain, and can localize to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes. Among these, any of the polynucleotides listed above (f) to (i), below (k'), and above (l) to (o) are preferred, and any of the polynucleotides listed above (f) to (i) and below (k') are more preferred. (k') A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (i) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., early endosomes or late endosomes) or exosomes.

[0043] The following polynucleotides are examples of polynucleotides that encode GOSR1. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 3; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 3, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (h) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 3, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (i) A polynucleotide that is a splicing variant of the polynucleotide sequence of SEQ ID NO: 3, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, has a transmembrane domain, and can localize to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (j) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 9; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 9, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 9, and which can localize to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any polypeptide described in (l) to (n) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes. Among these, any of the polynucleotides listed above (f) to (i), below (k'), and above (l) to (o) are preferred, and any of the polynucleotides listed above (f) to (i) and below (k') are more preferred. (k') A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (i) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes.

[0044] The following polynucleotides are examples of polynucleotides that encode SEC22B. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 6; (g) A polynucleotide comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 6, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (h) A polynucleotide comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted from the nucleotide sequence of SEQ ID NO: 6, having a transmembrane domain, and encoding a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (j) A polynucleotide encoding a polypeptide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, has a transmembrane domain, and can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (k) A polynucleotide that is a fragment of any of (f) to (h) and (j) above, has a transmembrane domain, and encodes a polypeptide that can localize to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12; (m) A polynucleotide encoding a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of Sequence ID No. 12, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (n) A polynucleotide encoding a polypeptide having a transmembrane domain, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 12, and capable of localizing to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network, or trans-Golgi network) or exosomes; (o) A polynucleotide encoding a polypeptide that is a fragment of any polypeptide described in (l) to (n) above, and has a transmembrane domain, and can be localized to intracellular vesicles (e.g., endoplasmic reticulum, endoplasmic reticulum-Golgi intermediate section, Golgi apparatus, cis-Golgi network or trans-Golgi network) or exosomes. Among these, any of the polynucleotides listed above (f) to (i), below (k'), and above (l) to (o) are preferred, and any of the polynucleotides listed above (f) to (i) and below (k') are more preferred. (k') A polynucleotide that is a fragment of any of the polynucleotides described in (f) to (i) above, and has a transmembrane domain, and encodes a polypeptide that can be localized to intracellular vesicles (e.g., the Golgi apparatus or trans-Golgi network) or exosomes.

[0045] The method for obtaining the polynucleotide encoding the SNARE protein of the present invention is not particularly limited and can be obtained by conventional chemical synthesis methods or genetic engineering techniques. For example, the polynucleotide encoding the SNARE protein can be artificially synthesized based on any of the nucleotide sequences of SEQ ID NOs: 3, 4, and 6. For artificial synthesis, commercially available DNA synthesis services provided by companies such as GenScript can be used. Alternatively, for example, the nucleotide sequences of SEQ ID NOs: 3, 4, and 6 can be cloned from a human-derived sample according to the method described in Molecular Cloning - A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001).

[0046] The polynucleotide encoding the SNARE protein of the present invention can also be produced by introducing a mutation into DNA consisting of, for example, any of the nucleotide sequences of SEQ ID NOs: 3, 4, and 6. Methods for introducing the mutation include, for example, ultraviolet irradiation and site-directed mutagenesis. Site-directed mutagenesis methods include methods utilizing Splicing overlap extension (SOE) PCR (Horton et al., Gene, 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and the Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, the Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio), Transformer TMCommercially available site-directed mutagenesis kits, such as the Site-Directed Mutagenesis Kit (Clonetech) and the KOD-Plus-Mutagenesis Kit (Toyobo), can also be used. By selecting from the mutated DNA that possesses a transmembrane domain and can localize to intracellular vesicles, polynucleotides encoding the SNARE protein of the present invention can be obtained. Whether or not the polypeptide encoded by the mutated DNA has a transmembrane domain can be determined, for example, by using prediction tools such as SOSUI (Hirokawa et al., Bioinformatics, 14(4):378-379, 1998) or TMHMM (Krogh et al., J Mol Biol. 305(3):567-580, 2001) based on the amino acid sequence of the polypeptide encoded by the DNA.

[0047] Alternatively, methods for deleting, substituting, adding, or inserting nucleotides into a nucleotide sequence are described, for example, by Dieffenbach et al. (Cold Spring Harbor Laboratory Press, New York, 581-621, 1995).

[0048] Alternatively, the polynucleotide encoding the SNARE protein of the present invention can also be obtained, for example, by subjecting DNA consisting of any of the nucleotide sequences of SEQ ID NOs: 3, 4, and 6 to genome editing using artificial DNA nucleases (or programmable nucleases).

[0049] The hepatitis B virus core protein antigen used in the present invention is not particularly limited, as long as it is an antigen derived from the hepatitis B virus core protein. In a preferred example, the hepatitis B virus core protein antigen is the hepatitis B virus core antigen (HBc antigen), which is the core protein of the hepatitis B virus. The HBc antigen used in the present invention includes a protein consisting of the amino acid sequence of SEQ ID NO: 13, encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 31, and a polypeptide having equivalent immunogenicity.

[0050] In the present invention, polypeptides having immunogenicity equivalent to that of HBc antigen include, for example, polypeptides that contain part or all of the core protein of hepatitis B virus and have immunogenicity capable of eliciting an immune response to hepatitis B virus. Whether or not a polypeptide is immunogenic can be confirmed by means known in the art, for example, by administering the polypeptide to mice and observing the immune response, or by in vitro T cell assay using cultured cells or human peripheral blood mononuclear cells (PBMCs). Specific examples of polypeptides having immunogenicity equivalent to that of HBc antigen include the polypeptides (q) to (s) below.

[0051] The following polypeptides are examples of HBc antigens, which are core protein antigens of the hepatitis B virus. (p) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (q) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 13, and which is immunogenic; (r) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 13, and which is immunogenic; (s) A polypeptide fragment of any of the polypeptides (p) to (r) above, and which is immunogenic (immunogenic fragment). Among these, either polypeptide (p) above or (s') below is preferred. (s') A polypeptide fragment of the polypeptide described in (p) above, and which is immunogenic (immunogenic fragment).

[0052] In a preferred example, the core protein antigen of the hepatitis B virus is the HBc antigen. The polynucleotides encoding the HBc antigen used in the present invention include polynucleotides encoding the HBc antigen and polynucleotides encoding polypeptides having immunogenicity equivalent to that of the HBc antigen. Specific examples of polynucleotides encoding polypeptides having immunogenicity equivalent to that of the HBc antigen include the following polynucleotides (u) to (x) and (z) to (ab).

[0053] The following polynucleotides are examples of polynucleotides that encode the HBc antigen. (t) A polynucleotide consisting of the nucleotide sequence of sequence number 31; (u) A polynucleotide encoding an immunogenic polypeptide, comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of Sequence ID No. 31; (v) A polynucleotide encoding an immunogenic polypeptide, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted into the nucleotide sequence of SEQ ID NO: 31; (w) A polynucleotide encoding an immunogenic polypeptide that hybridizes under stringent conditions to the complementary chain of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 31; (x) A polynucleotide that is a fragment of any of the polynucleotides (t) to (w) above and encodes an immunogenic polypeptide (immunogenic fragment); (y) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13; (z) A polynucleotide encoding an immunogenic polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 13; (aa) A polynucleotide encoding an immunogenic polypeptide, which consists of an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from the amino acid sequence of SEQ ID NO: 13; (ab) A polynucleotide encoding a polypeptide that is a fragment of any of the polypeptides (y) to (aa) above and is immunogenic (immunogenic fragment). Among these, any of the polynucleotides (t) to (v) above, (x') below, and (y) to (ab) above are preferred, and any of the polynucleotides (t) to (v) above and (x') below are more preferred. (x') A polynucleotide that is a fragment of the polynucleotides described in (t) to (v) above, and that encodes an immunogenic polypeptide (immunogenic fragment).

[0054] The method for obtaining the polynucleotide encoding the hepatitis B virus core protein antigen of the present invention is not particularly limited and can be obtained by conventional chemical synthesis methods or genetic engineering methods, similar to the method for obtaining the polynucleotide encoding the SNARE protein described above. The polynucleotide encoding the hepatitis B virus core protein antigen may be a polynucleotide obtained by linking multiple polynucleotides encoding one hepatitis B virus core protein antigen in a manner that allows for expression, or a polynucleotide obtained by linking two or more polynucleotides encoding hepatitis B virus core protein antigens in a manner that allows for expression. The hepatitis B virus core protein antigen used in the nucleic acid construct of the present invention is at least one type, preferably five types or less, more preferably three types or less, and even more preferably two types or less. Furthermore, the hepatitis B virus core protein antigen used in the nucleic acid construct of the present invention may be five types, four types, three types, two types, or one type.

[0055] From the viewpoint of improving immunogenicity, the nucleic acid construct of the present invention is preferably a nucleic acid construct in which a polynucleotide encoding the core protein antigen of hepatitis B virus is expressably linked downstream of a polynucleotide encoding the SNARE protein, more preferably a nucleic acid construct in which a polynucleotide encoding the core protein antigen of hepatitis B virus is expressably linked downstream of a polynucleotide encoding the SNARE protein via a polynucleotide encoding a linker and / or a polynucleotide encoding a proprotein convertase recognition sequence, and even more preferably a nucleic acid construct in which a polynucleotide encoding the core protein antigen of hepatitis B virus is expressably linked downstream of a polynucleotide encoding the SNARE protein via a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence. When the nucleic acid construct of the present invention includes a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence, the linking order is not particularly limited, and the polynucleotide encoding the proprotein convertase recognition sequence may be linked downstream of the polynucleotide encoding the linker, or the polynucleotide encoding the linker may be linked downstream of the polynucleotide encoding the proprotein convertase recognition sequence. Furthermore, a sequence in which the proprotein convertase recognition sequence is repeated approximately 1 to 5 times may be linked, or a polynucleotide encoding the proprotein convertase recognition sequence may be linked between multiple polynucleotides encoding the linker. In addition, any polynucleotide other than the polynucleotide encoding the linker and the polynucleotide encoding the proprotein convertase recognition sequence may be included between the polynucleotide encoding the SNARE protein and the polynucleotide encoding the hepatitis B virus core protein antigen, as long as the expression of both is not impaired.

[0056] In the present invention, "linker" refers to a peptide linker that links two polypeptides. The linker is not particularly limited as long as it can enable the SNARE protein and the hepatitis B virus core protein antigen to function normally. The length of the linker is preferably 3 amino acid residues or more, more preferably 4 amino acid residues or more, even more preferably 5 amino acid residues or more, and preferably 30 amino acid residues or less, more preferably 25 amino acid residues or less, and even more preferably 20 amino acid residues or less. Alternatively, the length of the linker is preferably 3 to 30 amino acid residues, more preferably 4 to 25 amino acid residues, and even more preferably 5 to 20 amino acid residues. Examples of such linkers include linkers having G, GS, GGS, GGGS (SEQ ID NO: 33), GGGGS (SEQ ID NO: 34), EAAAK (SEQ ID NO: 35), or XP as constituent elements. Here, X represents any amino acid residue. Specific examples include linkers consisting of sequences in which each constituent element is repeated 1 to 5 times, preferably 3 or 4 times, and a preferred specific example is a linker consisting of a sequence in which GGGGS is repeated 3 times (SEQ ID NO: 15). If the sequence components are GGGGS, an amino acid residue S may be added before the repeat sequence, or if they are EAAAK, an amino acid residue A may be added before and after the repeat sequence.

[0057] In the present invention, the proprotein convertase recognition sequence refers to an amino acid sequence recognized by proprotein convertase, a serine protease that converts proproteins into physiologically active proteins or peptides in the Golgi apparatus, and specifically refers to an amino acid sequence consisting of XRX(R / K)R. A preferred example is RRARR (SEQ ID NO: 17), where X represents any amino acid residue. Examples of proprotein convertases include furin, PC2, PC4, PC5 / 6, PC7, and PACE4, and these enzymes are known to commonly recognize the RX(R / K)R motif (Remacle AG, et al. Journal of Biological Chemistry 2008, 283(30): 20897-20906). Therefore, the proprotein convertase recognition sequence can be RX(R / K)R, where X represents any amino acid residue. The proprotein convertase recognizes the above amino acid sequence and cleaves the C-terminal side of the C-terminal arginine residue. In the present invention, preferably, the proprotein convertase is furin, and the furin recognition sequence is the same as the proprotein convertase recognition sequence described above.

[0058] There are no particular limitations on the method for obtaining the polynucleotide encoding the linker or the polynucleotide encoding the proprotein convertase recognition sequence, and they can be obtained by conventional chemical synthesis methods or genetic engineering techniques.

[0059] The polynucleotides encoding SNARE proteins, the polynucleotides encoding the hepatitis B virus core protein antigen, the polynucleotides encoding linkers, or the polynucleotides encoding proprotein convertase recognition sequences contained in the nucleic acid construct of the present invention may be codon-optimized as needed to suit the target species of the nucleic acid construct. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0060] In a preferred example, the nucleic acid construct of the present invention is an expression cassette comprising a regulatory region for controlling the expression of a polynucleotide encoding a SNARE protein and a polynucleotide encoding the core protein antigen of the hepatitis B virus. In the expression cassette, the polynucleotide encoding the SNARE protein and the polynucleotide encoding the core protein antigen of the hepatitis B virus are operably ligated to the regulatory region. Examples of the regulatory region include promoters, terminators, and enhancers. Preferably, the expression cassette comprises a promoter ligated upstream of the polynucleotide encoding the SNARE protein and the polynucleotide encoding the core protein antigen of the hepatitis B virus.

[0061] The nucleic acid construct of the present invention may have restriction enzyme recognition sites at one or both ends. The nucleic acid construct of the present invention can be introduced into a vector using these restriction enzyme recognition sites. For example, the nucleic acid construct can be introduced into a vector by cleaving a vector with a restriction enzyme and then adding the nucleic acid construct of the present invention having restriction enzyme recognition sites at its ends.

[0062] The type of vector is not particularly limited and may be any vector such as plasmid vectors, phages, phagemids, cosmids, or viral vectors. In one example, the vector into which the nucleic acid construct of the present invention is to be incorporated may be an expression vector, but on the other hand, if the nucleic acid construct to be incorporated is an expression cassette, it does not need to be an expression vector. In one example, the nucleic acid construct of the present invention is an expression cassette including a control region, and is incorporated into any vector to construct an expression vector. In another example, by incorporating the nucleic acid construct of the present invention into an expression vector including a control region, the expression cassette of the present invention is constructed on the expression vector.

[0063] In a preferred example, the nucleic acid construct of the present invention is a plasmid vector. Plasmid vectors include, but are not limited to, plasmid vectors such as pVAX1. Plasmid vectors generally have drug resistance genes incorporated into their sequence for the purpose of selectively retaining the plasmid vector in bacterial culture. However, because there is a risk of transmission of such genes to the bacterial flora in vivo, or activation and expression of such genes via mammalian promoters, plasmid vectors from which such gene sequences have been removed are more preferred.

[0064] In a preferred example, the nucleic acid construct of the present invention is a viral vector. Examples of viral vectors, but not limited to these, include adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, Sendai virus vectors, and herpesvirus vectors.

[0065] In a preferred example, the nucleic acid construct of the present invention is an mRNA construct. For example, using DNA containing a polynucleotide encoding a SNARE protein and a polynucleotide encoding the core protein antigen of hepatitis B virus as a template, Takara IVTpro TM mRNA Synthesis System (Takara Bio), mMESSAGE mMACHINE TM T7 mRNA Kit with CleanCap TM The nucleic acid construct of the present invention can be obtained as mRNA by using commercially available in vitro transcription kits such as Reagent AG (Invitrogen).

[0066] As shown in the examples below, nucleic acid constructs comprising a polynucleotide encoding the SNARE protein STX7, GOSR1, or SEC22B of the present invention and a polynucleotide encoding the core protein antigen of the hepatitis B virus exhibit higher immunogenicity and can induce or enhance a hepatitis B virus core protein antigen-specific cellular immune response compared to nucleic acid constructs comprising only a polynucleotide encoding the hepatitis B virus core protein antigen or nucleic acid constructs comprising a polynucleotide encoding a SNARE protein other than STX7, GOSR1, and SEC22B and a polynucleotide encoding the hepatitis B virus core protein antigen. In other words, by selecting and combining a specific SNARE protein and a specific hepatitis B virus antigen, a hepatitis B virus core protein antigen-specific cellular immune response can be efficiently induced or enhanced. This is presumed to be because, in the antigen presentation process in which nucleic acid constructs taken up into cells are translated or transcribed and expressed as antigen proteins, transported to intracellular vesicles where a portion of the antigen binds to major histocompatibility complex (MHC) molecules and is then transported to the cell surface, the nucleic acid construct of the present invention expresses the hepatitis B virus core protein antigen as a fusion polypeptide of the above-mentioned specific SNARE protein and the hepatitis B virus core protein antigen. This targets the hepatitis B virus core protein antigen to intracellular vesicles, increasing the probability of association with MHC molecules and thus improving the efficiency of antigen presentation. In addition, it has been reported that antigen-containing exosomes enhance antigen-specific Th1-type immune responses in vivo (Qazi KR, et al. Blood. 2009, 113(12): 2673-83). Therefore, it is possible that the hepatitis B virus core protein antigen is expressed as a fusion polypeptide of the aforementioned specific SNARE protein and antigen, thereby targeting the hepatitis B virus core protein antigen to exosomes and improving its immunogenicity. Thus, since the nucleic acid construct of the present invention efficiently induces or enhances a cellular immune response specific to the core protein antigen of the hepatitis B virus, it is expected that the nucleic acid construct of the present invention can be used to efficiently eliminate hepatitis B virus-infected cells, making it possible to prevent or treat hepatitis B caused by hepatitis B virus infection.

[0067] Furthermore, the production levels of cytokines such as IFNγ and IL-4, which can serve as indicators for determining whether an immune response is cellular or humoral, can be measured by conventionally known methods. Examples of such methods include ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linked immunosorbent spot) assay, immunohistochemistry, in situ hybridization, RT-PCR, microarrays, and flow cytometry. Reagents and kits for measuring cytokine production, such as the ELISPOT assay kit (CTL) used in the examples below, are commercially available and may be used for measurement. Cellular immune responses can be evaluated using cytokine production levels as indicators. For example, an increase in IFNγ production or the IFNγ production-to-IL-4 ratio (IFNγ / IL-4) can be considered an induction or enhancement of the cellular immune response, while a decrease in IFNγ production or IFNγ / IL-4 can be considered a weakening of the cellular immune response.

[0068] Therefore, the nucleic acid construct of the present invention can serve as an IFNγ production enhancer specific to the core protein antigen of the hepatitis B virus, an inducer or enhancer of a cellular immune response specific to the core protein antigen of the hepatitis B virus, or a preventive or therapeutic agent for hepatitis B (hereinafter referred to as "cellular immune response inducer or enhancer, etc."), and the nucleic acid construct can be used to manufacture the cellular immune response inducer or enhancer, etc. Furthermore, the nucleic acid constructs of the present invention can be used to enhance hepatitis B virus core protein antigen-specific IFNγ production, to induce or enhance hepatitis B virus core protein antigen-specific cellular immune responses, and to prevent or treat hepatitis B.

[0069] Here, "use" can refer to administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, treatment, or diagnosis performed on humans, and more specifically, methods of surgery, treatment, or diagnosis performed on humans by a physician or a person under the direction of a physician.

[0070] In this invention, "hepatitis B" refers to a liver disease caused by infection with the hepatitis B virus, and includes acute hepatitis B caused by transient infection and chronic hepatitis B caused by persistent infection. Hepatitis B is preferably chronic hepatitis B.

[0071] In this invention, "prevention" means preventing, suppressing, or delaying the onset of a disease, symptom, or condition in an individual, or reducing the risk of developing a disease, symptom, or condition in an individual. Furthermore, "treatment" includes improving a disease, symptom, or condition, preventing, suppressing, or delaying its deterioration, or reversing, preventing, suppressing, or delaying the progression of a disease, symptom, or condition.

[0072] The cellular immune response inducer or enhancer of the present invention may, on its own, be a pharmaceutical or quasi-drug for the prevention or treatment of hepatitis B, for enhancing IFNγ production specific to the core protein antigen of hepatitis B virus, for inducing or enhancing a cellular immune response specific to the core protein antigen of hepatitis B virus, or it may be a material or formulation used in combination with such pharmaceutical or quasi-drug.

[0073] When the cellular immune response inducer or enhancer of the present invention is used as a pharmaceutical product (including quasi-drugs), the pharmaceutical product may be administered in any dosage form. Examples of dosage forms include oral administration in the form of tablets, capsules, granules, powders, syrups, etc., or parenteral administration in the form of injections, suppositories, inhalants, transdermal agents, topical agents, etc. Parenteral administration is preferred, and parenteral administration by injection is more preferred. Such various dosage forms of pharmaceutical preparations can be prepared by appropriately combining the nucleic acid construct of the present invention with other pharmaceutically acceptable excipients, binders, bulking agents, disintegrants, diluents, thickeners, emulsifiers, lubricants, dispersants, coating agents, surfactants, coating agents, osmotic pressure regulators, buffers, pH adjusters, preservatives, stabilizers, antioxidants, colorants, flavoring agents, deodorizers, fragrances, etc.

[0074] The content of the nucleic acid construct of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) varies depending on the target and route of administration, and is therefore not particularly limited and can be appropriately selected over a wide range. For example, the nucleic acid construct may be contained in an amount between 0.00001% and 100% by mass of the total composition.

[0075] The dosage of the cellular immune response inducer or enhancer of the present invention may vary depending on the species, weight, sex, age, condition, or other factors of the subject. The dosage, route, and interval of administration can be appropriately determined by those skilled in the art. For example, the dosage is determined as the amount of nucleic acid construct of the present invention, between 1 ng and 10 mg per day for one adult (weighing 60 kg). If the nucleic acid construct is a viral vector, the dosage is, for example, 10 to 1 × 10 per day for one adult (weighing 60 kg). 15 It could be a virus particle.

[0076] The cellular immune response inducer or enhancer of the present invention can be administered to either humans or non-human animals. Examples of non-human animals include non-human mammals, such as great apes, other primates, mice, rats, horses, cattle, pigs, sheep, dogs, cattle, hamsters, and companion animals. The cellular immune response inducer or enhancer of the present invention is preferably administered to humans, more preferably to humans who need or desire induction or enhancement of a hepatitis B virus core protein antigen-specific cellular immune response, prevention or treatment of hepatitis B, and even more preferably to patients with hepatitis B or humans suspected of having hepatitis B.

[0077] Furthermore, the nucleic acid construct of the present invention can serve as a nucleic acid vaccine, and the nucleic acid construct can be used to manufacture a nucleic acid vaccine. Furthermore, the nucleic acid constructs of the present invention can be used to prevent or treat hepatitis B by inducing or enhancing a cellular immune response specific to the core protein antigen of the hepatitis B virus. Hereinafter, such use may be administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic.

[0078] The nucleic acid vaccine of the present invention can, on its own, induce or enhance a cellular immune response specific to the core protein antigen of the hepatitis B virus, and thus can serve as a pharmaceutical product for preventing or treating hepatitis B, or as a material or formulation used in combination with such pharmaceutical product.

[0079] In one embodiment, the nucleic acid vaccine is a DNA vaccine. The DNA vaccine includes the nucleic acid construct of the present invention, which is a plasmid vector. The plasmid vector is not particularly limited, but examples include the pVAX1 vector. Among these, plasmid vectors that do not contain drug resistance genes are preferred from the viewpoint of safety.

[0080] In another embodiment, the nucleic acid vaccine is an mRNA vaccine. The mRNA vaccine comprises the nucleic acid construct of the present invention, which is mRNA, and the nucleic acid construct may be mRNA that has been treated to add a Cap structure or polyA to stabilize the mRNA, improve translation efficiency or prevent an excessive immune response, and / or mRNA in which some bases have been modified (for example, uridine is replaced with pseudouridine or 1-methylpseudridine). It may also be a self-amplifying RNA comprising the nucleic acid construct of the present invention and containing the sequence of a virus-derived RNA-dependent RNA polymerase (RdRP) complex and its replication origins (5'CSE, 3'CSE), or a trans-amplifying RNA obtained by mixing RNA containing the nucleic acid construct of the present invention and the replication origins of the RdRP complex with mRNA containing the sequence of the RdRP complex. Preferably, the mRNA vaccine further comprises a construct responsible for drug delivery, such as liposomes or lipid nanoparticles composed of lipids, or polymer nanoparticles such as PLGA nanoparticles composed of high molecular weight polymers, as a carrier for stabilizing and delivering mRNA, and more preferably the mRNA is encapsulated in the construct.

[0081] In yet another embodiment, the nucleic acid vaccine is a viral vector vaccine. The viral vector vaccine comprises the nucleic acid construct of the present invention, which is a viral vector. The viral vector is not particularly limited, but examples include adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, Sendai virus vectors, herpesvirus vectors, and the like.

[0082] The nucleic acid vaccine of the present invention may contain, in addition to the nucleic acid construct, a pharmaceutically acceptable carrier as appropriate, and may be formulated in a predetermined form. Here, examples of carriers include carriers commonly used in vaccine production, specifically buffers, emulsifiers, preservatives (e.g., thimerosal), isotonic agents, pH adjusters, viscosity modifiers, adjuvants, or immunostimulants. An adjuvant is a substance that enhances the immune response to an antigen when administered together with that antigen. However, since the nucleic acid vaccine of the present invention can function as an adjuvant itself, the addition of an adjuvant is not necessarily required, and the composition may not contain an adjuvant.

[0083] The nucleic acid vaccine of the present invention is preferably in liquid form and is appropriately formulated to suit the intended route of administration. Routes of administration include oral and parenteral administration, such as intramuscular, intradermal, subcutaneous, transdermal, intranasal, sublingual, oral, and inhalation, but intramuscular, intradermal, or subcutaneous administration is preferred. Injectable formulations include, for example, liquid formulations, emulsion formulations, water-soluble or hydrophobic suspension formulations, and dry powder formulations that are dissolved or suspended by adding a liquid. Alternatively, the nucleic acid vaccine of the present invention can also be administered as dendritic cells into which the nucleic acid vaccine of the present invention has been introduced. Specifically, such administration can be carried out by collecting peripheral blood from a target organism, separating dendritic cell progenitor cells, differentiating the progenitor cells into dendritic cells in the presence of appropriate cytokines, introducing the nucleic acid vaccine of the present invention to the dendritic cells to present antigens, and administering the dendritic cells to the target organism. The administered dendritic cells are referred to as a dendritic cell vaccine. The dendritic cell vaccine can improve the efficiency of antigen presentation and enhance immune induction.

[0084] The content of the nucleic acid construct of the present invention in the nucleic acid vaccine of the present invention varies depending on the target recipient and route of administration, and is therefore not particularly limited and can be appropriately selected from a wide range. For example, the nucleic acid construct may be contained in an amount between 0.00001% and 100% by mass of the total nucleic acid vaccine.

[0085] The dosage of the nucleic acid vaccine of the present invention may vary depending on the species, weight, sex, age, condition, or other factors of the target. The dosage, route, and interval of administration may be appropriately determined by those skilled in the art. For example, the dosage is determined as the amount of nucleic acid construct of the present invention, between 1 ng and 10 mg per dose unit. If the nucleic acid construct is a viral vector, the dosage is, for example, 10 to 1 × 10 per dose unit. 15 It could be a virus particle.

[0086] The nucleic acid vaccine of the present invention can be administered to either humans or non-human animals. Examples of non-human animals are those described above. The nucleic acid vaccine of the present invention is preferably administered to humans, more preferably to humans who need or desire induction or enhancement of a hepatitis B virus core protein antigen-specific cellular immune response, or prevention or treatment of hepatitis B, and even more preferably to patients with hepatitis B or humans suspected of having hepatitis B.

[0087] The number of doses of the nucleic acid vaccine of the present invention may be set appropriately depending on the application, and is at least once, but may be two or more times from the viewpoint of effectiveness. Further administration is sometimes called booster immunization, and this can provide a more effective infection prevention or therapeutic effect. An interval of at least one week is recommended for booster immunization, and an interval of 1 to 4 weeks is preferred.

[0088] Nucleic acid vaccines, unlike the hepatitis B virus itself, are non-pathogenic and are considered safer than live vaccines or inactivated vaccines. Furthermore, because they are nucleic acid-based, they can be manufactured quickly and at low cost.

[0089] In one preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for the prevention or treatment of hepatitis B. In a more preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for the prevention or treatment of chronic hepatitis B. The vaccine for the prevention or treatment of hepatitis B can be administered directly to the body. It is believed that such a vaccine for the prevention or treatment of hepatitis B can exert preventive or therapeutic effects against hepatitis B, particularly chronic hepatitis B, in which conventional vaccines do not provide sufficient preventive or therapeutic effects, by efficiently inducing or enhancing a cellular immune response specific to the core protein antigen of the hepatitis B virus.

[0090] Exemplary embodiments of the present invention are further disclosed herein, including the following substances, manufacturing methods, uses, and methods. However, the present invention is not limited to these embodiments.

[0091] [1] A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of STX7, GOSR1, and SEC22B, and a polynucleotide encoding the core protein antigen of the hepatitis B virus. [2] The nucleic acid construct according to [1], wherein the core protein antigen is an HBc antigen. [3] The nucleic acid construct according to [1] or [2], wherein the polynucleotide encoding the core protein antigen is preferably any polynucleotide selected from the group consisting of (t) and polynucleotides having equivalent function, more preferably any polynucleotide selected from the group consisting of (t) to (ab), even more preferably any polynucleotide selected from the group consisting of (t) to (v), (x') and (y) to (ab), even more preferably any polynucleotide selected from the group consisting of (t) to (v) and (x'), and even more preferably the polynucleotide of (t). [4] A nucleic acid construct according to any one of [1] to [3], wherein a polynucleotide encoding the core protein antigen is linked downstream of a polynucleotide encoding the SNARE protein. [5] The nucleic acid construct according to any one of [1] to [4], wherein the polynucleotide encoding the SNARE protein and the polynucleotide encoding the core protein antigen are linked via a polynucleotide encoding a linker and / or a polynucleotide encoding a proprotein convertase recognition sequence, preferably a polynucleotide encoding a linker and a polynucleotide encoding a proprotein convertase recognition sequence. [6] The nucleic acid construct according to [5], wherein the proprotein convertase recognition sequence is an amino acid sequence consisting of RX(R / K)R (where X represents any amino acid residue), preferably an amino acid sequence consisting of XRX(R / K)R (where X represents any amino acid residue), and more preferably an amino acid sequence shown in Sequence ID No. 17. [7] A nucleic acid construct described in any one of items [1] to [6], which is a plasmid vector, mRNA, or viral vector. [8] The nucleic acid construct according to any one of [1] to [7], wherein the SNARE protein is preferably a mammalian SNARE protein, and more preferably a human SNARE protein. [9] The nucleic acid construct according to any one of [1] to [8], wherein the polynucleotide encoding the SNARE protein is preferably any polynucleotide selected from the group consisting of (f) and polynucleotides having equivalent functions thereto, more preferably any polynucleotide selected from the group consisting of (f) to (o), even more preferably any polynucleotide selected from the group consisting of (f) to (i), (k') and (l) to (o) (provided that if the SNARE protein is SEC22B, then any polynucleotide selected from the group consisting of (f) to (i) and (k') (provided that if the SNARE protein is SEC22B, then any polynucleotide selected from the group consisting of (f) to (h), (k')), and even more preferably the polynucleotide of (f).

[10] The nucleic acid construct according to any one of [1] to [9], wherein the SNARE protein is preferably selected from the group consisting of STX7 and GOSR1, and more preferably STX7.

[0092] A pharmaceutical composition comprising a nucleic acid construct as described in any one of items

[11] , [1], to

[10] . A hepatitis B virus core protein antigen-specific IFNγ production enhancer comprising a nucleic acid construct described in any one of items

[12] , [1], to

[10] as an active ingredient. An agent for inducing or enhancing a hepatitis B virus core protein antigen-specific cellular immune response, comprising a nucleic acid construct described in any one of items [1] to

[10] as an active ingredient.

[14] A prophylactic or therapeutic agent for hepatitis B comprising any one of the nucleic acid constructs described in item [1] to

[10] as an active ingredient.

[15] The agent according to any one of

[12] to

[14] , wherein the agent preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0093]

[16] Use of any one of the nucleic acid constructs described in [1] to

[10] for the production of a hepatitis B virus core protein antigen-specific IFNγ production enhancer.

[17] Use of any one of the nucleic acid constructs described in [1] to

[10] for the production of an agent for inducing or enhancing a core protein antigen-specific cellular immune response to hepatitis B virus.

[18] Use of any one of the nucleic acid constructs described in [1] to

[10] for the manufacture of a prophylactic or therapeutic agent for hepatitis B.

[19] The use according to any one of

[16] to

[18] , wherein the agent preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0094]

[20] Use of any one of the nucleic acid constructs described in [1] to

[10] for enhancement of core protein antigen-specific IFNγ production of hepatitis B virus.

[21] Use of any one of the nucleic acid constructs described in [1] to

[10] for inducing or enhancing a core protein antigen-specific cellular immune response to hepatitis B virus.

[22] Use of any one of the nucleic acid constructs described in [1] to

[10] for the prevention or treatment of hepatitis B.

[0095]

[23] A nucleic acid construct according to any one of items [1] to

[10] for use in enhancing core protein antigen-specific IFNγ production of hepatitis B virus.

[24] A nucleic acid construct according to any one of [1] to

[10] for use in inducing or enhancing a core protein antigen-specific cellular immune response to hepatitis B virus.

[25] A nucleic acid construct according to any one of items [1] to

[10] for use in the prevention or treatment of hepatitis B.

[0096] A method for enhancing hepatitis B virus core protein antigen-specific IFNγ production, comprising administering a nucleic acid construct described in any one of items [1] to

[10] to a subject requiring it in an effective amount. A method for inducing or enhancing a core protein antigen-specific cellular immune response to hepatitis B virus, comprising administering a nucleic acid construct described in any one of items [1] to

[10] to a subject requiring it in an effective amount. A method for preventing or treating hepatitis B, comprising administering an effective amount of a nucleic acid construct described in any one of items [1] to

[10] (28) to a subject in need thereof.

[29] The dose of the nucleic acid construct is preferably 1 ng to 10 mg / 60 kg body weight per day, and when the nucleic acid construct is a viral vector, preferably 10 to 1 × 10 per day. 15 The method described in any one of items

[26] to

[28] , wherein the virus particle is equal to 60 kg of body weight.

[0097] A nucleic acid vaccine containing a nucleic acid construct described in any one of items

[30] , [1], to

[10] as an active ingredient.

[31] The nucleic acid vaccine described in

[30] , which induces or enhances a core protein antigen-specific cellular immune response to the hepatitis B virus.

[32] A nucleic acid vaccine as described in

[30] or

[31] , which is a vaccine for the prevention or treatment of hepatitis B.

[33] The nucleic acid vaccine according to any one of

[30] to

[32] , wherein the nucleic acid vaccine preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0098]

[34] Use of any one of the nucleic acid constructs described in [1] to

[10] for the manufacture of nucleic acid vaccines.

[35] The use described in

[34] , wherein the nucleic acid vaccine is a vaccine for the prevention or treatment of hepatitis B.

[36] The use according to

[34] or

[35] , wherein the nucleic acid vaccine preferably contains 0.00001 to 100% by mass of the nucleic acid construct.

[0099]

[37] Use of a nucleic acid vaccine as described in any one of paragraphs

[30] to

[33] for induction or enhancement of a core protein antigen-specific cellular immune response to hepatitis B virus.

[38] Use of any one of the nucleic acid vaccines described in

[30] to

[33] for the prevention or treatment of hepatitis B.

[0100]

[39] A nucleic acid vaccine according to any one of items

[30] to

[33] , for use in inducing or enhancing a core protein antigen-specific cellular immune response to hepatitis B virus.

[40] A nucleic acid vaccine as described in any one of items

[30] to

[33] , for use in the prevention or treatment of hepatitis B.

[0101] A method for inducing or enhancing a core protein antigen-specific cellular immune response to hepatitis B virus, comprising administering a nucleic acid vaccine described in any one of items

[41] ,

[30] to

[33] in an effective dose to a target requiring it. A method for preventing or treating hepatitis B, comprising administering an effective dose of a nucleic acid vaccine described in any one of items

[42] ,

[30] , to a person in need of it.

[43] The dosage of the nucleic acid vaccine is preferably 1 ng to 10 mg per dose unit if the nucleic acid vaccine is a DNA vaccine or an mRNA vaccine, and preferably 10 to 1 × 10 per dose unit if the nucleic acid vaccine is a viral vector vaccine.15 The method according to

[41] or

[42] , wherein the particles are virus particles.

[0102] In

[44] ,

[14] ,

[15] ,

[18] ,

[19] ,

[22] ,

[25] ,

[28] ,

[29] ,

[32] ,

[33] ,

[35] ,

[36] ,

[38] ,

[40] ,

[42] and

[43] , hepatitis B is chronic hepatitis B. In

[45] ,

[30] to

[43] , nucleic acid vaccines are administered parenterally. In

[46] ,

[26] to

[29] ,

[41] to

[43] , the subjects are patients with hepatitis B or people who are suspected of having hepatitis B. [Examples]

[0103] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0104] Example 1 ELISPOT Six types of human SNARE proteins (VAMP7, VAMP8, GOSR1, STX7, STX10, SEC22B, nucleotide sequences: SEQ ID NOs: 1-6, amino acid sequences: SEQ ID NOs: 7-12) were linked to the C-terminus of hepatitis B virus core antigen (HBc antigen, amino acid sequence: SEQ ID NO: 13) as an antigen using a linker (nucleotide sequence: SEQ ID NO: 14, amino acid sequence: SEQ ID NO: 15) and a proprotein convertase recognition sequence (pc, nucleotide sequence: SEQ ID NO: 16, amino acid sequence: SEQ ID NO: 17). mRNA encoding either the amino acid sequence of the fusion polypeptide (SEQ ID NOs: 18-23) or the amino acid sequence of the HBc antigen alone was constructed. The nucleotide sequences encoding the antigen and the fusion polypeptide were codon-optimized before being introduced into plasmids (In-Fusion® HD Cloning Kit, Takara). The nucleotide sequences of the plasmids into which each polypeptide was introduced are shown in SEQ ID NOs: 24-30. The nucleotide sequence of the HBc antigen before codon optimization is shown in SEQ ID NO: 31. For mRNA synthesis, PrimeCap® T7 RNA Polymerase (low dsRNA) (Takara Bio) was used. Specifically, plasmid DNA with each sequence inserted was treated with restriction enzymes to create linear DNA, which was used as a template for in vitro transcription using T7 polymerase and 5' end capping with CleanCap Reagent AG (3'OMe) (Trilink). The obtained mRNA was purified by LiCl precipitation and formulated by encapsulating it in LNPs consisting of four lipids: ALC-0315 (MedChemexpress), ALC-0159 (MedChemexpress), DSPC (Avanti), and cholesterol (Merck) using NanoAssemblr Ignite (Precision NanoSystems). The formulated mRNA-LNPs were adjusted to the appropriate concentration by replacing the buffer with DPBS (wako) by dialysis and ultrafiltration. A 10 μg mRNA-LNP / 50 μL PBS preparation was intramuscularly administered to the thigh of a healthy C57BL / 6J. After performing the above procedure on days 0 and 14, the spleen was removed and blood was drawn on day 21, followed by euthanasia.The excised spleen was mashed using a 40-μm cell strainer (AS ONE) while adding Stain Buffer (FBS) (BD). The resulting suspension was centrifuged at 250×g for 5 minutes and washed with Stain Buffer. 5×10. 6 Individual spleen cells were collected, suspended in 50 μL of CTL-Test Medium (C.T.L.), and seeded onto an ELISpot plate. Furthermore, antigen stimulation was performed at a final concentration of 10 μg / mL by adding 50 μL of Test Medium supplemented with HBc protein (abcam). As a negative control, a group without the addition of HBc protein (N.T.) was prepared. After 24 hours, an ELISPOT assay was performed using an ELISPOT assay kit (C.T.L.) according to the attached protocol to detect IFNγ and IL4 produced specifically against the antigen (HBc).

[0105] The results are shown in Fig. 1. When GOSR1-pc-HBc and STX7-pc-HBc were used, the number of spleen cells secreting IFNγ and IL4 specifically against HBc increased significantly compared with HBc alone (antigen). Also, when SEC22B-pc-HBc was used, the number of spleen cells secreting IFNγ and IL4 specifically against HBc increased compared with HBc alone. The fold increases were 20-fold for GOSR1, 13-fold for STX7, and 6-fold for SEC22B. Generally, IFNγ is known to be secreted by Th1 cells that induce cellular immunity or cytotoxic T cells (CD8 + cells) involved in cellular immunity. Therefore, this result indicates that cellular immunity can be enhanced by fusing GOSR1, STX7, or SEC22B to the HBc antigen. Also, IL4 is generally known to be secreted from Th2 cells and induce antibody production. This result suggests that antibody production can be induced by fusing GOSR1, TX7, or SEC22B to the HBc antigen. This experiment was performed with n = 5 in each group, and a significant difference was determined when p < 0.05 was obtained in the Dunnett test for each group under unstimulated conditions (N.T.) or HBc-stimulated conditions (HBc). The above results will be explained in detail in comparison with the results obtained in the embodiment of Patent Document 1. Examples in Patent Document 1 disclose the results of an ELISPOT assay using fusion polypeptides of specific SNARE proteins (VAMP7, GOSR2, STX10, STX18, BNIP1, STX7, VTI1A, STX16, STX5, GOSR1, STX8, STX12, VAMP8, or SEC22B) and specific antigens. Specific antigens include the allergen ovalbumin (OVA), the cancer antigen human Wilm Tumor 1 (WT1), and the autoantigen mouse myelin oligodendrocyte glycoprotein peptide repeat sequence (MOG35-55) (see paragraphs

[0134] to

[0135] and Figure 1, paragraphs

[0138] to

[0139] and Figure 3(A), paragraphs

[0150] to

[0151] and Figure 9(A), (E), etc., in Patent Document 1). Based on the graphs illustrating the results of these fusion polypeptides in enhancing the immune response, the increase in IFNγ-producing cells compared to the baseline results for antigen-only polypeptides without SNARE protein fusion was approximately 2 to 5 times in all cases. Therefore, based on the results of the example in Patent Document 1, the increase in IFNγ-producing cells (20 times, 13 times, and 6 times) of GOSR1-pc-HBc, STX7-pc-HBc, and SEC22B-pc-HBc in this example is remarkably high and difficult to predict even for those skilled in the art.

[0106] Example 2: Measurement of cytotoxic T cells by FACS From the splenocyte suspension obtained in Example 1, 5 × 10⁶ 6After isolating individual cells and removing the supernatant, the cells were suspended in 2 mL of Lysing Buffer (BD) and allowed to stand at room temperature for 3 minutes to induce hemolysis. After centrifugation and removal of the supernatant, the cells were washed with Stain Buffer and suspended in 200 μL of Fixable Viability Stain 660 (BD), and the dead cells were stained at 4°C for 10 minutes. After two washes, 1 μg of Purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block;, BD) was added and blocking was performed. 8 μL of MBL MHC Class I Custom tetramer (amino acid sequence: SEQ ID NO: 32, MGLKIRQL, MBL) and 10 μL of Anti-CD8 (Mouse) mAb-FITC (MBL) were added and stained at 4°C for 30 minutes. 100 μL of CYTOFIX BUFFER (BD) was added and the cells were fixed at 4°C for 15 minutes. The sample was then washed, suspended in Stain Buffer, and detected using a flow cytometer.

[0107] The results are shown in Figure 2. When using STX7-pc-HBc, the proportion of CD8-positive cells with HBc antigen-specific TCRs (T cell receptors) was significantly increased compared to HBc alone (antigen). This result suggests that STX7-pc-HBc is effective in increasing the proportion of cytotoxic T cells (CD8). + This indicates an increase in cells. In this experiment, n=5 was used for each group, and a statistically significant difference was considered to exist if p<0.05 was determined when performing the Dunnett test.

[0108] Reference Example 1: Measurement of Antibody Titer Plasma was separated from the blood collected in Example 1, and antibody titer measurements were performed. For antibody titer measurement, 100 μL / well of DPBS solution containing 0.1 μg / mL of HBc protein was added to a 96-well ELISA plate (IWAKI) to immobilize HBc. The following day, after washing the plate, blocking was performed for 1 hour with 100 μL / well of 1% BSA-containing DPBS. Subsequently, the plate was washed, and the separated serum was added at 100 μL / well, diluting it stepwise twofold. After 2 hours, the serum was washed away, and 1 μg / mL of anti-Mouse IgG (abcam) was added. After standing for 1 hour and washing, 50 μL / well of TMB solution (abcam) was added, and a color reaction was performed. After 10 minutes, 50 μL / well of Stop solution (abcam) was added to stop the color reaction, and absorbance measurement (450 nm) was performed. At this time, the antibody titer of each sample was defined as the value at the highest dilution ratio in which the absorbance was more than twice that of the control sample to which DPBS was added instead of serum.

[0109] The results are shown in Figure 3. No significant changes in IgG antibody titers were observed when using GOSR1-pc-HBc, STX7-pc-HBc, or SEC22B-pc-HBc compared to HBc antigen alone. This experiment was conducted with n=5 in each group, and a p<0.05 result in the Dunnett test in each group was considered statistically significant.

Claims

1. A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of STX7, GOSR1, and SEC22B, and a polynucleotide encoding the core protein antigen of the hepatitis B virus.

2. The nucleic acid construct according to claim 1, wherein the core protein antigen is an HBc antigen.

3. The nucleic acid construct according to claim 1, wherein a polynucleotide encoding the core protein antigen is linked downstream of the polynucleotide encoding the SNARE protein.

4. The nucleic acid construct according to claim 1, wherein the polynucleotide encoding the SNARE protein and the polynucleotide encoding the core protein antigen are linked via a polynucleotide encoding a linker and / or a polynucleotide encoding a proprotein convertase recognition sequence.

5. The nucleic acid construct according to claim 1, which is a plasmid vector, mRNA, or viral vector.

6. An agent for inducing or enhancing a hepatitis B virus core protein antigen-specific cellular immune response, comprising a nucleic acid construct according to any one of claims 1 to 5 as an active ingredient.

7. A preventive or therapeutic agent for hepatitis B comprising a nucleic acid construct according to any one of claims 1 to 5 as an active ingredient.

8. A nucleic acid vaccine comprising a nucleic acid construct according to any one of claims 1 to 5 as an active ingredient.

9. The nucleic acid vaccine according to claim 8, which induces or enhances a core protein antigen-specific cellular immune response to the hepatitis B virus.

10. The nucleic acid vaccine according to claim 8, which is a vaccine for the prevention or treatment of hepatitis B.

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