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

Abstract

Providing nucleic acid constructs that enhance antigen-specific immune responses against enveloped viruses. [Solution] A nucleic acid construct comprising a polynucleotide encoding a SNARE protein selected from the group consisting of VAMP7, STX7, GOSR1, and SEC22B, and a polynucleotide encoding a surface protein antigen of an enveloped virus.

Description

[Technical Field]

[0001] This invention relates to a nucleic acid construct comprising a polynucleotide encoding a SNARE protein and a polynucleotide encoding a virus-derived antigen, and to the use thereof. [Background technology]

[0002] 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.

[0003] 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 combining an allergen with a specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein enhanced the antigen-specific cellular immune response.

[0004] 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]

[0005] [Patent Document 1] Japanese Patent Publication No. 2023-24408 [Non-patent literature]

[0006] [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]

[0007] The present invention relates to providing nucleic acid constructs that enhance antigen-specific immune responses against enveloped viruses and the use thereof. [Means for solving the problem]

[0008] The inventors have discovered that, surprisingly, using a nucleic acid construct containing a polynucleotide encoding the SNARE protein VAMP7, STX7, GOSR1, or SEC22B and a polynucleotide encoding the surface protein antigen of an enveloped virus can efficiently induce or enhance an antigen-specific humoral immune response compared to nucleic acid constructs containing only a polynucleotide encoding the surface protein antigen of an enveloped 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 surface protein antigen of an enveloped virus.

[0009] Therefore, the present invention provides the following 1) to 5). 1) A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of VAMP7, STX7, GOSR1, and SEC22B, and a polynucleotide encoding an enveloped virus surface protein antigen. 2) An agent for inducing or enhancing an enveloped virus surface protein antigen-specific humoral immune response, comprising the nucleic acid construct described in 1) as an active ingredient. 3) An agent for inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses, comprising the nucleic acid construct described in 1) as an active ingredient. 4) A prophylactic or therapeutic agent for enveloped virus infections, comprising the nucleic acid construct described in 1) as an active ingredient. 5) A nucleic acid vaccine containing the nucleic acid construct described in 1) as the active ingredient. [Effects of the Invention]

[0010] The nucleic acid construct of the present invention can efficiently induce or enhance an antigen-specific humoral immune response against enveloped viruses. Such a nucleic acid construct is useful as a nucleic acid vaccine. [Brief explanation of the drawing]

[0011] [Figure 1] Antibody titers of IgG subclasses when mice were administered mRNA encoding a SNARE protein-HBs antigen fusion polypeptide. (A) Represents the IgG antibody titer of each group. (B) Average value of IgG antibody titer. Antigen, VAMP7, VAMP8, GOSR1, STX7, STX10, and SEC22B represent mRNA encoding HBs antigen alone, VAMP7-pc-HBs, VAMP8-pc-HBs, GOSR1-pc-HBs, STX7-pc-HBs, STX10-pc-HBs, and SEC22B-pc-HBs, respectively. [Figure 2] Evaluation of immunogenicity of mRNA encoding SNARE protein-HBs antigen fusion polypeptide. (A) IL4 production is shown by the number of spot-forming cells (SFCs). NT represents the control without antigen restimulation, and HBs represents the results with HBs antigen restimulation. (B) Average number of IL4-producing cells. Antigen, VAMP7, VAMP8, GOSR1, STX7, STX10, and SEC22B represent mRNA encoding HBs antigen alone, VAMP7-pc-HBs, VAMP8-pc-HBs, GOSR1-pc-HBs, STX7-pc-HBs, STX10-pc-HBs, and SEC22B-pc-HBs, respectively. [Figure 3] Antibody titers of IgG subclasses when mice were administered plasmid vectors encoding the SNARE protein VAMP7-HA1 antigen fusion polypeptide. (A) Shows the IgG antibody titer for each group. (B) Average value of IgG antibody titer. Empty, HA1, and V7-pc-HA1 represent a pVAX1 vector without the antigen sequence, a pVAX1 vector with only the HA1 antigen sequence introduced, and a pVAX1 vector with the VAMP7-pc-HA1 antigen fusion sequence introduced, respectively. [Figure 4]Evaluation of immunogenicity of plasmid vectors encoding the SNARE protein VAMP7-HA1 antigen fusion polypeptide. (A) IL4 production is shown by the number of spot-forming cells (SFCs). NT represents the control without antigen restimulation, and HA1 represents the result with HA1 antigen restimulation. (B) Mean, standard error, and p-value of IL4-producing cell count. Empty, HA1, and V7-pc-HA1 represent a pVAX1 vector without the antigen sequence, a pVAX1 vector with only the HA1 antigen sequence introduced, and a pVAX1 vector with the VAMP7-pc-HA1 antigen fusion sequence introduced, respectively. [Modes for carrying out the invention]

[0012] 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, while RNA includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA. mRNA, in particular, is synthesized by an in vitro transcription reaction for reasons such as initiating translation in vivo, stabilizing mRNA, and suppressing degradation. After synthesis, a 5' cap (methylated guanosine) is added by a cap-forming enzyme, and poly(A) polymerase is added. The poly(A) sequence may be incorporated into the template DNA used in the in vitro transcription reaction. This includes mRNA with a cap structure or polyA added, as well as RNA with some base modifications (for example, uridine being replaced with pseudouridine or 1-methylpseudridine).

[0013] In this specification, the term "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 a DNA in which some biological information is contained in the sequence information of the bases constituting the DNA. Furthermore, the "gene" includes not only the "gene" represented by a specific nucleotide sequence, but also nucleic acids encoding homologs (i.e., 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 / ]).

[0014] In this specification, the terms "peptide", "polypeptide" or "protein" are used interchangeably.

[0015] In this specification, the term "amino acid residue" means the 20 amino acid residues constituting a protein, 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).

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The present invention provides a nucleic acid construct comprising a polynucleotide encoding a SNARE protein selected from the group consisting of VAMP7, STX7, GOSR1, and SEC22B, and a polynucleotide encoding a surface protein antigen of an enveloped virus.

[0030] In the present invention, the SNARE protein is a protein belonging to the protein family having a SNARE motif, and specifically, it is any protein selected from the group consisting of VAMP7, STX7, GOSR1, and SEC22B (hereinafter sometimes simply referred to as the SNARE protein). From the viewpoint of immune induction or enhancement, any of the proteins selected from the group consisting of VAMP7, STX7, and GOSR1 is preferred as the SNARE protein, with VAMP7 and STX7 being more preferred. The SNARE protein has a transmembrane domain and is localized to intracellular vesicles.

[0031] VAMP7 (vesicle-associated membrane protein 7) is a type of SNARE protein that has a transmembrane domain and is expected to be localized to late endosomes, lysosomes, and cell membranes. In a preferred example, VAMP7 is mammalian VAMP7. In a more preferred example, VAMP7 is human VAMP7, which is a protein consisting of the amino acid sequence of SEQ ID NO: 7, encoded by a gene (Gene ID: 6845) consisting of the nucleotide sequence of SEQ ID NO: 1. The VAMP7 used in this invention includes VAMP7 and polypeptides having equivalent function.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Specifically, the following polypeptides are examples of VAMP7. (a) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (b) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 7, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) 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: 7, and which has a transmembrane domain and can be localized to intracellular vesicles (e.g., late endosomes or lysosomes) 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: 1, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) 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., late endosomes or lysosomes) or exosomes. Splicing variants of the VAMP7 gene consisting of the nucleotide sequence of SEQ ID NO: 1 include variants that are registered in NCBI's RefSeq (Reference Sequence) as NM_001185183.2 or NM_001145149.3 and encode proteins registered as NP_001172112.1 or NP_001138621.1, respectively.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] The following polynucleotides are examples of polynucleotides that encode VAMP7. (f) A polynucleotide consisting of the nucleotide sequence of sequence number 1; (g) A polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical to the nucleotide sequence of Sequence ID No. 1, and having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) 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: 1, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) or exosomes; (i) A polynucleotide that is a splicing variant of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and which has a transmembrane domain and encodes a polypeptide that can localize to intracellular vesicles (e.g., late endosomes or lysosomes) 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: 1, has a transmembrane domain, and can localize to intracellular vesicles (e.g., late endosomes or lysosomes) 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., late endosomes or lysosomes) or exosomes; (l) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (m) A polynucleotide encoding a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 7, having a transmembrane domain, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) 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: 7, and capable of localizing to intracellular vesicles (e.g., late endosomes or lysosomes) 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., late endosomes or lysosomes) or exosomes. Among these, any of the polynucleotides (f) to (i) above, (k') below, and (l) to (o) above are preferred, and any of the polynucleotides (f) to (i) above and (k') below 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., late endosomes or lysosomes) or exosomes.

[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 (f) to (i) above, (k') below, and (l) to (o) above are preferred, and any of the polynucleotides (f) to (i) above and (k') below 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 (f) to (i) above, (k') below, and (l) to (o) above are preferred, and any of the polynucleotides (f) to (i) above and (k') below 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 (f) to (h) above, (k') below, and (l) to (o) above are preferred, and any of the polynucleotides (f) to (h) above and (k') below are more preferred. (k') A polynucleotide that is a fragment of any of the polynucleotides (f) to (h) above, 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: 1, 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: 1, 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: 1, 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), ODA method (Hashimoto-Gotoh et al., Gene, 152, 271-276, 1995), and Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488). Alternatively, 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: 1, 3, 4, and 6 to genome editing using artificial DNA nucleases (or programmable nucleases).

[0049] The surface protein antigen of an enveloped virus used in the present invention is not particularly limited as long as it is an antigen derived from the surface protein of an enveloped virus. Here, an enveloped virus refers to a virus that has a lipid bilayer envelope on the outermost part of the virus particle, and the surface protein of an enveloped virus refers to a protein present on the surface of the virus particle of an enveloped virus. Enveloped viruses include viruses that have RNA as their nucleic acid and viruses that have DNA as their nucleic acid. Examples of enveloped viruses that have RNA as their nucleic acid include influenza virus, coronavirus, SARS coronavirus, SARS coronavirus-2, RSV, mumps virus, Lassa virus, dengue virus, rubella virus, and human immunodeficiency virus, while examples of enveloped viruses that have DNA as their nucleic acid include hepatitis B virus, human herpesvirus, and vaccinia virus. Of these, hepatitis B virus and influenza virus are preferred. Examples of surface proteins of enveloped viruses include hepatitis B virus surface proteins (HBs) in the case of hepatitis B virus, and hemagglutinin (HA) and neuraminidase (NA) in the case of influenza virus.

[0050] In a preferred example, the surface protein antigen of an enveloped virus is the HBs antigen, which is the surface protein of the hepatitis B virus. The HBs antigen used in the present invention includes proteins consisting of the amino acid sequence of SEQ ID NO: 13, encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 31 (large HBs (LHBs); including preS1, preS2, and S regions), proteins consisting of the amino acid sequence of SEQ ID NO: 38, encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 40 (middle HBs (MHBs); including preS2, and S regions), proteins consisting of the amino acid sequence of SEQ ID NO: 39, encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 41 (small HBs (SHBs); consisting only of the S region), and polypeptides having equivalent immunogenicity.

[0051] In the present invention, polypeptides having immunogenicity equivalent to that of HBs antigen include, for example, polypeptides that contain part or all of the surface 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 HBs antigen include the polypeptides (q) to (s) below.

[0052] The following polypeptides are examples of HBs antigens, which are surface protein antigens of the hepatitis B virus. (p) A polypeptide consisting of any of the amino acid sequences of SEQ ID NOs: 13, 38, and 39, preferably any of SEQ ID NOs: 13 and 39; (q) A polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of any of SEQ ID NOs: 13, 38, and 39, preferably any of SEQ ID NOs: 13 and 39, and which is immunogenic; (r) A polypeptide having immunogenic properties, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted to any of the amino acid sequences of SEQ ID NOs. 13, 38, and 39, preferably any of SEQ ID NOs. 13 and 39; (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).

[0053] In a preferred example, the surface protein antigen of an enveloped virus is the HBs antigen. The polynucleotides encoding the HBs antigen used in the present invention include polynucleotides encoding the HBs antigen and polynucleotides encoding polypeptides having immunogenicity equivalent to that of the HBs antigen. Specific examples of polynucleotides encoding polypeptides having immunogenicity equivalent to that of the HBs antigen include the following polynucleotides (u) to (x) and (z) to (ab).

[0054] The following polynucleotides are examples of polynucleotides that encode the HBs antigen. (t) A polynucleotide consisting of any of sequence numbers 31, 40, and 41, preferably any of sequence numbers 31 and 41; (u) A polynucleotide encoding an immunogenic polypeptide, comprising a nucleotide sequence having at least 80% identity with any of SEQ ID NOs: 31, 40, and 41, preferably any of SEQ ID NOs: 31 and 41; (v) Polynucleotides comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted to any of the nucleotide sequences of SEQ ID NOs: 31, 40, and 41, preferably SEQ ID NOs: 31 and 41, and which encode an immunogenic polypeptide; (w) A polynucleotide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of any of SEQ ID NOs: 31, 40, and 41, preferably any of SEQ ID NOs: 31 and 41, and encodes an immunogenic polypeptide; (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 any of SEQ ID NOs: 13, 38, and 39, preferably any of SEQ ID NOs: 13 and 39; (z) A polynucleotide encoding an immunogenic polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of any of SEQ ID NOs: 13, 38, and 39, preferably any of SEQ ID NOs: 13 and 39; (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 to any of the amino acid sequences of SEQ ID NOs. 13, 38, and 39, preferably any of SEQ ID NOs. 13 and 39; (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).

[0055] In another preferred example, the surface protein antigen of an enveloped virus is the HA antigen, which is the surface protein of the influenza virus. The HA antigen used in the present invention includes the protein consisting of the amino acid sequence of SEQ ID NO: 32 (influenza hemagglutinin HA1 subunit), encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 36; the protein consisting of the amino acid sequence of SEQ ID NO: 42 (influenza hemagglutinin HA), encoded by the gene consisting of the nucleotide sequence of SEQ ID NO: 43; and polypeptides having equivalent immunogenicity.

[0056] In the present invention, polypeptides having immunogenicity equivalent to that of the HA antigen include, for example, polypeptides that contain part or all of the surface protein of the influenza virus and have immunogenicity capable of eliciting an immune response against the influenza 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 the HA antigen include the polypeptides (q) to (s) below.

[0057] The following polypeptides are examples of HA antigens, which are surface protein antigens of the influenza virus. (p) A polypeptide comprising either SEQ ID NO: 32 or 42, preferably the amino acid sequence of SEQ ID NO: 32; (q) A polypeptide comprising either SEQ ID NO: 32 or 42, preferably an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 32, and which is immunogenic; (r) A polypeptide having immunogenic properties, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, added, or inserted from either SEQ ID NO: 32 or 42, preferably the amino acid sequence of SEQ ID NO: 32; (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).

[0058] In another preferred example, the surface protein antigen of an enveloped virus is the HA antigen. The polynucleotides encoding the HA antigen used in the present invention include polynucleotides encoding the HA antigen and polynucleotides encoding polypeptides having immunogenicity equivalent to that of the HA antigen. Specific examples of polynucleotides encoding polypeptides having immunogenicity equivalent to that of the hemagglutinin antigen include the following polynucleotides (u) to (x) and (z) to (ab).

[0059] The following polynucleotides are examples of polynucleotides that encode the HA antigen. (t) A polynucleotide consisting of either SEQ ID NOs. 36 or 43, preferably the nucleotide sequence of SEQ ID NO. 36; (u) A polynucleotide encoding an immunogenic polypeptide, comprising a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO: 36 or 43, preferably SEQ ID NO: 36; (v) A polynucleotide encoding an immunogenic polypeptide, comprising a nucleotide sequence in which one or more nucleotides are deleted, substituted, added, or inserted to the nucleotide sequence of either SEQ ID NO: 36 or 43, preferably SEQ ID NO: 36; (w) A polynucleotide that hybridizes under stringent conditions to a complementary chain of a polynucleotide consisting of either SEQ ID NO: 36 or 43, preferably SEQ ID NO: 36, and which encodes an immunogenic polypeptide; (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 either SEQ ID NOs. 32 or 42, preferably the amino acid sequence of SEQ ID NO. 32; (z) A polynucleotide encoding an immunogenic polypeptide, which consists of either SEQ ID NO: 32 or 42, preferably an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 32; (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 either SEQ ID NO: 32 or 42, preferably the amino acid sequence of SEQ ID NO: 32; (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).

[0060] The method for obtaining the polynucleotide encoding the surface protein antigen of the enveloped virus 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 surface protein antigen of the enveloped virus may be a polynucleotide obtained by linking multiple polynucleotides encoding the surface protein antigen of one enveloped virus in a manner that allows for expression, or a polynucleotide obtained by linking two or more polynucleotides encoding the surface protein antigens of two or more enveloped viruses in a manner that allows for expression. The surface protein antigen of the enveloped virus 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 surface protein antigen of the enveloped virus used in the nucleic acid construct of the present invention may be five types, four types, three types, two types, or one type.

[0061] 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 an enveloped virus surface protein antigen is expressably linked downstream of a polynucleotide encoding a SNARE protein, more preferably a nucleic acid construct in which a polynucleotide encoding an enveloped virus surface protein antigen is expressably linked downstream of a polynucleotide encoding a 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 an enveloped virus surface protein antigen is expressably linked downstream of a polynucleotide encoding a 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 surface protein antigen of the enveloped virus, as long as the expression of both is not impaired.

[0062] 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 surface protein antigen of an enveloped virus 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: 44), GGGGS (SEQ ID NO: 45), EAAAK (SEQ ID NO: 46), 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.

[0063] 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.

[0064] 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.

[0065] The polynucleotides encoding SNARE proteins, the polynucleotides encoding surface protein antigens of enveloped viruses, 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 / ]).

[0066] 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 an enveloped virus surface protein antigen. In the expression cassette, the polynucleotide encoding the SNARE protein and the polynucleotide encoding the enveloped virus surface protein antigen 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 enveloped virus surface protein antigen.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 an enveloped virus surface protein antigen 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).

[0072] As shown in the examples below, nucleic acid constructs containing a polynucleotide encoding the SNARE protein VAMP7, STX7, GOSR1, or SEC22B of the present invention and a polynucleotide encoding the surface protein antigen of an enveloped virus exhibit higher immunogenicity compared to nucleic acid constructs containing only a polynucleotide encoding the surface protein antigen of an enveloped virus, or nucleic acid constructs containing a polynucleotide encoding a SNARE protein other than VAMP7, STX7, GOSR1, and SEC22B and a polynucleotide encoding the surface protein antigen of an enveloped virus. Surprisingly, these constructs can induce or enhance a humoral immune response specific to the surface protein antigen of an enveloped virus, and can induce or enhance the production of antibodies specific to the surface protein antigen. In other words, by selecting and combining a specific SNARE protein with a specific antigen of an enveloped virus, it is possible to efficiently induce or enhance a humoral immune response specific to the surface protein antigen of an enveloped virus, and to induce or enhance the production of antibodies specific to the surface protein antigen. 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 surface protein antigen of the enveloped virus as a fusion polypeptide of the above-mentioned specific SNARE protein and the surface protein antigen of the enveloped virus. This targets the surface protein antigen of the enveloped virus to intracellular vesicles, increasing the probability of association with MHC molecules and thus improving the efficiency of antigen presentation. Thus, the nucleic acid construct of the present invention efficiently induces or enhances a humoral immune response specific to the surface protein antigen of enveloped viruses, and induces or enhances the production of surface protein antigen-specific antibodies. Therefore, by using the nucleic acid construct of the present invention, it becomes possible to prevent or treat enveloped virus infections caused by enveloped virus infection.

[0073] 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. Furthermore, antibody production can be measured using conventionally known methods. Examples of such methods include ELISA and immunoturbidimetric analysis. Humoral immune responses can be evaluated using cytokine production and / or antibody production as indicators. For example, an increase in IL-4 production or the IL-4 / IFNγ ratio indicates that the humoral immune response has been induced or enhanced, while a decrease in IL-4 production or IL-4 / IFNγ indicates that the humoral immune response has been weakened. Alternatively, an increase in IgG antibody production indicates that the humoral immune response has been induced or enhanced, while a decrease in IgG antibody production indicates that the humoral immune response has been weakened.

[0074] Therefore, the nucleic acid construct of the present invention can serve as an agent for inducing or enhancing a humoral immune response specific to the surface protein antigen of an enveloped virus, an agent for inducing or enhancing antibody production specific to the surface protein antigen of an enveloped virus, or an agent for preventing or treating enveloped virus infections (hereinafter referred to as humoral immune response inducers or enhancers, etc.), and the nucleic acid construct can be used to manufacture humoral immune response inducers or enhancers, etc. Furthermore, the nucleic acid constructs of the present invention can be used to induce or enhance a humoral immune response specific to the surface protein antigen of enveloped viruses, to induce or enhance antibody production specific to the surface protein antigen of enveloped viruses, and to prevent or treat enveloped virus infections.

[0075] 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.

[0076] In this invention, "enveloped virus infection" refers to a disease caused by an enveloped virus infection. Examples include hepatitis B caused by hepatitis B virus infection and influenza caused by influenza virus infection.

[0077] 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.

[0078] The humoral immune response inducer or enhancer of the present invention may, on its own, be a pharmaceutical or quasi-drug for inducing or enhancing a humoral immune response specific to the surface protein antigen of an enveloped virus, for inducing or enhancing antibody production specific to the surface protein antigen of an enveloped virus, or for the prevention or treatment of an enveloped virus infection. It may also be a material or formulation used in combination with such pharmaceutical or quasi-drug.

[0079] When the humoral 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.

[0080] The content of the nucleic acid construct of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) varies depending on the target antigen, the recipient, and the 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.

[0081] The dosage of the humoral 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.

[0082] The humoral immune response inducer or enhancer of the present invention can be administered to both humans and 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 humoral immune response inducer or enhancer of the present invention is preferably administered to humans, more preferably to induce or enhance a humoral immune response specific to the surface protein antigen of enveloped viruses, induce or enhance the production of antibodies specific to the surface protein antigen of enveloped viruses, or to humans who need or desire the prevention or treatment of enveloped virus infections, and even more preferably to patients with enveloped virus infections or humans who are suspected of having an enveloped virus infection.

[0083] 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 induce or enhance a humoral immune response specific to the surface protein antigen of enveloped viruses, to induce or enhance antibody production specific to the surface protein antigen of enveloped viruses, and to prevent or treat enveloped virus infections. Here, such use may be administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic.

[0084] The nucleic acid vaccine of the present invention can, on its own, be a pharmaceutical product for preventing or treating enveloped virus infections by inducing or enhancing a humoral immune response specific to the surface protein antigen of an enveloped virus, or by inducing or enhancing antibody production specific to the surface protein antigen of an enveloped virus, or it can be a material or formulation used in combination with such pharmaceutical product.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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 antigen, the recipient, and the 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 nucleic acid vaccine.

[0091] 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.

[0092] 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 an enveloped virus surface protein antigen-specific humoral immune response, induction or enhancement of an enveloped virus surface protein antigen-specific antibody production, or prevention or treatment of an enveloped virus infection, and even more preferably to patients with an enveloped virus infection or humans who are at risk of having an enveloped virus infection.

[0093] 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.

[0094] Nucleic acid vaccines, unlike viruses themselves, are non-pathogenic and are considered safer than live vaccines or inactivated vaccines. Furthermore, by changing the nucleic acid encoding the antigen, they can be adapted to a wide variety of antigens, and because they are nucleic acid-based, they can be manufactured quickly and at low cost.

[0095] In a preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for the prevention or treatment of enveloped virus infections. This vaccine for the prevention or treatment of enveloped virus infections can be administered directly to the body. It is believed that such a vaccine for the prevention or treatment of enveloped virus infections can exert a preventive or therapeutic effect against enveloped virus infections by efficiently inducing or enhancing a humoral immune response specific to the surface protein antigens of enveloped virus infections. In particular, it is expected to be a vaccine for the prevention or treatment of chronic infections for which conventional vaccines do not provide sufficient preventive or therapeutic effects. In a more preferred embodiment, the nucleic acid vaccine of the present invention is a vaccine for the prevention or treatment of hepatitis B or influenza.

[0096] 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.

[0097] [1] A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of VAMP7, STX7, GOSR1, and SEC22B, and a polynucleotide encoding an enveloped virus surface protein antigen. [2] The nucleic acid construct according to [1], wherein the enveloped virus is hepatitis B virus or influenza virus. [3] The nucleic acid construct according to [1] or [2], wherein the surface protein antigen of the enveloped virus is the HBs antigen of hepatitis B virus or the HA antigen of influenza virus. [4] The nucleic acid construct according to any one of [1] to [3], wherein the polynucleotide encoding the surface protein antigen of the enveloped virus 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). [5] A nucleic acid construct according to any one of [1] to [4], wherein a polynucleotide encoding the surface protein antigen of the enveloped virus is linked downstream of the polynucleotide encoding the SNARE protein. [6] The nucleic acid construct according to any one of [1] to [5], wherein the polynucleotide encoding the SNARE protein and the polynucleotide encoding the surface protein antigen of the enveloped virus 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. [7] The nucleic acid construct according to [6], 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. [8] A nucleic acid construct described in any one of [1] to [7], which is a plasmid vector, mRNA, or viral vector. [9] The nucleic acid construct according to any one of [1] to [8], wherein the SNARE protein is preferably a mammalian SNARE protein, and more preferably a human SNARE protein.

[10] The nucleic acid construct according to any one of [1] to [9], wherein the polynucleotide encoding the SNARE protein is preferably any polynucleotide selected from the group consisting of (f) and polynucleotides having equivalent function, 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).

[11] The nucleic acid construct according to any one of [1] to

[10] , wherein the SNARE protein is preferably selected from the group consisting of VAMP7, STX7, and GOSR1, and more preferably selected from the group consisting of VAMP7 and STX7.

[0098] A pharmaceutical composition comprising a nucleic acid construct as described in any one of items

[12] , [1], to

[11] . An agent for inducing or enhancing an enveloped virus surface protein antigen-specific humoral immune response, comprising a nucleic acid construct described in any one of items [1] to

[11] as an active ingredient. An agent for inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses, comprising a nucleic acid construct described in any one of items [1] to

[11] as an active ingredient.

[15] A prophylactic or therapeutic agent for enveloped virus infections comprising a nucleic acid construct described in any one of items [1] to

[11] as an active ingredient.

[16] The agent according to

[15] , wherein the enveloped virus infection is hepatitis B or influenza.

[17] The agent according to any one of

[13] to

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

[0099]

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

[11] for the production of an antigen-specific humoral immune response inducer or enhancer for the surface protein of an enveloped virus.

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

[11] for the production of an agent for inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses.

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

[11] for the manufacture of a prophylactic or therapeutic agent for enveloped virus infections.

[21] The use described in

[20] , wherein the enveloped virus infection is hepatitis B or influenza.

[22] The use according to any one of

[18] to

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

[0100]

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

[11] for inducing or enhancing an antigen-specific humoral immune response to the surface protein of an enveloped virus.

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

[11] for inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses.

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

[11] for the prevention or treatment of an enveloped virus infection.

[26] The use described in

[25] , wherein the enveloped virus infection is hepatitis B or influenza.

[0101]

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

[11] for use in inducing or enhancing an antigen-specific humoral immune response to the surface protein of an enveloped virus.

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

[11] for use in inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses.

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

[11] for use in the prevention or treatment of an enveloped virus infection.

[30] The use described in

[29] , wherein the enveloped virus infection is hepatitis B or influenza.

[0102] A method for inducing or enhancing an enveloped virus surface protein antigen-specific humoral immune response, comprising administering a nucleic acid construct described in any one of items [1] to

[11] to a subject requiring it in an effective amount. A method for inducing or enhancing the production of an enveloped virus surface protein antigen-specific antibody, comprising administering a nucleic acid construct described in any one of items [1] to

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

[11] to a subject in need thereof.

[34] The method according to

[33] , wherein the enveloped virus infection is hepatitis B or influenza.

[35] 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

[31] to

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

[0103] A nucleic acid vaccine containing a nucleic acid construct described in any one of items

[36] [1] to

[11] as an active ingredient.

[37] A nucleic acid vaccine according to

[36] that induces or enhances an antigen-specific humoral immune response to the surface protein of an enveloped virus.

[38] A nucleic acid vaccine according to

[36] that induces or enhances the production of surface protein antigen-specific antibodies of enveloped viruses.

[39] A vaccine for the prevention or treatment of enveloped virus infections, A nucleic acid vaccine as described in any one of items

[36] to

[38] .

[40] The nucleic acid vaccine according to

[39] , wherein the enveloped virus infection is hepatitis B or influenza.

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

[36] to

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

[0104]

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

[11] for the manufacture of nucleic acid vaccines.

[43] The use described in

[42] , wherein the nucleic acid vaccine is a vaccine for the prevention or treatment of an enveloped virus infection.

[44] The use described in

[43] , wherein the enveloped virus infection is hepatitis B or influenza.

[45] The use according to any one of

[42] to

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

[0105]

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

[36] to

[41] for induction or enhancement of an antigen-specific humoral immune response to the surface protein of an enveloped virus.

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

[36] to

[41] for the induction or enhancement of surface protein antigen-specific antibody production of an enveloped virus.

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

[36] to

[41] for the prevention or treatment of enveloped virus infections.

[49] The use described in

[48] , wherein the enveloped virus infection is hepatitis B or influenza.

[0106]

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

[36] to

[41] , for use in inducing or enhancing an antigen-specific humoral immune response to the surface protein of an enveloped virus.

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

[36] to

[41] , for use in inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses.

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

[36] to

[41] , for use in the prevention or treatment of an enveloped virus infection.

[53] The nucleic acid vaccine according to

[52] , wherein the enveloped virus infection is hepatitis B or influenza.

[0107] A method for inducing or enhancing an enveloped virus surface protein antigen-specific humoral immune response, comprising administering a nucleic acid vaccine described in any one of items

[54] ,

[36] , to

[41] in an effective dose to a subject in need thereof. A method for inducing or enhancing the production of surface protein antigen-specific antibodies of an enveloped virus, comprising administering an effective dose of a nucleic acid vaccine described in any one of items

[55] ,

[36] , to a subject in need thereof. A method for preventing or treating enveloped virus infections, comprising administering an effective dose of a nucleic acid vaccine described in any one of items

[56] ,

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

[57] The method according to

[56] , wherein the enveloped virus infection is hepatitis B or influenza.

[58] 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 described in any one of the items

[54] to

[57] , wherein the particles are virus particles.

[0108] In

[59] ,

[36] to

[58] , nucleic acid vaccines are administered parenterally. In paragraphs

[60] ,

[31] to

[35] , and

[54] to

[58] , the subjects are patients with enveloped virus infection or persons suspected of having an enveloped virus infection. [Examples]

[0109] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0110] Example 1: Measurement of anti-HBs IgG antibody titer 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 surface antigen (HBs antigen, amino acid sequence: SEQ ID NOs: 13) as an antigen using a linker (nucleotide sequence: SEQ ID NOs: 14, amino acid sequence: SEQ ID NOs: 15) and a proprotein convertase recognition sequence (pc, nucleotide sequence: SEQ ID NOs: 16, amino acid sequence: SEQ ID NOs: 17). mRNA encoding either the amino acid sequence of the fusion polypeptide (SEQ ID NOs: 18-23) or the amino acid sequence of the HBs 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 sequence of the HBs antigen before codon optimization is shown in SEQ ID NOs: 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 encapsulated in LNPs consisting of four lipids: ALC-0315 (MedChemexpress), ALC-0159 (MedChemexpress), DSPC (Avanti), and cholesterol (Merck) using NanoAssemblr Ignite to formulate the mRNA-LNPs. The formulated mRNA-LNPs were adjusted to the appropriate concentration by replacing the buffer with DPBS (wako) by dialysis and ultrafiltration. The 10 μg mRNA-LNP / 50 μL PBS formulation was intramuscularly administered to the thigh of a healthy C57BL / 6J patient. After performing the above procedures on days 0 and 14, the spleen was removed and blood was drawn on day 21, followed by euthanasia. Plasma was separated from the collected blood, and antibody titers were measured.For antibody titer measurement, 100 μL / well of a DPBS solution containing 0.1 μg / mL of HBs protein (abcam) was added to a 96-well ELISA plate (IWAKI) to immobilize HBs. 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 to perform a color reaction. 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.

[0111] The results are shown in Figure 1. A significant increase in IgG antibody titers was observed when using STX7-pc-HBs compared to when using HBs mono-sequence sequences (antigen). Increases in IgG antibody titers were also observed with VAMP7-pc-HBs, GOSR1-pc-HBs, and SEC22B-pc-HBs. These results indicate that the fusion of specific SNAREs to the HBs antigen induces enhanced humoral immunity. 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. 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 IgG antibody titer measurements 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

[0136] to

[0137] and Figure 2(A), paragraphs

[0140] to

[0141] and Figure 4(A), paragraphs

[0148] to

[0149] and Figure 8(A), (E), etc., in Patent Document 1). The results indicate that the IgG antibody titers of these fusion polypeptides did not increase compared to the results of antigen-only polypeptides without SNARE protein fusion. On the other hand, the results of the ELISPOT assay (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., of Patent Document 1) indicate that the cellular immune response was significantly enhanced with these fusion polypeptides compared to antigen-only polypeptides without SNARE protein fusion. Therefore, the examples in Patent Document 1 showed that fusion polypeptides, which fuse specific SNARE proteins with antigen proteins, enhanced the cellular immune response compared to polypeptides of the antigen alone, while none of the fusion polypeptides increased IgG antibody titers. These results indicate that the increase in IgG antibody titers for STX7-pc-HBs, VAMP7-pc-HBs, GOSR1-pc-HBs, and SEC22B-pc-HBs in these examples is an unexpected result that would be difficult for even those skilled in the art to predict, and is also different from the description in Patent Document 1.

[0112] Example 2 ELISpot Assay The spleen collected in Example 1 was ground using a 40-μm cell strainer (AS ONE) while adding Stain Buffer (FBS). The obtained suspension was centrifuged at 250×g for 5 minutes and washed with Stain Buffer. 5×10 6 spleen cells were collected, suspended in 50 μL of CTL-Test Medium, and seeded onto an ELISpot plate. Further, 50 μL of Test Medium (C.T.L.) supplemented with HBs protein was added to perform antigen stimulation at a final concentration of 10 μg / mL. As a negative control, a group (N.T.) without added HBs protein 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 IL4 specifically produced against the antigen (HBs).

[0113] The results are shown in Figure 2. A significant increase in the number of spleen cells secreting IL4 was observed when using STX7-pc-HBs and SEC22B-pc-HBs, compared with the case of using the HBs single sequence (antigen), and an increasing trend was also observed with VAMP7-pc-HBs and GOSR1-pc-HBs. Since IL4 is known to induce antibody production, these results indicate that antibody production is promoted by the fusion of specific SNAREs to the HBs antigen.

[0114] Measurement of anti-HA1 IgG antibody titer in Example 3 We created mRNA encoding either the amino acid sequence of a fusion polypeptide (SEQ ID NO: 33) or the amino acid sequence of HA1 alone by linking the Influenza Hemagglutinin HA1 subunit antigen (HA1 antigen, amino acid sequence: SEQ ID NO: 32) as an antigen to the C-terminus of the human SNARE protein VAMP7 with a linker (nucleotide sequence: SEQ ID NO: 14, amino acid sequence: SEQ ID NO: 15) and a proprotein convertase recognition sequence (nucleotide sequence: SEQ ID NO: 16, amino acid sequence: SEQ ID NO: 17). The nucleotide sequences encoding the antigen and fusion polypeptide were codon-optimized before being introduced into plasmid DNA (pVAX1 vector, Thermo Fisher Scientific). The nucleotide sequences of the plasmids into which each polypeptide was introduced are shown in SEQ ID NOs: 34 and 35. The sequence of the HA1 antigen before codon optimization is shown in SEQ ID NO: 36. Each of the prepared vectors, along with 50 μg of a pVAX1 vector without the antigen sequence (negative control, nucleotide sequence: SEQ ID NO: 37), was intramuscularly administered into the thigh muscle of healthy female BALB / c mice. Gene transfer was then performed by applying voltage using an electroporator (NEPA21, Neppageen Co., Ltd.). After performing the above procedure on days 0, 7, and 14 from the start of the experiment, the spleen and blood were collected on day 28, and the mice were euthanized. Serum was separated from the collected blood, and antibody titer was measured. For antibody titer measurement, 100 μL / well of a DPBS solution containing 1 μg / mL of HA1 recombinant protein (Cosmo Bio Co., Ltd.) was added to a 96-well ELISA plate to immobilize HA1. 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 0.5 μg / mL of anti-Mouse IgG was added. After standing for 1 hour and washing, 50 μL / well of TMB solution was added and a color reaction was performed. After 10 minutes, 50 μL / well of Stop solution was added to stop the color reaction, and absorbance was measured (450 nm).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.

[0115] The results are shown in Figure 3. When VAMP7-pc-HA1 (V7-pc-HA1) was used, a significant increase in IgG antibody titers was observed compared to the HA1 antigen mono-sequence (HA1). A significant difference was also observed when compared to the negative control. These results indicate that the fusion of a specific SNARE to the HA1 antigen induces enhancement of humoral immunity. 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.

[0116] Example 4 ELISpot Assay The spleen collected in Example 3 was ground using a 40 μm cell strainer while adding Stain Buffer (FBS). The resulting suspension was centrifuged at 250 × g for 5 minutes, the supernatant was removed, and 2 mL of Pharm Lyse™ solution (BD) was added and gently vortexed. The suspension was then allowed to stand at room temperature for 3 minutes to induce hemolysis. After hemolysis, the suspension was centrifuged again and washed with Stain Buffer. 5 × 10 6 Individual splenocytes were isolated, suspended in 50 μL of CTL-Test Medium, and seeded onto an ELISPOT plate. Further antigen stimulation was performed by adding 50 μL of Test Medium containing recombinant HA1 protein (Cosmo Bio Co., Ltd.) to achieve a final concentration of 10 μg / mL. A negative control group (NT) without HA1 protein was prepared. After 24 hours, the ELISPOT assay was performed using the ELISPOT assay kit according to the provided protocol to detect IL4 specifically produced by the antigen (HA1).

[0117] The results are shown in Figure 4. An increasing trend in the number of IL4-secreting cells was observed due to the fusion of the VAMP7 sequence with the HA1 antigen.

Claims

1. A nucleic acid construct comprising a polynucleotide encoding one of the SNARE proteins selected from the group consisting of VAMP7, STX7, GOSR1, and SEC22B, and a polynucleotide encoding an enveloped virus surface protein antigen.

2. The nucleic acid construct according to claim 1, wherein the enveloped virus is hepatitis B virus or influenza virus.

3. The nucleic acid construct according to claim 1, wherein the surface protein antigen of the enveloped virus is the HBs antigen of hepatitis B virus or the HA antigen of influenza virus.

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

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

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

7. An agent for inducing or enhancing an enveloped virus surface protein antigen-specific humoral immune response, comprising a nucleic acid construct according to any one of claims 1 to 6 as an active ingredient.

8. An agent for inducing or enhancing the production of surface protein antigen-specific antibodies of enveloped viruses, comprising a nucleic acid construct according to any one of claims 1 to 6 as an active ingredient.

9. A prophylactic or therapeutic agent for enveloped virus infections comprising a nucleic acid construct according to any one of claims 1 to 6 as an active ingredient.

10. A nucleic acid vaccine comprising a nucleic acid construct according to any one of claims 1 to 6 as an active ingredient.

11. The nucleic acid vaccine according to claim 10, which induces or enhances an antigen-specific humoral immune response to the surface protein of an enveloped virus.

12. The nucleic acid vaccine according to claim 10, which induces or enhances the production of surface protein antigen-specific antibodies of enveloped viruses.

13. The nucleic acid vaccine according to claim 10, which is a vaccine for the prevention or treatment of enveloped virus infections.

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