Lipid complex

Abstract

[Problem] A composition and a method that can be used to induce an immune response to HTLV-1 are needed. [Solution] The present invention relates to a lipid complex in which at least one nucleic acid selected from a nucleic acid containing a polynucleotide that encodes an immunogenic fragment of a human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein, a nucleic acid containing a polynucleotide that encodes an immunogenic fragment of an HTLV-1 antigenic Tax protein, and a nucleic acid containing a polynucleotide that encodes an immunogenic fragment an HTLV-1 antigenic HBZ protein is encapsulated in a lipid.

Description

lipid complex

[0001] The present disclosure relates to a lipid complex in which a nucleic acid is encapsulated in a lipid, and a pharmaceutical composition comprising the same. In particular, the present disclosure relates to a lipid complex that can be used to induce an immune response against HTLV-1, and a pharmaceutical composition comprising the same.

[0002] The retrovirus human T-cell leukemia virus type-I (HTLV-1) is known to cause diseases such as adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated inflammatory diseases, such as HTLV-1-associated myelopathy (HAM) and HTLV-1-associated uveitis (HU). These HTLV-1-associated diseases develop in HTLV-1-infected individuals (carriers). It is estimated that there are approximately 800,000 HTLV-1-infected individuals (carriers) in Japan. Furthermore, HTLV-1 infection rarely causes any noticeable symptoms.

[0003] Because ATL is resistant to treatment and has a poor prognosis, prevention of HTLV-1 infection is considered important for preventing the onset of ATL. Furthermore, ATL patients who have undergone hematopoietic stem cell transplantation may experience a relapse of ATL after transplantation. For these reasons, inducing an immune response against HTLV-1 is also important for improving the prognosis of ATL treatment. However, effective treatments and prevention methods for these HTLV-1-related diseases have not yet been established.

[0004] Cook, Lucy B et al. “Revised Adult T-Cell Leukemia-Lymphoma International Consensus Meeting Report.” Journal of clinical oncology : official journal of the American Society of Clinical Oncology vol. 37,8 (2019): 677-687. doi:10.1200 / JCO.18.00501

[0005] There is a need for compositions and methods that can be used to induce an immune response against HTLV-1.

[0006] The present disclosure is, for example, as follows.

[0007] [1] A lipid complex in which at least one type of nucleic acid selected from the group consisting of a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an antigenic Gag protein of human T-cell leukemia virus 1 (HTLV-1), a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an antigenic Tax protein of HTLV-1, and a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an antigenic HBZ protein of HTLV-1 is encapsulated in a lipid. [2] The lipid complex of [1], wherein the nucleic acid is at least one selected from: a nucleic acid comprising a polynucleotide encoding HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding HTLV-1 antigenic Gag protein p19 (Gag p19) or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding HTLV-1 antigenic Gag protein p24 (Gag p24) or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding HTLV-1 antigenic Tax protein or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding HTLV-1 antigenic HBZ protein or an immunogenic fragment thereof. [3] The lipid complex of [1] or [2], wherein the lipid comprises a cationic lipid and at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol. [4] The nucleic acid comprises a nucleic acid comprising a polynucleotide encoding at least one selected from the group consisting of Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof. The lipid complex according to any one of [1] to [3].[5] The nucleic acid is: (i) at least one amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 8, 11 to 14; (ii) an amino acid sequence having at least 80% homology to at least one amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 8, 11 to 14; (iii) an amino acid sequence in which 1 to 31 amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NOs: 1, 4, 7, 8, 11 to 14; (iv) an amino acid sequence comprising at least one amino acid sequence selected from SEQ ID NOs: 11 or 12, SEQ ID NO: 13, or SEQ ID NO: 14, and having at least 80% homology to SEQ ID NO: 1; (v) an amino acid sequence comprising at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1, the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1, or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1; (vi) an amino acid sequence comprising at least one of the amino acid sequences of positions 1 to 59 of SEQ ID NO: 1, the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1, or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1, and having at least 80% homology to SEQ ID NO: 1; (vii) an amino acid sequence comprising at least one of the amino acid sequences of positions 151 to 208 of SEQ ID NO: 4, or the amino acid sequence of positions 251 to 308 of SEQ ID NO: 4; (viii) an amino acid sequence comprising at least one of the amino acid sequences of positions 151 to 208 of SEQ ID NO: 4, or the amino acid sequence of positions 251 to 308 of SEQ ID NO: 4, and having at least 80% homology to SEQ ID NO: 4; (ix) an amino acid sequence of positions 1 to 58 of SEQ ID NO: 8; or (x) an amino acid sequence comprising the amino acid sequence of positions 1 to 58 of SEQ ID NO: 8, and having at least 80% homology to SEQ ID NO: 8. The lipid complex according to any one of [1] to [4], which is a nucleic acid comprising a polynucleotide encoding an amino acid sequence. [6] The lipid complex according to any one of [1] to [5], wherein the nucleic acid is a nucleic acid comprising a polynucleotide encoding an antigenic Gag protein.

[0008] [7] The lipid complex according to any one of [1] to [6], wherein the lipid complex is a lipid nanoparticle (LNP). [8] The lipid complex according to any one of [1] to [7], wherein the nucleic acid is mRNA. [9] The nucleic acid is: (i) a polynucleotide consisting of at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 9, 10, and 15 to 17; (ii) a polynucleotide consisting of a nucleotide sequence in which 1 to 94 nucleotides have been deleted, inserted, substituted, and / or added in the nucleotide sequence of (i); (iii) a polynucleotide consisting of a nucleotide sequence having 80% or more identity to the nucleotide sequence of (i); and (iv) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of (i); (v) a polynucleotide consisting of at least one nucleotide sequence selected from the nucleotide sequence of bases 1 to 177 of SEQ ID NO: 3, the nucleotide sequence of bases 154 to 327 of SEQ ID NO: 3, or the nucleotide sequence of bases 304 to 390 of SEQ ID NO: 3; (vi) a polynucleotide comprising at least one of the nucleotide sequence of positions 1 to 177 of SEQ ID NO: 3, the nucleotide sequence of positions 154 to 327 of SEQ ID NO: 3, or the nucleotide sequence of positions 304 to 390 of SEQ ID NO: 3, and having 80% or more identity to the nucleotide sequence of SEQ ID NO: 3; (vii) a polynucleotide comprising at least one of the nucleotide sequence of positions 451 to 624 of SEQ ID NO: 6 or the nucleotide sequence of positions 751 to 924 of SEQ ID NO: 6; (viii) a polynucleotide comprising at least one of the nucleotide sequence of positions 451 to 624 of SEQ ID NO: 6 or the nucleotide sequence of positions 751 to 924 of SEQ ID NO: 6, and having 80% or more identity to the nucleotide sequence of SEQ ID NO: 6; (ix) a polynucleotide comprising the nucleotide sequence of positions 1 to 174 of SEQ ID NO: 10; (x) a polynucleotide comprising the nucleotide sequence of positions 1 to 174 of SEQ ID NO: 10, and having 80% or more identity to the nucleotide sequence of SEQ ID NO: 10;

[10] The lipid complex according to [8], wherein the cationic lipid is a nucleic acid comprising at least one selected from the following formula (I): [In the formula, L １ and L ２each independently represents an alkylene group having 3 to 10 carbon atoms; R １ and R ２ each independently represents an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms; X １ represents a single bond or —CO—O—, and ring P represents any one of the following formulae (P-1) to (P-6). [In the formula, R ３ represents an alkyl group having 1 to 3 carbon atoms.] or a pharmaceutically acceptable salt thereof.

[11] The lipid complex according to any one of [1] to [9], wherein the cationic lipid is represented by the following formula (II): 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate or a pharmaceutically acceptable salt thereof, [1] to

[10] . The lipid complex according to any one of the above.

[0009]

[12] A pharmaceutical composition comprising the lipid complex of any one of [1] to

[11] .

[13] The pharmaceutical composition of

[12] , wherein the pharmaceutical composition is a vaccine.

[14] The pharmaceutical composition of any one of

[11] or

[12] , for use in the prevention and / or treatment of an HTLV-1-associated disease.

[15] Use of the lipid complex of any one of [1] to

[11] for the production of a pharmaceutical composition.

[0010]

[16] A method for preventing and / or treating an HTLV-1-associated disease, comprising administering to a subject the pharmaceutical composition of

[12] or

[13] .

[17] Use of the lipid complex of any of [1] to

[11] or the pharmaceutical composition of

[12] or

[13] for use in the prevention and / or treatment of an HTLV-1-associated disease.

[0011] According to the present disclosure, lipid complexes and pharmaceutical compositions capable of inducing an immune response against HTLV-1 are provided. Because the pharmaceutical compositions of the present disclosure can induce an immune response against HTLV-1, they can be used for the prevention and / or treatment of HTLV-1-associated diseases. In some embodiments, the pharmaceutical compositions of the present disclosure can be used as mRNA vaccines for the prevention of HTLV-1-associated diseases. In some embodiments, the lipid complexes (particularly lipid nanoparticles) of the present disclosure have excellent stability.

[0012] FIG. 1 is a graph showing the results of the ELISPOT assay in Example 2. FIG. 1 shows the results showing the number of spots in each peptide pool. FIG. 2 is a graph showing the results of the ELISPOT assay in Example 2. FIG. 2 shows the results showing the number of spots in each peptide pool. FIG. 3 is a graph showing the results of the ELISPOT assay in Example 2. FIG. 3 shows the results showing the number of spots in each peptide pool. FIG. 4 is a graph showing the results of the LIPS assay in Reference Example 1. FIG. 5 is a graph showing the results of the RT-qPCR in Reference Example 1. FIG. 6 is a photograph showing the results of the proximity ligation assay in Reference Example 1. FIG. 7 is a photograph showing the results of the ELISPOT assay in Reference Example 1. FIG. 8 is a photograph showing the results of the ELISPOT assay in Reference Example 1.

[0013] The present disclosure will be described in detail below with reference to embodiments and examples, but the present disclosure is not limited to the embodiments and examples shown below and can be modified as desired without departing from the gist of the present disclosure. All documents and publications described in this specification are incorporated herein by reference in their entirety, regardless of their purpose.

[0014] <Terminology> The meanings of terms used in this specification will be explained below, and the present disclosure will be described in detail.

[0015] "Cationic lipid" refers to an amphipathic molecule having a lipophilic region containing one or more hydrocarbon groups and a hydrophilic region containing polar groups that protonate at a specific pH. "Neutral lipid" refers to a lipid that exists at physiological pH in either an uncharged or neutral zwitterionic form. "Polyethylene glycol-modified lipid" (PEG lipid) refers to a lipid containing a polyethylene glycol (PEG) group. "Sterol" refers to an alcohol with a steroid backbone.

[0016] "Alkyl" means a linear, cyclic, or branched saturated aliphatic hydrocarbon group having the specified number of carbon atoms. "Alkenyl" means a linear or branched hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond. Examples include, but are not limited to, monoenes, dienes, trienes, and tetraenes. "Alkylene" means a linear, cyclic, or branched divalent saturated aliphatic hydrocarbon group having the specified number of carbon atoms.

[0017] A "lipid complex" refers to an assembly containing multiple lipids physically bound to each other by intermolecular forces. Typically, a "lipid complex" is a "lipid particle." A "lipid particle" is a particle consisting of an assembly of lipid molecules. A "particle" typically has an average particle size of nano- to micron-size (1 nm to 1 μm or um), and is called a nanosphere, microsphere, nanocapsule, microcapsule, etc. Particles can be, for example, a dispersed phase in an emulsion or an internal phase in a suspension. Typically, a "lipid particle" is a microparticle consisting of a membrane-like molecular assembly formed by the association of lipids. A "lipid nanoparticle" (LNP) refers to a lipid particle having an average particle size of nano-size (e.g., about 1 nm to 1000 nm).

[0018] "Protein" refers to a polymer of peptides composed of unmodified (naturally occurring), modified, and / or artificial amino acids. The polymer may be linear, branched, or cyclic. The protein may also be referred to as a peptide or polypeptide.

[0019] "Retrovirus" refers to a type of RNA virus that possesses reverse transcriptase. Retroviruses are known to infect and replicate in host cells as follows: Retroviruses infect target host cells, synthesize double-stranded DNA by reverse transcription of viral RNA, and integrate the viral DNA as a provirus into the genomic DNA of the host cell. The host cell then produces viral RNA and viral proteins, and retroviral protease cleaves the immature viral proteins, allowing the mature viral proteins to assemble into viral particles. The viral particles bud from the surface of the host cell.

[0020] "HTLV-1" refers to a type of retrovirus, human T-cell leukemia virus type 1. HTLV-1 is a virus that causes adult T-cell leukemia / lymphoma (ATL (or ATLL): Adult T-cell leukemia / lymphoma), HTLV-1 associated myelopathy (HAM: HTLV-1 associated myelopathy, TSP: Tropical spastic paraparesis), and HTLV-1 uveitis (HU: HTLV-1 HTLV-1 is known as a virus that causes diseases such as uveitis. HTLV-1 is a virus belonging to the Oncovirinae subfamily of the Retroviridae family. HTLV-1 has viral structural genes such as Gag, Pol, and Env. HTLV-1 also has regulatory genes such as Tax and Rex. HTLV-1 also has accessory genes such as p12, p13, p30, and HBZ. An example of the HTLV-1 provirus is a protein consisting of the amino acid sequence registered in Genbank under accession number AB513134.1.

[0021] "HTLV-1 associated disease" is a general term for diseases caused by HTLV-1 infection. Examples of "HTLV-1 associated disease" include 1) ATL, 2) HAM, and 3) HU. "ATL" or "ATLL" refers to adult T-cell leukemia / lymphoma. It is known that in HTLV-1-infected individuals (carriers), HTLV-1 infects CD4+ T cells and the like, causing the infected cells to become cancerous, resulting in the development of ATL. "HAM" refers to HTLV-1 associated myelopathy (TSP: tropical spastic paraparesis). "HU" refers to HTLV-1 uveitis or HTLV-1 associated uveitis.

[0022] "Processing" refers to a reaction in which a polypeptide translated as a precursor is cleaved or modified by a protease or the like to be processed into a more mature protein. "Gag protein (hereinafter also referred to as Gag)" refers to a retroviral capsid precursor protein. Gag is known to play a role in the assembly and budding of virus particles. Gag is processed by a retroviral protease. HTLV-1 Gag is processed, for example, by a protease into p15 (nucleocapsid protein), p19 (matrix protein), p24 (capsid protein), and the like. An example of the HTLV-1 Gag is a protein (SEQ ID NO: 1) consisting of the amino acid sequence registered in Genbank under accession number AAA85841.1.

[0023] Amino acid sequence of HTLV-1 Gag (SEQ ID NO: 1): MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVLPVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAET RGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHT NSPLGDMLRACQTWTPKDKTKVLVVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNSIGGEV

[0024] "Tax protein (hereinafter also referred to as Tax)" refers to a transcriptional activator that activates both the long terminal repeat (LTR) and intracellular promoter of a retrovirus. Tax is known to play an important role in viral infectivity and canceration of infected cells. An example of the HTLV-1 TAX is a protein consisting of the amino acid sequence registered in Genbank under accession number P03409.2 (SEQ ID NO: 4).

[0025] Amino acid sequence of HTLV-1 TAX (SEQ ID NO: 4) MAHFPGFGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0026] "HBZ protein (hereinafter also referred to as HBZ)" refers to a retroviral bZIP factor. HBZ is known to contribute to the transcriptional regulation of viral RNA. An example of the HTLV-1 HBZ is a protein consisting of the amino acid sequence registered in Genbank under accession number BAE06226.1 (SEQ ID NO: 7). The HTLV-1 HBZ may also be a protein consisting of the amino acid sequence represented by SEQ ID NO: 8.

[0027] Amino acid sequence of HTLV-1 HBZ (SEQ ID NO: 7) MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQTAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ

[0028] Amino acid sequence of HTLV-1 HBZ (SEQ ID NO: 8) MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQIAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ

[0029] "T cells" refer to a type of white blood cell, classified as lymphocytes, that express a T cell receptor (TCR). Known examples of T cells include CD4+ helper T cells and CD8+ cytotoxic T cells.

[0030] "Antigenicity" refers to the property of an antigen to induce immune responses such as antibody production and cellular immunity.

[0031] "Immunogenic" refers to the property of an antigen to induce an immune response, such as the production of antibodies or cell-mediated immunity. "Immunogenic fragment," also referred to as an immunogenic portion, refers to a fragment or truncation of a protein or polypeptide that induces an immune response. "Vaccine" refers to a composition that generates an immune response for the prevention and / or treatment of a disease or condition. Thus, a vaccine is a pharmaceutical product containing an immunogenic agent and intended for use in humans or animals to generate specific defense and protection by vaccination.

[0032] The term "pharmaceutically acceptable carrier" refers to a solvent and / or additive that can be commonly used in the formulation technology of pharmaceutical compositions. Preferably, the pharmaceutically acceptable carrier is one that is almost or completely non-toxic to living organisms.

[0033] "Nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "nucleic acid" is used in conjunction with a specific protein herein, the "nucleic acid" refers to a polymer of nucleotides encoding the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA. The nucleic acid may be, for example, single-stranded or double-stranded. The nucleic acid can be interchangeably referred to as "polynucleotide" or "oligonucleotide." In the present disclosure, when the nucleic acid is DNA, the base sequence of the DNA can be, for example, the same as in the examples of the base sequences of RNA polynucleotides shown in the respective SEQ ID NOs described below, with uracil (u) replaced with thymine (t).

[0034] "Treatment" means reversing, alleviating, delaying the onset, or inhibiting the progression of a disease described herein. "Prevention" means reducing the likelihood of onset of a disease or pathological condition, suppressing, delaying, or stopping the onset of a disease or pathological condition, suppressing, alleviating, delaying, or stopping the progression of a pathological condition, suppressing, reducing, delaying, or stopping the worsening of a condition, or suppressing, delaying, or stopping the recurrence of a disease or pathological condition. The "prevention" may be, for example, treatment of a subject (patient) who develops a target disease, or treatment of an animal model of the target disease. "Aggravation" means an exacerbation of the degree (severity) of a disease or pathological condition.

[0035] "Isolated" means identified and separated and / or recovered from components of its natural state. The "isolation" can be achieved, for example, by at least one purification step.

[0036] The upper and lower limit values ​​of the numerical ranges described herein can be combined in any way. For example, if "A to B" and "C to D" are described, the ranges "A to D" and "C to B" are also included as numerical ranges in the present disclosure. Furthermore, a numerical range described herein, "from the lower limit value to the upper limit value," means a range that is equal to or greater than the lower limit value and equal to or less than the upper limit value. In this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B." In this specification, numerical values ​​used to indicate component contents, numerical ranges, etc. should be understood to be modified by the term "about" unless otherwise specified.

[0037] 1. Lipid Complex One aspect of the present disclosure relates to a lipid complex (hereinafter also referred to as the "lipid complex of the present disclosure" or "lipid complex") in which at least one type of nucleic acid selected from a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein, a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a HTLV-1 antigenic Tax protein, and a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of a HTLV-1 antigenic HBZ protein is encapsulated in a lipid.

[0038] (Nucleic Acid) The lipid complex of the present disclosure may contain, for example, any one, more than one, or all of the following: a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of Gag; a nucleic acid comprising a polynucleotide encoding Tax or an immunogenic fragment thereof; or a nucleic acid comprising a polynucleotide encoding HBZ or an immunogenic fragment thereof.

[0039] The lipid complexes of the present disclosure are characterized by comprising a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic Gag protein, a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Tax protein or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic HBZ protein or an immunogenic fragment thereof. Because the lipid complexes of the present disclosure comprise these nucleic acids, when administered to a subject, they can induce an immune response against HTLV-1, particularly an immune response against HTLV-1 Gag, Tax, and / or HBZ. Therefore, the lipid complexes of the present disclosure can be used to induce an immune response against HTLV-1. Furthermore, the lipid complexes of the present disclosure can be used for the prevention and / or treatment of HTLV-1-associated diseases.

[0040] In some embodiments, the nucleic acid is at least one selected from the following: a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Gag protein p19 (Gag p19) or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Gag protein p24 (Gag p24) or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Tax protein or an immunogenic fragment thereof, a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic HBZ protein or an immunogenic fragment thereof.

[0041] In some embodiments, the nucleic acid comprises a nucleic acid comprising a polynucleotide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof.

[0042] A nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both. Furthermore, the nucleic acid may be composed of natural nucleic acid residues, unnatural nucleic acid residues, or both. Specific examples of the nucleic acid include DNA, RNA, and / or DNA / RNA composed of natural and / or unnatural nucleic acid residues. Examples of the unnatural nucleic acid residue include modified nucleotide residues or modified ribonucleotide residues in which the base, sugar residue, or sugar phosphate backbone of the nucleotide residue is modified. When the sugar residue is modified, examples of the unnatural nucleic acid residue include cEt (constrained ethyl bicyclic nucleic acid, manufactured by Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), and ENA (registered trademark, 2'-O,4'-C-Ethylenebridged Nucleic Acid). The nucleic acid may have, for example, a 5' cap at the 5' end.

[0043] The nucleic acid may be a single-stranded or double-stranded nucleic acid molecule.

[0044] In the lipid complex, the nucleic acid containing the polynucleotide encoding each protein can be designed, for example, by substituting the corresponding codon based on the amino acid sequence of each protein. In the pharmaceutical composition of the present disclosure, the base sequence of the nucleic acid may be, for example, codon-optimized.

[0045] In the lipid complexes, various nucleic acids and proteins can be synthesized, for example, by genetic engineering techniques or organic synthesis techniques, and can also be referred to as synthetic DNA such as cDNA or synthetic RNA. In some embodiments, the nucleic acid can be RNA or DNA. In certain embodiments, the nucleic acid is mRNA. In the present disclosure, when the nucleic acid is mRNA, the nucleic acid may include a 5' cap structure, a polyA sequence, or the like at the end. The following describes nucleic acids that can be contained in the lipid complexes of the present disclosure.

[0046] (I) Nucleic Acid Comprising a Polynucleotide Encoding an Immunogenic Fragment of an HTLV-1 Antigenic Gag Protein The nucleic acid may be a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic Gag protein (Gag). The polynucleotide encoding the immunogenic fragment of Gag is a polynucleotide essentially comprising a region encoding the immunogenic fragment of Gag. The immunogenic fragment of Gag may be any protein as long as it is immunogenic to HTLV-1. The nucleic acid may comprise a polynucleotide encoding the immunogenic fragment of Gag. Alternatively, the nucleic acid may be composed of a polynucleotide encoding the immunogenic fragment of Gag. For example, when the nucleic acid is mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, etc. in addition to the polynucleotide encoding the immunogenic fragment of Gag. The immunogenic fragment of Gag may be, for example, at least 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 amino acids in length.

[0047] In some embodiments, the polynucleotide encoding an immunogenic fragment of Gag comprises a polynucleotide encoding at least one selected from HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and Gag protein p24 (Gag p24) or an immunogenic fragment thereof. That is, in some embodiments, the nucleic acid is a nucleic acid comprising a polynucleotide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof.

[0048] The nucleic acid may comprise a polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof. The nucleic acid may also be a polynucleotide encoding Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof. For example, when the nucleic acid is mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, etc. in addition to the polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof.

[0049] Furthermore, Gag p15, Gag p19, and Gag p24 may be further processed to a length of, for example, 50 amino acids or 9 amino acids. In some embodiments, the lipid membrane complex may encapsulate a nucleic acid comprising a polynucleotide encoding the 50 amino acids or 9 amino acids obtained by further processing Gag p15, Gag p19, or Gag p24.

[0050] (a) Gag p15 or an immunogenic fragment thereof Examples of the Gag p15 include the following proteins (a1), (a2), and (a3): (a1) a protein consisting of the amino acid sequence of SEQ ID NO: 11 or 12; (a2) a protein consisting of the amino acid sequence of SEQ ID NO: 11 or 12 in which one or several amino acids have been deleted, inserted, substituted, or added; and (a3) ​​a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 11 or 12.

[0051] Amino acid sequence of Gag p15 (SEQ ID NO: 11) VVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNLHRGGGLTSPPTLQQVLPNQDPASIL

[0052] Amino acid sequence of Gag p15 (SEQ ID NO: 12) VVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNSIGGEV

[0053] In (a1), the amino acid sequence shown in SEQ ID NO: 11 or 12 is an amino acid sequence derived from HTLV-1. Furthermore, in (a1), the amino acid sequence shown in SEQ ID NO: 11 is, for example, the sequence of endogenous mature Gag p15 that can be expressed in infected cells, which is generated when ribosomal frameshifting occurs. The amino acid sequence shown in SEQ ID NO: 12 is, for example, the sequence of Gag p15 that is generated when ribosomal frameshifting does not occur. The amino acid sequence of (a1) is preferably, for example, the amino acid sequence of SEQ ID NO: 12, which is predicted to be expressed in large amounts in vivo when made into a vaccine composition.

[0054] In the protein (a2), "one or several" may be within a range in which (a2) is a protein immunogenic against HTLV-1, for example. "One or several" in (a2) means, for example, 1 to 31, 1 to 26, 1 to 25, 1 to 21, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (a1). In the present disclosure, a numerical range of the number discloses, for example, all positive integers falling within that range. That is, for example, the description "1 to 5" means the disclosure of all of "1, 2, 3, 4, and 5" (the same applies hereinafter).

[0055] In the protein (a2), the substitution is preferably a conservative substitution. The conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Examples of the conservative substitution include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0056] With respect to the protein (a3), the "identity" may be within a range such that the protein (a3) ​​is a peptide immunogenic to HTLV-1. The "identity" of the protein (a3) ​​is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of the protein (a1). The "identity" can be calculated, for example, by using default parameters in the homology algorithm BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Center for Biotechnology Information (NCBI) (the same applies hereinafter).

[0057] (b) Gag p19 or an Immunogenic Fragment thereof The Gag p19 may be, for example, the following protein (b1), (b2), or (b3):

[0058] (b1) A protein consisting of the amino acid sequence of SEQ ID NO: 13. (b2) A protein consisting of the amino acid sequence of SEQ ID NO: 13 in which one or more amino acids have been deleted, inserted, substituted, or added. (b3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 13.

[0059] Amino acid sequence of Gag p19 (SEQ ID NO: 13) MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVL

[0060] In the protein (b2), "one or several" may be within a range in which the protein (b2) is immunogenic against HTLV-1. The "one or several" in (b2) may be, for example, 1 to 38, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (b1).

[0061] In the protein (b3), the "identity" may be within a range such that the protein (b3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (b3) to the amino acid sequence of the protein (b1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0062] (c) Gag p24 or an Immunogenic Fragment thereof Examples of the Gag p24 include the following proteins (c1), (c2), and (c3):

[0063] (c1) A protein consisting of the amino acid sequence of SEQ ID NO: 14. (c2) A protein consisting of the amino acid sequence of SEQ ID NO: 14 in which one or more amino acids have been deleted, inserted, substituted, or added. (c3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 14.

[0064] Amino acid sequence of Gag p24 (SEQ ID NO: 14) PVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAETRGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHTNSPLGDMLRACQTWTPKDKTKVL

[0065] In the protein of (c2), "one or several" may be within a range in which the protein of (c2) is immunogenic against HTLV-1. The "one or several" in (c2) may be, for example, 1 to 64, 1 to 53, 1 to 42, 1 to 32, 1 to 21, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (c1).

[0066] In the protein (c3), the "identity" may be within a range such that the protein (c3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (c3) to the amino acid sequence of the protein (c1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0067] The immunogenic fragment of p15, p19, or p24 may be, for example, any protein fragment that is 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0068] The lipid complexes of the present disclosure may comprise, for example, a nucleic acid comprising a polynucleotide encoding any one of the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof, or may comprise a nucleic acid comprising a polynucleotide encoding two of them, or may comprise a nucleic acid comprising a polynucleotide encoding all of them. For example, the lipid complexes of the present disclosure preferably comprise nucleic acids comprising polynucleotides encoding the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof. By using such a configuration, the lipid complexes can, for example, efficiently induce an immune response against HTLV-1.

[0069] (d) Gag or Immunogenic Fragment Thereof The Gag p15, Gag p19, and Gag p24 are processed Gag proteins. Therefore, the Gag protein or its immunogenic fragment can also be referred to as a precursor or precursor protein of, for example, Gag p15 or its immunogenic fragment, Gag p19 or its immunogenic fragment, and / or Gag p24 or its immunogenic fragment. In some embodiments, the lipid membrane complex may encapsulate a nucleic acid including a polynucleotide encoding a Gag protein or its immunogenic fragment, etc.

[0070] Examples of the Gag (protein before processing) include the following proteins (d1), (d2), and (d3): (d1) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (d2) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, inserted, substituted, or added; and (d3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 1.

[0071] In the protein (d2), "one or several" may be within a range in which the protein (d2) is immunogenic against HTLV-1, for example. The "one or several" in the (d2) may be, for example, 1 to 128, 1 to 107, 1 to 85, 1 to 64, 1 to 42, 1 to 31, 1 to 21, 1 to 14, 1 to 12, 1 to 8, 1 to 4, 1 or 2, or 1 in the amino acid sequence of the (d1).

[0072] The "identity" of the protein (d3) may be within a range such that the protein (d3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (d3) to the amino acid sequence of the protein (d1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0073] The immunogenic fragment of Gag may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1, and may be, for example, any protein fragment of the protein that is 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0074] (A) Polynucleotide Encoding Gag p15 The polynucleotide encoding Gag p15 is, for example, a polynucleotide selected from the group consisting of the following (A1) to (A6) and (A7): (A1) a polynucleotide consisting of the base sequence of SEQ ID NO: 15; (A2) a polynucleotide consisting of the base sequence of (A1) in which one or several bases have been deleted, inserted, substituted, and / or added; (A3) a polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (A1); (A4) a polynucleotide consisting of a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (A1) to (A3); (A5) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 11 or 12; (A6) a polynucleotide encoding a protein consisting of the amino acid sequence of (A5) in which one or several amino acids have been deleted, inserted, substituted, and / or added; (A7) a polynucleotide encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of (A5);

[0075] In the polynucleotide (A1), the nucleotide sequence of SEQ ID NO: 15 is as follows: The nucleotide sequence of SEQ ID NO: 15 is the coding sequence (mRNA) of Gag p15 consisting of the amino acid sequence of SEQ ID NO: 12. The polynucleotide (A1) of SEQ ID NO: 15 can be obtained from, for example, HTLV-1.

[0076] Polynucleotide of (A1) (SEQ ID NO: 15) UGGUGCAGCCCAAGAAGCCUCCUCCUAACCAGCCUUGCUUUCGCUGCGGCAAAGCUGGACACUGGAGCCGCGAUUGCACACAGCCUCGCCCUCCUCCUGGACCUUGCCCUCUUUGCCAGGAUCCUAC CCACUGGAAGCGCGACUGCCCUCGCCUGAAACCUACCAUCCCUGAACCUGAACCUGAAGAAGACGCUCUGCUGCUGGAUCUGCCCGCUGAUAUCCCCCACCCCAAGAACAGCAUCGGCGGCGAGGUG

[0077] When the nucleic acid is DNA, the polynucleotide encoding Gag p15 is, for example, SEQ ID NO: 15, in which u is replaced with t.

[0078] In (A2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (A2) has immunogenicity against HTLV-1. The "one or several" in (A2) may be, for example, 1 to 94, 1 to 78, 1 to 76, 1 to 63, 1 to 51, 1 to 47, 1 to 31, 1 to 25, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 7, 1 to 6, 1 to 5, 1 or 2, or 1 in the base sequence of (A1).

[0079] In (A3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (A3) has immunogenicity against HTLV-1. The "identity" in (A3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (A1).

[0080] In (A4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (A1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used.

[0081] In (A4), "stringent conditions" may be, for example, low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Those skilled in the art can set the degree of stringency by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" are, for example, those described in the aforementioned "Molecular Cloning: A Laboratory Manual 2nd Edition" edited by Sambrook et al. nd Ed.) (Cold Spring Harbor Laboratory Press (1989)) or the like can also be used.

[0082] The polynucleotide (A5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 11 or 12, by substituting the corresponding codons.

[0083] In (A6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (A6) has immunogenicity against HTLV-1. The "one or several" in (A6) may be, for example, 1 to 31, 1 to 26, 1 to 25, 1 to 21, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (A5).

[0084] In (A7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (A7) has immunogenicity against HTLV-1. The "identity" in (A7) to the amino acid sequence of (A5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0085] (B) Polynucleotide Encoding Gag p19 The polynucleotide encoding Gag p19 is, for example, a polynucleotide selected from the group consisting of the following (B1) to (B6) and (B7): (B1) A polynucleotide consisting of the base sequence of SEQ ID NO: 16; (B2) A polynucleotide consisting of the base sequence of (B1) in which one or several bases have been deleted, inserted, substituted, and / or added; (B3) A polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (B1); (B4) A polynucleotide consisting of a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (B1) to (B3); (B5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 13; (B6) A polynucleotide encoding a protein consisting of the amino acid sequence of (B5) in which one or several amino acids have been deleted, inserted, substituted, and / or added; (B7) A polynucleotide encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of (B5).

[0086] In (B1), the nucleotide sequence of SEQ ID NO: 16 is as follows: The nucleotide sequence of SEQ ID NO: 16 is the coding sequence (mRNA) of Gag p19 consisting of the amino acid sequence of SEQ ID NO: 13. The polynucleotide (B1) of SEQ ID NO: 16 can be obtained from, for example, HTLV-1.

[0087] Polynucleotide of (B1) (SEQ ID NO: 16) GGCCAGAUCUUCUCUCGCUCUGCUUCUCCUAUUCCUCGCCCUCCGCGGACUUGCUGCUCACCACUGGCUUAACUUCCUGCAGGCUGCUUACCGCCUGGAACCUGGCCCUAGCAGCUACGACUUCCACCAGCUGAAGAAGUUCCUGAAGAUCGCCCUGGAGACCCCCGUGGAUCUGCCCCAUCAACUACA GCCUGCUGGCUAGCCUGCUGCCUAAAGGCUACCCUGGCCGCGUGAACGAGAUCCUGCACAUCCUGAUCCAGACCCAGGCCCAGAUCCCUUCUCGCCCUGCUCCUCCUCCCUUCUUCUCCUACCCACGAUCCCCCCGAUAGCGAUCCCCAGAUCCCCCCUCCUUACGUGGAACCUACCGCUCCUCAGGUGCUG

[0088] When the nucleic acid is DNA, the polynucleotide encoding Gag p19 is, for example, SEQ ID NO: 16, in which u is replaced with t.

[0089] In (B2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (B2) has immunogenicity against HTLV-1. The "one or several" in (B2) may be, for example, 1 to 116, 1 to 96, 1 ​​to 94, 1 to 77, 1 to 58, 1 to 38, 1 to 19, 1 to 15, 1 to 11, 1 to 7, 1 to 3, 1 or 2, or 1 in the base sequence of (B1).

[0090] In (B3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (B3) has immunogenicity against HTLV-1. The "identity" in (B3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (B1).

[0091] In (B4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (B1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (B4), the stringent conditions can be the same as those described in (A4).

[0092] The polynucleotide (B5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 13, by substituting the corresponding codons.

[0093] In (B6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (B6) has immunogenicity against HTLV-1. The "one or several" in (B6) may be, for example, 1 to 38, 1 to 32, 1 to 31, 1 to 25, 1 to 19, 1 to 12, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (B5).

[0094] In (B7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (B7) has immunogenicity against HTLV-1. The "identity" in (B7) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of (B5).

[0095] (C) Polynucleotide Encoding Gag p24 The polynucleotide encoding Gag p24 is, for example, a polynucleotide selected from the group consisting of the following (C1) to (C6) and (C7): (C1) A polynucleotide consisting of the base sequence of SEQ ID NO: 17; (C2) A polynucleotide consisting of the base sequence of (C1) in which one or several bases have been deleted, inserted, substituted, and / or added; (C3) A polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (C1); (C4) A polynucleotide consisting of a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (C1) to (C3); (C5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14; (C6) A polynucleotide encoding a protein consisting of the amino acid sequence of (C5) in which one or several amino acids have been deleted, inserted, substituted, and / or added; (C7) A polynucleotide encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of (C5).

[0096] In the polynucleotide (C1), the nucleotide sequence of SEQ ID NO: 17 is as follows: The nucleotide sequence of SEQ ID NO: 17 is a coding sequence (mRNA) of Gag p24 consisting of the amino acid sequence of SEQ ID NO: 14. The polynucleotide (C1) of SEQ ID NO: 17 can be obtained from, for example, HTLV-1.

[0097] (C1) polynucleotide (SEQ ID NO: 17) CCUGUGAUGCACCCUCACGGCGCUCCUCCUAAUCACCGCCCUUGGCAGAUGAAGGACCUGCAGGCCAUCAAACAGGAAGUGAGCCAGGCUGCUCCCGGCAGCCCAGUUUUAUGCAGACCAUCCGCCUGGCUGUGCAGCAGUUCGAUCCCACCGCUAAAG AUCUGCAGGACCUGCUGCAGUACCUGUGCAGCAGCCUGGUGGCUAGCCUGCACCACCAGCAGCUGGAUAGCCUGAUCAGCGAAGCCGAAACCCGCGCAUCACCGGCUACAAUCCUCUGGCUGGCCCUCUGCGCGUGCAGGCUAACAACCCUCAGCAGCAG GGACUGCGCCGCGAAUACCAGCAGCUGUGGCUGGCUGCUUUUGCUGCUCUGCCCGGCAGCGCUAAAGAUCCCAGCUGGGCUAGCAUCCUGCAGGGCCUGGAAGAACCCUACCACGCCUUCGUGGAGCGCCUGAACAUCGCCCUGGACAACGGCCUGCCUGA AGGCACCCCUAAAGAUCCUAUCCUGCGCAGCCUGGCCUACAGCAACGCCAACAAGGAGUGCCAGAAGCUGCUGCAGGCUCGCGGACACACCAAUAGCCCUCUGGGAGAUAUGCUGCGCGCUUGCCAGACCUGGACCCCCAAGGACAAGACCAAGGUGCUGG

[0098] When the nucleic acid is DNA, the polynucleotide encoding Gag p24 is, for example, SEQ ID NO: 17, in which u is replaced with t.

[0099] In (C2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (C2) has immunogenicity against HTLV-1. The "one or several" in (C2) may be, for example, 1 to 192, 1 to 160, 1 to 128, 1 to 96, 1 ​​to 94, 1 to 64, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, or 1 or 2 in the base sequence of (C1).

[0100] In (C3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (C3) has immunogenicity against HTLV-1. The "identity" in (C3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (C1).

[0101] In (C4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (C1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (C4), the stringent conditions can be the same as those described in (A4).

[0102] The polynucleotide (C5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 14, by substituting the corresponding codons.

[0103] In (C6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (C6) has immunogenicity against HTLV-1. The "one or several" in (C6) may be, for example, 1 to 64, 1 to 53, 1 to 42, 1 to 32, 1 to 31, 1 to 21, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (C5).

[0104] In (C7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (C7) has immunogenicity against HTLV-1. The "identity" in (C7) with respect to the amino acid sequence of (C5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0105] (Polynucleotides Encoding Immunogenic Fragments of Gag p15, p19, or p24) The polynucleotides encoding the immunogenic fragments of Gag p15, p19, or p24 may be any polynucleotides as long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1.

[0106] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1 (p19-3 in the Examples described below), and more preferably encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below).

[0107] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of positions 52 to 109 (p19-2 in the Examples described below), or the amino acid sequence of positions 102 to 130 (p19-3 in the Examples described below), and it is preferable that the region other than the polynucleotide consists of an amino acid sequence other than the polynucleotide in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the polynucleotide in SEQ ID NO: 1. More preferably, it encodes at least one of the amino acid sequence of amino acids 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) or the amino acid sequence of amino acids 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), and the region other than the polynucleotide preferably consists of an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence of SEQ ID NO: 1 other than the polynucleotide, or the amino acid sequence of SEQ ID NO: 1 other than the polynucleotide.

[0108] The nucleic acid encapsulated in the lipid complex of the present disclosure is preferably a polynucleotide encoding at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1 (p19-3 in the Examples described below), and more preferably a polynucleotide encoding at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below).

[0109] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably comprises at least one of the nucleotide sequences of bases 1 to 177 of SEQ ID NO: 3 (polynucleotide encoding p19-1 in the Examples described below), bases 154 to 327 of SEQ ID NO: 3 (polynucleotide encoding p19-2 in the Examples described below), or bases 304 to 390 of SEQ ID NO: 3 (polynucleotide encoding p19-3 in the Examples described below). More preferably, the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably comprises at least one of the nucleotide sequences of bases 1 to 177 of SEQ ID NO: 3 (polynucleotide encoding p19-1 in the Examples described below), or bases 154 to 327 of SEQ ID NO: 3 (polynucleotide encoding p19-2 in the Examples described below).

[0110] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is, in particular, a polynucleotide consisting of at least one of the nucleotide sequences of bases 1 to 177 of SEQ ID NO: 3 (a polynucleotide encoding p19-1 in the Examples described below), bases 154 to 327 of SEQ ID NO: 3 (a polynucleotide encoding p19-2 in the Examples described below), or bases 304 to 390 of SEQ ID NO: 3 (a polynucleotide encoding p19-3 in the Examples described below), and the region of the nucleic acid other than the polynucleotide is preferably a nucleotide sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence other than the polynucleotide of SEQ ID NO: 3. More preferably, the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is at least one of the base sequences of bases 1 to 177 of SEQ ID NO: 3 (the polynucleotide encoding p19-1 in the Examples described below) or base sequences of bases 154 to 327 of SEQ ID NO: 3 (the polynucleotide encoding p19-2 in the Examples described below), and the region of the nucleic acid other than the polynucleotide is preferably a base sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence other than the polynucleotide of SEQ ID NO: 3 or the base sequence other than the polynucleotide of SEQ ID NO: 3.

[0111] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1 (p19-3 in the Examples described below), and the region of the nucleic acid other than the polynucleotide preferably consists of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 1. More preferably, the polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure encodes at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), and the region of the nucleic acid other than the polynucleotide preferably consists of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 1.

[0112] In some embodiments, the lipid complexes of the present disclosure may comprise a nucleic acid comprising a polynucleotide encoding any one, two, or all of the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof. In certain embodiments, the lipid complexes of the present disclosure preferably comprise, for example, a nucleic acid comprising a polynucleotide encoding the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and a nucleic acid comprising a polynucleotide encoding the Gag p24 or an immunogenic fragment thereof. By employing such a configuration, the lipid complexes of the present disclosure can, for example, efficiently induce an immune response against HTLV-1.

[0113] (D) Polynucleotide Encoding Gag Examples of nucleic acids encoding Gag (protein before processing) include nucleic acids containing a polynucleotide selected from the group consisting of (D1) to (D6) and (D7) below. (D1) A polynucleotide consisting of the base sequence of SEQ ID NO: 2 or 3; (D2) A polynucleotide consisting of the base sequence of (D1) in which one or several bases have been deleted, inserted, substituted, and / or added; (D3) A polynucleotide consisting of a base sequence that is 80% or more identical to any of the base sequences of (D1); (D4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the base sequence of any of (D1) to (D3); (D5) A polynucleotide that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 1; (D6) A polynucleotide that encodes a protein consisting of the amino acid sequence of (D5) in which one or several amino acids have been deleted, inserted, substituted, and / or added; (D7) A polynucleotide that encodes a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (D5).

[0114] In (D1), the nucleotide sequences of SEQ ID NOs: 2 and 3 are as follows: The nucleotide sequences of SEQ ID NOs: 2 and 3 are coding sequences (mRNA) of unprocessed Gag consisting of the amino acid sequence of SEQ ID NO: 1. The polynucleotides (D1) of SEQ ID NOs: 2 and 3 can be obtained from, for example, HTLV-1.

[0115]

[0116] In (D2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (D2) has immunogenicity against HTLV-1. The "one or several" in (D2) may be, for example, 1 to 387, 1 to 322, 1 to 258, 1 to 192, 1 to 129, 1 to 94, 1 to 64, 1 to 51, 1 to 42, 1 to 38, 1 to 25, 1 to 12, 1 to 6, 1 to 3, 1 or 2, or 1 in the base sequence of (D1).

[0117] In (D3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (D3) has immunogenicity against HTLV-1. The "identity" in (D3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (D1).

[0118] In (D4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (D1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (D4), the stringent conditions can be the same as those described in (A4).

[0119] The polynucleotide (D5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 1, by substituting the corresponding codons.

[0120] In (D6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (D6) has immunogenicity against HTLV-1. The "one or several" in (D6) may be, for example, 1 to 128, 1 to 107, 1 to 85, 1 to 64, 1 to 42, 1 to 21, 1 to 14, 1 to 12, 1 to 8, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (D5).

[0121] In (D7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (D7) has immunogenicity against HTLV-1. The "identity" of (D7) to the amino acid sequence of (D5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0122] (Polynucleotide Encoding Immunogenic Fragment of Gag) In the present disclosure, the polynucleotide of the immunogenic fragment of Gag may be any polynucleotide, as long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1, for example.

[0123] (II) Nucleic Acid Comprising a Polynucleotide Encoding an Immunogenic Fragment of HTLV-1 Antigenic Tax The nucleic acid may be a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of HTLV-1 antigenic Tax protein (Tax). The polynucleotide encoding the immunogenic fragment of Tax is a polynucleotide essentially comprising a region encoding the immunogenic fragment of Tax. The immunogenic fragment of Tax may be any protein that is immunogenic to HTLV-1. The nucleic acid may comprise a polynucleotide encoding Tax or an immunogenic fragment thereof. Alternatively, the nucleic acid may be a polynucleotide encoding Tax or an immunogenic fragment thereof. For example, when the nucleic acid is mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, etc. in addition to the polynucleotide encoding the immunogenic fragment of Tax. The immunogenic fragment of Tax can be, for example, at least 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0124] In some embodiments, the polynucleotide encoding an immunogenic fragment of Tax comprises a polynucleotide encoding Tax or an immunogenic fragment thereof, i.e., in some embodiments, the nucleic acid is a nucleic acid comprising a polynucleotide encoding Tax or an immunogenic fragment thereof.

[0125] (e) Tax or an immunological fragment thereof Examples of the Tax include the following proteins (e1), (e2), and (e3): (e1) a protein consisting of the amino acid sequence of SEQ ID NO: 4 (e2) a protein consisting of the amino acid sequence of SEQ ID NO: 4 in which one or several amino acids have been deleted, inserted, substituted, or added (e3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 4.

[0126] In the protein (e2), "one or several" may be within a range in which (e2) is a protein having immunogenicity against HTLV-1. The "one or several" in (e2) means, for example, 1 to 105, 1 to 88, 1 to 70, 1 to 52, 1 to 35, 1 to 17, 1 to 14, 1 to 10, 1 to 7, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (e1).

[0127] In the protein (e3), the "identity" may be within a range such that the protein (e3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (e3) to the amino acid sequence of the protein (e1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0128] In the present disclosure, the immunogenic fragment of Tax may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1, and may be, for example, any protein fragment of the protein that is 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0129] (E) Polynucleotide Encoding Tax The polynucleotide encoding Tax may be, for example, a polynucleotide selected from the group consisting of the following (E1) to (E6) and (E7): (E1) A polynucleotide consisting of the base sequence of SEQ ID NO: 5 or 6; (E2) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, inserted, substituted, and / or added in the base sequence of (E1); (E3) A polynucleotide consisting of a base sequence that is 80% or more identical to any of the base sequences of (E1); (E4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the base sequence of any of (E1) to (E3); (E5) A polynucleotide that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 4; (E6) A polynucleotide that encodes a protein consisting of the amino acid sequence of (E5) in which one or more amino acids have been deleted, inserted, substituted, and / or added; (E7) A polynucleotide that encodes a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (E5).

[0130] In (E1), the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 are as follows: The nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 are coding sequences (mRNA) of Tax consisting of the amino acid sequence of SEQ ID NO: 4. The polynucleotides (E1) of SEQ ID NO: 5 and SEQ ID NO: 6 can be obtained from, for example, HTLV-1.

[0131]

[0132] In (E2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (E2) has immunogenicity against HTLV-1. The "one or several" in (E2) may be, for example, 1 to 318, 1 to 265, 1 to 212, 1 to 159, 1 to 106, 1 to 53, 1 to 42, 1 to 31, 1 to 21, 1 to 10, 1 to 6, 1 or 2, or 1 in the base sequence of (E1).

[0133] In (E3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (E3) has immunogenicity against HTLV-1. The "identity" in (E3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (E1).

[0134] In (E4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (E1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (E4), the stringent conditions can be the same as those described in (A4).

[0135] The polynucleotide (E5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 4 by substituting the corresponding codons.

[0136] In (E6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (E6) has immunogenicity against HTLV-1. The "one or several" in (E6) may be, for example, 1 to 105, 1 to 88, 1 to 70, 1 to 52, 1 to 35, 1 to 31, 1 to 17, 1 to 14, 1 to 10, 1 to 7, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (E5).

[0137] In (E7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (E7) has immunogenicity against HTLV-1. The "identity" of (E7) to the amino acid sequence of (E5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0138] (Polynucleotide encoding an immunogenic fragment of Tax) The polynucleotide encoding the immunogenic fragment of Tax may be any polynucleotide, as long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1, for example.

[0139] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably encodes at least one of the amino acid sequence from 151 to 208 of SEQ ID NO: 4 (Tax-4 in the Examples described below) or the amino acid sequence from 251 to 308 of SEQ ID NO: 4 (Tax-6 in the Examples described below).

[0140] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes at least one of the amino acid sequences from 151 to 208 of SEQ ID NO: 4 (Tax-4 in the Examples described below) or the amino acid sequence from 251 to 308 of SEQ ID NO: 4 (Tax-6 in the Examples described below), and it is preferable that the region other than the polynucleotide consists of an amino acid sequence other than the polynucleotide in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the polynucleotide in SEQ ID NO: 4.

[0141] The nucleic acid encapsulated in the lipid complex of the present disclosure is preferably one that encodes at least one of the amino acid sequence from 151 to 208 of SEQ ID NO: 4 (Tax-4 in the Examples below) or the amino acid sequence from 251 to 308 of SEQ ID NO: 4 (Tax-6 in the Examples below).

[0142] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably contains at least one base sequence selected from the base sequence 451 to 624 of SEQ ID NO: 6 (the polynucleotide encoding Tax-4 in the Examples described below) and the base sequence 751 to 924 of SEQ ID NO: 6 (the polynucleotide encoding Tax-6 in the Examples described below).

[0143] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is, in particular, a polynucleotide consisting of at least one base sequence of bases 451 to 624 of SEQ ID NO: 6 (a polynucleotide encoding Tax-4 in the Examples described below) or bases 751 to 924 of SEQ ID NO: 6 (a polynucleotide encoding Tax-6 in the Examples described below), and the region other than the polynucleotide in the nucleic acid is preferably a base sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the base sequence other than the polynucleotide of SEQ ID NO: 6.

[0144] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes at least one of the amino acid sequence from 151 to 208 of SEQ ID NO: 4 (Tax-4 in the Examples described below) or the amino acid sequence from 251 to 308 of SEQ ID NO: 4 (Tax-6 in the Examples described below), and the region of the nucleic acid other than the polynucleotide preferably consists of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 4.

[0145] (III) Nucleic Acid Comprising a Polynucleotide Encoding an Immunogenic Fragment of an HTLV-1 Antigenic HBZ Protein The nucleic acid may be a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic HBZ protein (HBZ). The polynucleotide encoding the immunogenic fragment of HBZ is a polynucleotide essentially comprising a region encoding the immunogenic fragment of HBZ. The immunogenic fragment of HBZ may be any protein immunogenic to HTLV-1. The nucleic acid may comprise a polynucleotide encoding HBZ or an immunogenic fragment thereof. Alternatively, the nucleic acid may be a polynucleotide encoding HBZ or an immunogenic fragment thereof. For example, when the nucleic acid is mRNA, the nucleic acid may comprise a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, etc. in addition to the polynucleotide encoding the immunogenic fragment of HBZ. The HBZ immunogenic fragment may, for example, be at least 5, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 amino acids in length.

[0146] In some embodiments, the polynucleotide encoding an immunogenic fragment of HBZ comprises a polynucleotide encoding HBZ or an immunogenic fragment thereof, i.e., in some embodiments, the nucleic acid is a nucleic acid comprising a polynucleotide encoding HBZ or an immunogenic fragment thereof.

[0147] (f) HBZ or an immunological fragment thereof The HBZ can be, for example, the following protein (f1), (f2), or (f3): (f1) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 7 (f2) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 7 in which one or several amino acids have been deleted, inserted, substituted, or added (f3) a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 7 or 7.

[0148] In the protein (f2), "one or several" may be within a range in which the protein (f2) is immunogenic against HTLV-1. The "one or several" in the (f2) may be, for example, 1 to 62, 1 to 52, 1 to 41, 1 to 31, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 or 2 amino acids in the amino acid sequence of the (f1).

[0149] In the protein (f3), the "identity" may be within a range such that the protein (f3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (f3) to the amino acid sequence of the protein (f1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0150] The immunogenic fragment of HBZ may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1, and may be, for example, any protein fragment of the protein that is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0151] (F) Polynucleotide Encoding HBZ The polynucleotide encoding HBZ is, for example, a polynucleotide selected from the group consisting of (F1) to (F6) and (F7) below. (F1) A polynucleotide consisting of the base sequence of SEQ ID NO: 9 or 10; (F2) A polynucleotide consisting of the base sequence of (F1) in which one or several bases have been deleted, inserted, substituted, and / or added; (F3) A polynucleotide consisting of a base sequence that is 80% or more identical to the base sequence of any of (F1) to (F3); (F4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the base sequence of any of (F1); (F5) A polynucleotide that encodes a protein consisting of the amino acid sequence of SEQ ID NO: 7 or 8; (F6) A polynucleotide that encodes a protein consisting of the amino acid sequence of (F5) in which one or several amino acids have been deleted, inserted, substituted, and / or added; (F7) A polynucleotide that encodes a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (F5).

[0152] In (F1), the nucleotide sequences of SEQ ID NOs: 9 and 10 are as follows: The nucleotide sequences of SEQ ID NOs: 9 and 10 are coding sequences (mRNA) of HBZ consisting of the amino acid sequence of SEQ ID NO: 8. The polynucleotides (F1) of SEQ ID NOs: 9 and 10 can be obtained from, for example, HTLV-1.

[0153] (F1) Polynucleotide (SEQ ID NO: 9) AGGAAAUAAGAGAGAAAGAGUAAGAAAGAAAUAUAAAGACCCACAUGGCUGCUAGCGCCUGUUUCGCUGCCUGCCUGUGAGCUGCCUGAAGACCUGCUGGAGGAACUGGGAGGAACUGGGACGGCCUGUGACCGCGUGAGCCUGGAAGCAAGAAACUCUCGCGGACGCCUUGCUGCCUGGAGAGAAGAAUCUCGCGCUUCGCCGCGACCUUCCUGCCUGGACCUUGGAAGAAGAAUCUCGCGGCCUUCGCCUUGGAGAAAAAGCUCCUCUCUCUCUCUCUCUCUUAGGAGGAAAAACACACAGAGUAGACAAAGAAAGCUGAGGGAAGCGCAAAGAAAAAGCUGCGCGCGCGCCGAGGAAAAAAAAGCUGCUGAUGUGGCCGCGCCGCAAACAGGAAGAAACAGG AACGCCGCGAACGCAAAUGGCGCCAGGGCGCUGAAAAAAGCUAAGCAGCACAGCGCCGCAAGGAGAAGAUGCAGGAACUGGGCAUCGACGGCUACACCCGCCAGCUGGAAGGCGAAGUGGAAAGCCUGGAAGCCGAACGCCGCAAGCUGCUGCAGGAGAAGGAGGACCUGAUGGCGAGGUGACUACUGGCAGGCCGCCUGGAAGCCAUGUGGCUGCAGU GAUAAGCUGCCUUCUGCGGGGGCUUGCCUUCUGGCCAUGCCCUUCUCUCUCCCCUUGCACCUGUACCUCUGGUCUUUGAAUAAAGCCUGAGUAGGGAAGGCGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (F1)の polynucleotideAUGGCUGCUAGCGGCCUGUUUCGCUGCCUGCCUGUGAGCUGCCCUGAAGACCUGCUGGUGGAGGAACUGGUGGACGGCCUGCUGAGCCUGGAAGAAGAACUGAAGGACAAGGAGGAGGAAGAAGCCGUGCUGGACGGCCUGCUGAGCCUGGAAG AAGAAUCUCGCGGACGCCUUCGCCGCGGACCUCCUGGAGAAAAAAGCUCCUCCUAGAGGAGAAACACACAGAGAUAGACAAAGAAAAGCUGAGGAGAAGCGCAAGCGCAAGAAGGAGCGCGAGAAGGAGGAGGAGAAGCAGAUCGCCGAGUACCUG AAGCGCAAAGAAGAAGAAAAAAGCUCGCCGCCGCCGCCGCGCUGAAAAAAAAAGCUGCUGAUGUGGCCCGCCGCAAACAGGAAGAACAGGAACGCCGCGAACGCAAAUGGCGCCAGGGCGCUGAAAAAAGCUAAGCAGCACAGCGCCCGCAAGGAGA AGAUGCAGGAACUGGGCAUCGACGGCUACACCCGCCAGCUGGAAGGCGAAGUGGAAAGCCUGGAAGCCGAACGCCGCAAGCUGCUGCAGGAGAAGGAGGACCUGAUGGGCGAGGUGAACUACUGGCAGGGCCGCCUGGAAGCCAUGUGGCUGCAG

[0154] In (F2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (F2) has immunogenicity against HTLV-1. The "one or several" in (F2) may be, for example, 1 to 186, 1 to 156, 1 to 123, 1 to 94, 1 to 93, 1 to 60, 1 to 30, 1 to 24, 1 to 18, 1 to 12, 1 to 6, 1 to 3, 1 or 2, or 1 in the base sequence of (F1).

[0155] In (F3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (F3) has immunogenicity against HTLV-1. The "identity" in (F3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (F1).

[0156] In (F4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (F1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (F4), the stringent conditions can be the same as those described in (A4).

[0157] The polynucleotide (F5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 7 or 8 by substituting the corresponding codons.

[0158] In (F6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (F6) has immunogenicity against HTLV-1. The "one or several" in (F6) may be, for example, 1 to 62, 1 to 52, 1 to 41, 1 to 31, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 or 2 amino acids in the amino acid sequence of (F5).

[0159] In (F7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (F7) has immunogenicity against HTLV-1. The "identity" of (F7) with respect to the amino acid sequence of (F5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0160] (Polynucleotide encoding an immunogenic fragment of HBZ) The polynucleotide encoding the immunogenic fragment of HBZ may be any polynucleotide, as long as the protein encoded by the polynucleotide has immunogenicity against HTLV-1.

[0161] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is preferably one that encodes the amino acid sequence from positions 1 to 58 of SEQ ID NO: 8 (HBZ-1 in the Examples described below).

[0162] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure particularly encodes the amino acid sequence from 1 to 58 of SEQ ID NO: 8 (HBZ-1 in the Examples described below), and it is preferable that the region other than the polynucleotide consists of an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the polynucleotide in SEQ ID NO: 1 or the amino acid sequence other than the polynucleotide in SEQ ID NO: 8.

[0163] It is particularly preferable that the nucleic acid encapsulated in the lipid complex of the present disclosure encodes the amino acid sequence of amino acids 1 to 58 of SEQ ID NO: 8 (HBZ-1 in the Examples below).

[0164] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure preferably contains the base sequence from bases 1 to 174 of SEQ ID NO: 10 (the polynucleotide encoding HBZ-1 in the Examples described below).

[0165] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure is, in particular, a polynucleotide consisting of the 1st to 174th base sequence of SEQ ID NO: 10 (the polynucleotide encoding HBZ-1 in the Examples described below), and the region of the nucleic acid other than the polynucleotide is preferably a base sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to a base sequence other than the polynucleotide in SEQ ID NO: 10 or a base sequence other than the polynucleotide in SEQ ID NO: 10.

[0166] The polynucleotide contained in the nucleic acid encapsulated in the lipid complex of the present disclosure encodes the amino acid sequence of amino acids 1 to 58 of SEQ ID NO: 1 (HBZ-1 in the Examples described below), and the region of the nucleic acid other than the polynucleotide preferably consists of a base sequence encoding an amino acid sequence other than the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence other than the amino acid sequence of SEQ ID NO: 10.

[0167] It is more preferable that the nucleic acid encapsulated in the lipid complex of the present disclosure comprises a polynucleotide encoding the nucleotide sequence from bases 1 to 174 of SEQ ID NO: 10 (the polynucleotide encoding HBZ-1 in the Examples described below).

[0168] (Method for producing each protein) Each protein for obtaining nucleic acid can be synthesized by genetic engineering techniques, specifically, by using the transformant described below. The protein may also be produced by in vitro synthesis, for example.

[0169] For example, the transformant is cultured, and Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure is collected from the culture. The culture may be a culture supernatant, or a transformant such as cultured cells or cultured bacterial cells, or a processed or disrupted product thereof.

[0170] After the culture, if the Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure is produced within the host, the production method of the present disclosure involves, for example, disrupting the host to extract Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof.Furthermore, if the Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure is produced or secreted outside the host, the production method of the present disclosure involves, for example, using the culture medium as is or removing the host by centrifugation or the like. Thereafter, the production method of the present disclosure can isolate or purify proteins such as Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof by using, for example, a general biochemical method used for isolating and purifying proteins, specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography, either alone or in appropriate combination.

[0171] (Structure of Lipid Complex) The lipid complex of the present disclosure has a structure in which at least one type of nucleic acid is encapsulated in a lipid. In some embodiments, the lipid complex is a lipid particle. The lipid particle has a structure in which the nucleic acid is encapsulated in a fine particle composed of lipids. In some embodiments, the lipid complex is a lipid nanoparticle (LNP). The LNP has a structure in which the nucleic acid is encapsulated in a particle composed of lipids and having a nano-sized average particle diameter (e.g., about 1 nm to 1000 nm).

[0172] In some embodiments, the lipids constituting the lipid complex comprise (i) a cationic lipid and (ii) at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol. Examples of the form of the lipid complex comprising (i) and (ii) include a complex of a nucleic acid and a membrane (reverse micelle) consisting of a single lipid layer (single molecule), a complex of a nucleic acid and a liposome, and a complex of a nucleic acid and a micelle.

[0173] (i) Cationic lipid The cationic lipid is not particularly limited. For example, the cationic lipids described in International Publication Nos. 2015 / 105131, 2016 / 104580, 2017 / 222016, and 2019 / 131580 can be used. The cationic lipid may be in the form of a salt, a hydrate, or a solvate.

[0174] An example of the cationic lipid according to one embodiment is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: In the above formula (I), L １ and L ２ each independently represents an alkylene group having 3 to 10 carbon atoms (e.g., 3 to 8 carbon atoms). １ and L ２ are each independently a linear alkylene group having 3 to 10 carbon atoms (for example, 3 to 8 carbon atoms). １ and R ２ Each of X independently represents an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms. １ represents a single bond or -CO-O-. In the above formula (I), ring P represents any of the following formulas (P-1) to (P-6). In one embodiment, ring P represents any of the following formulas (P-1), (P-2), (P-4), (P-5), and (P-6). In one embodiment, ring P represents the following formula (P-1) or (P-6). In one embodiment, ring P represents the following formula (P-1). The above formulas (P-1), (P-2), (P-3), (P-4), (P-5), and (P-6), middle, R ３represents an alkyl group having 1 to 3 carbon atoms.

[0175] Examples of cationic lipids include, for example, 1-oxo-1-(undecan-5-yloxy)nonadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecan-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-(undecan-5-yloxy)heptadecan-8-yl-1-methylpiperidine 4-carboxylate, 21-oxo-21-(undecan-5-yloxy)henicosan-10-yl-1-methylpiperidine 4-carboxylate, 21-(octan-3-yloxy)-21-oxoheneicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, (Z)-1-((2-butylnon-3-en-1-yl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-oxo-1-((3-pentyloctyl)oxy)icosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((3,4-dipropylheptyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((6-(butyldisulfanyl)-3-(3-(butyldisulfanyl)propyl)hexyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 2-butyloctyl-10-((4-(dimethylamino)butanoyl)oxy)icosanoate, 2-{9-[(2-butyloctyl)oxy]-9-oxononyl}dodecyl 1-methylpiperidine-4-carboxylate, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, 2-nonyl-11-oxo-11-[(3-pentyloctyl)oxy]undecyl Examples of the hydroxybenzoate include 1-methylpiperidine-4-carboxylate, bis(3-pentyloctyl) 9-{[(1-methylpiperidine-4-carbonyl)oxy]methyl}heptadecanedioate, di[(Z)-2-nonen-1-yl] 9-{[(1-methylpiperidine-4-carbonyl)oxy]methyl}heptadecanedioate, 1-(2-octylcyclopropyl)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, (3S)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate, (3R)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate, and pharmaceutically acceptable salts thereof.

[0176] In some embodiments, the cationic lipid is 1-((2-butyloctyl)oxy)-1-oxoicosan-10-yl-1-methylpiperidine-4-carboxylate, 1-((2-butyloctyl)oxy)-1-oxononadecan-10-yl-1-methylpiperidine-4-carboxylate, 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate, 1-(2-octylcyclopropyl)heptadecan-8-yl-1-methylpiperidine-4-carboxylate, (3S)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpyrrolidine-3-carboxylate, or (3R)-2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl The composition contains at least one selected from 1-methylpyrrolidine-3-carboxylate and pharmaceutically acceptable salts thereof.

[0177] In certain embodiments, the cationic lipid is 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate or a pharmaceutically acceptable salt thereof. The structure of 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate is shown below.

[0178] The cationic lipids can be used alone or in combination of two or more.

[0179] (ii) Neutral lipids, polyethylene glycol-modified lipids, and sterols. The neutral lipids are not particularly limited, and examples thereof include dioleoylphosphatidylethanolamine (DOPE), phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), palmitoyloleoylphosphatidylcholine (POPC), stearyloleoylphosphatidylcholine (SOPC), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine ... dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoylphosphatidylcholine (DOPE-mal), dioleoyl phospholipids such as dioleoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), dilignoceroylphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylserine (DOPS), sphingomyelin, etc.; ceramide (Cer), etc.

[0180] In some embodiments, the neutral lipid comprises at least one selected from DOPE, HSPC, DPPC, DSPC, or DAPC. In certain embodiments, the neutral lipid comprises DPPC.

[0181] The neutral lipids can be used alone or in combination of two or more.

[0182] The polyethylene glycol-modified lipid is not particularly limited, and examples thereof include PEG2000-DMG (PEG2000-dimyristylglycerol), PEG2000-DPG (PEG2000-dipalmitoylglycerol), PEG2000-DSG (PEG2000-distearoylglycerol), PEG5000-DMG (PEG5000-dimyristylglycerol), PEG5000-DPG (PEG5000-dipalmitoylglycerol), PEG5000-DSG (PEG5000-distearoylglycerol), glycerol), PEG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine), PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxylpropyl-3-amine), PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEG-cholesterol, etc. Examples of PEG-DAA include PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, PEG-distearyloxypropyl, etc.

[0183] In some embodiments, the polyethylene glycol-modified lipid comprises at least one selected from PEG2000-DMG, PEG2000-DPG, PEG2000-DSG, PEG-cDMA, or PEG-C-DOMG. In certain embodiments, the polyethylene glycol-modified lipid comprises PEG2000-DMG.

[0184] The polyethylene glycol-modified lipids can be used alone or in combination of two or more. The polyethylene glycol-modified lipids may have methoxylated PEG terminals (MPEG; methoxypolyethylene glycol). For example, PEG2000-DMG includes MPEG2000-DMG.

[0185] The sterol is not particularly limited, but examples thereof include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocastol, fucosterol, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and the like.

[0186] In some embodiments, the sterol comprises at least one selected from cholesterol, dihydrocholesterol, lanosterol, or β-sitosterol, hi certain embodiments, the sterol comprises cholesterol.

[0187] The sterols can be used alone or in combination of two or more.

[0188] (Composition / Ratio of Lipids) The content of lipids in the lipid complexes and the ratio of each lipid component constituting the lipid are not particularly limited. In some embodiments, the lipid complexes contain, for example, 50 to 99.99 wt%, for example, 70 to 99.99 wt%, for example, 90 to 99 wt%, of lipid components relative to the total weight of the lipid complex. In some embodiments, the lipid complexes contain, for example, 10 to 99 mol%, 20 to 90 mol%, or 30 to 70 mol% of the above-mentioned cationic lipids, based on the total lipids contained in the lipid complexes. In some embodiments, the lipid complexes may contain, for example, 0 to 50 mol%, 0 to 40 mol%, or 0 to 30 mol% of neutral lipids, based on the total lipids contained in the lipid complexes. In some embodiments, the lipid complexes may contain, for example, 0 to 30 mol%, 0 to 20 mol%, or 0 to 10 mol% of polyethylene glycol-modified lipids, based on the total lipids contained in the lipid complexes. In some embodiments, the lipid complex may contain, for example, 0 to 90 mol %, 10 to 80 mol %, or 20 to 50 mol % of sterol based on the total lipid contained in the lipid complex.

[0189] The content of nucleic acid in the lipid complex is not particularly limited. In some embodiments, the lipid complex contains, for example, 0.01 to 50 wt %, for example, 0.1 to 30 wt %, for example, 1 to 10 wt % of nucleic acid relative to the total weight of the lipid complex.

[0190] In some embodiments, the lipid complexes comprise 0.01-50% by weight of nucleic acid and 50-99.99% by weight of lipid, based on the total weight of the lipid complex, and comprise 30-70 mol% of cationic lipid, 0-30 mol% of neutral lipid, 0-10 mol% of polyethylene glycol-modified lipid, and 20-50 mol% of sterol, based on the total lipid content of the lipid complex. In some embodiments, the lipid complexes comprise 0.01-50% by weight of nucleic acid and 50-99.99% by weight of lipid, based on the total weight of the lipid complex, and comprise 30-70 mol% of cationic lipid, 1-30 mol% of neutral lipid, 1-10 mol% of polyethylene glycol-modified lipid, and 20-50 mol% of sterol, based on the total lipid content of the lipid complex.

[0191] The combination of lipid components in the lipid complex is not particularly limited. In some embodiments, the lipid comprises a combination of a cationic lipid, a neutral lipid, and a sterol. In some embodiments, the lipid comprises a combination of the above-mentioned cationic lipid, a neutral lipid, a polyethylene glycol-modified lipid, and a sterol. It has been reported that the cationic lipid is necessary for encapsulating nucleic acid and efficiently delivering nucleic acid into target cells, and the polyethylene glycol-modified lipid is necessary for suppressing particle aggregation. Furthermore, the simultaneous presence of four types of lipids, including a neutral lipid and a sterol in addition to these two types of lipids, can form stable particles encapsulating nucleic acid.

[0192] In some embodiments, the lipid complexes are composed of cationic lipid / neutral lipid / polyethylene glycol-modified lipid / sterol, and the molar ratio of the lipids may be, for example, 10-99 / 0-50 / 0-30 / 0-90, 20-90 / 0-40 / 0-20 / 10-80, or 30-70 / 0-30 / 0-10 / 20-50. In some embodiments, the molar ratio of cationic lipid / neutral lipid / polyethylene glycol-modified lipid / sterol in the lipid complexes may be 10-99 / 1-50 / 1-30 / 1-90, 20-90 / 1-40 / 1-20 / 10-80, or 30-70 / 1-30 / 1-10 / 20-50. In a specific embodiment, the molar ratio of cationic lipid / neutral lipid / polyethylene glycol-modified lipid / sterol in the lipid complexes is 47 / 11 / 1.5 / 40.5.

[0193] In one embodiment of the lipid complex, the lipid comprises 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate as the cationic lipid, DPPC as the neutral lipid, (M)PEG2000-DMG as the polyethylene glycol-modified lipid, and cholesterol as the sterol.

[0194] (Characteristics of Lipid Complexes) The "average particle size" of the lipid complexes herein refers to the Z-average particle size, which is measured by dynamic light scattering. The average particle size (Z-average) of the lipid complexes may be, for example, 10 to 1000 nm, for example, 30 to 500 nm, for example, 30 to 200 nm.

[0195] The encapsulation rate of nucleic acid in lipid complexes was measured, for example, using Quant-iT RiboGreen RNA Reagent (Invitrogen, Cat# R11491). Specifically, the nucleic acid concentration (A) measured after dilution with RNase-Free Water was defined as the nucleic acid present in the lipid complex external solution, and the nucleic acid concentration (B) measured after dilution with 1% Triton X-100 was defined as the total nucleic acid concentration in the formulation. The encapsulation rate can be calculated as follows: Encapsulation rate (%) = 100 - (A / B) x 100. In some embodiments, the encapsulation rate (%) of nucleic acid in lipid complexes calculated by the above method is, for example, greater than 80%, 85%, or 90%. In one embodiment, the encapsulation rate (%) of nucleic acid in lipid complexes is greater than 90%.

[0196] In some embodiments, the polydispersity index (PDI) is less than 0.3 (particularly less than 0.2, especially less than 0.1).

[0197] (Method for Producing Lipid Complexes) Methods for encapsulating effective molecules into lipid complexes include, for example, reverse phase evaporation, Zwitterion (NaCl) hydration, cationic core hydration, and a method using ethanol and calcium (see also Biomembr., 1468, 239-252 (2000)). Lipid complexes encapsulating the nucleic acid of the present disclosure can be prepared by methods known in the technical field as described above.

[0198] In some embodiments, lipid complexes can be prepared by, for example, mixing a lipid solution containing a cationic lipid and at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol with an acidic buffer containing nucleic acid. This method results in lipid complexes whose interior is filled with a core of nucleic acid and lipid.

[0199] Examples of solvents for dissolving lipids include polar organic solvents such as alcohols, for example, ethanol, isopropanol, chloroform, or t-butanol. Examples of acidic buffers for dissolving nucleic acids include sulfate buffers, phosphate buffers, phthalate buffers, tartaric acid buffers, citrate buffers, formic acid buffers, oxalate buffers, and acetate buffers.

[0200] In one embodiment, the lipid complex can be produced by a method comprising, for example, a step (a) of mixing a polar organic solvent-containing aqueous solution containing a cationic lipid and at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols with an aqueous solution containing nucleic acid to obtain a mixed solution, and a step (b) of reducing the content of the polar organic solvent in the mixed solution. The electrostatic interaction between the water-soluble nucleic acid and the cationic lipid, and the hydrophobic interaction between the lipids, can form a lipid complex in which the nucleic acid is encapsulated within a particle composed of lipids. For example, by reducing the content of the polar organic solvent in the mixed solution, the solubility of the lipid components, including the cationic lipid and at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols, in the polar organic solvent-containing aqueous solution can be changed, thereby forming a lipid complex. Examples of polar organic solvents include alcohols such as ethanol. First, in step (a), a polar organic solvent-containing aqueous solution containing (I) the above-mentioned cationic lipid and (II) at least one lipid selected from the group consisting of neutral lipids, polyethylene glycol-modified lipids, and sterols is dissolved, and (III) an aqueous solution containing nucleic acid is mixed to obtain a mixed solution. The concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution is not particularly limited as long as it satisfies the conditions for dissolving the lipid molecules even after mixing with the aqueous solution of nucleic acid. For example, the concentration of the polar organic solvent in the polar organic solvent-containing aqueous solution in step (a) can be 0.1 to 60% by weight. The aqueous solution containing nucleic acid can be obtained, for example, by dissolving nucleic acid in an acidic buffer. Subsequently, in step (b), the content of the polar organic solvent is reduced by adding water or the like to the mixed solution. This allows the formation of a lipid complex. To efficiently form the lipid complex, it is preferable to rapidly reduce the content of the polar organic solvent. For example, the concentration of the polar organic solvent in the final polar organic solvent-containing aqueous solution in step (b) can be 0 to 5% by weight. Alternatively, the mixture obtained in step (a) may be subjected to dialysis to remove the polar organic solvent and replace the solvent with a pharmaceutically acceptable medium.The content of the polar organic solvent in the solution is reduced during the dialysis process, which allows the formation of lipid complexes. According to the production method of this embodiment, lipid complexes in which nucleic acids are efficiently encapsulated inside lipid particles can be obtained.

[0201] 2. Pharmaceutical Compositions One aspect of the present disclosure relates to a pharmaceutical composition (hereinafter also referred to as the "pharmaceutical composition of the present disclosure" or "pharmaceutical composition") comprising the lipid-conjugates of the present disclosure. In some embodiments, the present disclosure provides use of the lipid-conjugates for producing a pharmaceutical composition.

[0202] In some embodiments, pharmaceutical compositions may contain the lipid conjugates described above, a pharmaceutically acceptable carrier, and optionally other additives.

[0203] Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable vehicles, suspending agents, solubilizing agents, preservatives, fillers, extenders, binders, wetting agents, disintegrating agents, lubricants, dispersing agents, flavoring agents, stabilizers, isotonicity agents, preservatives, anti-adsorption agents, surfactants, diluents, pH adjusters, soothing agents, buffers, sulfur-containing reducing agents, antioxidants, and the like, which are added appropriately within a range that does not interfere with the effects of the present disclosure.

[0204] Pharmaceutically acceptable media include sterilized water; physiological saline; isotonic solutions containing adjuvants such as glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and the like; and buffer solutions such as phosphate buffer, citrate buffer, acetate buffer, and the like.

[0205] The suspending agent is not particularly limited, and examples thereof include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc. The solubilizing agent is not particularly limited, and examples thereof include alcohols such as ethanol, propylene glycol, and polyethylene glycol, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl esters, etc.

[0206] The stabilizer is not particularly limited, and examples thereof include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.

[0207] The isotonic agent is not particularly limited, and examples thereof include D-mannitol, sorbitol, and the like.

[0208] The preservative is not particularly limited, and examples thereof include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0209] The adsorption inhibitor is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, and polyethylene glycol.

[0210] The sulfur-containing reducing agent is not particularly limited, and examples thereof include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thiochitic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

[0211] The antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0212] Other additives include, for example, sugars such as sucrose, glucose, sorbitol, and lactose; amino acids such as glutamine, glutamic acid, sodium glutamate, and histidine; organic salts such as citric acid (e.g., sodium citrate and potassium citrate), phosphoric acid, acetic acid (e.g., sodium acetate), lactic acid, carbonic acid, and tartaric acid; and inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate.

[0213] The pharmaceutical composition can be formulated into various dosage forms, such as injections.

[0214] The pharmaceutical composition may be in a powder state from which the solvent has been removed, for example, by lyophilization or the like, or may be in a liquid state. Some embodiments of the pharmaceutical composition are powder compositions containing the lipid complexes of the above-described embodiments. The powder composition may be prepared by removing the solvent from a liquid composition (dispersion) containing the lipid complexes, for example, by filtration, centrifugation, or the like, or by lyophilizing the dispersion. When the pharmaceutical composition is in a powder state, it can be suspended or dissolved in a pharmaceutically acceptable medium before use and used as an injection. One embodiment of the composition of the present disclosure is a liquid composition containing the lipid complexes of the above-described embodiments and a pharmaceutically acceptable medium. When the composition is in a liquid state, it can be used as an injection as is or suspended or dissolved in a pharmaceutically acceptable medium.

[0215] The subject to which the pharmaceutical composition of the present disclosure is administered is not limited, and can be applied to various animals. The pharmaceutical composition of the present disclosure can be applied to, for example, humans or non-human mammals (monkeys, mice, rats, rabbits, cows, horses, goats, etc.), preferably humans and experimental animals in clinical trials, screening, and experiments.

[0216] The conditions for use (administration conditions) of the pharmaceutical composition of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of the active ingredient in the pharmaceutical composition, the type of subject to administration, etc.

[0217] The method of administering the pharmaceutical composition of the present disclosure to a subject (e.g., administration route, dosage, number of daily administrations, timing of administration, etc.) is not limited and can be appropriately determined by a person skilled in the art (e.g., a physician) depending on the subject's health condition, the severity of the disease, the type of concomitant drug, etc. For example, the pharmaceutical composition is administered one or more times. The multiple times may be, for example, two, three, four, five or more times. The number of administrations may be appropriately determined while confirming the preventive effect on the subject. In the case of multiple administrations, the administration interval can be appropriately determined while confirming the preventive effect on the subject, and examples include once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.

[0218] The administration of the pharmaceutical composition of the present disclosure to a subject is not particularly limited, and may be parenteral administration, for example, intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intradermal administration, intravenous administration, etc. For example, intramuscular administration or subcutaneous administration is preferred because it allows safe and stable administration regardless of the skill of the administerer.

[0219] The dosage of the pharmaceutical composition varies depending on the administration subject, target organ, symptoms, and administration method. In one embodiment, the pharmaceutical composition of the present disclosure can be administered in an amount sufficient to induce an immune response against HTLV-1, i.e., an effective dose, depending on the administration form. The dosage can be determined appropriately depending on, for example, the age, body weight, symptoms, etc. of the administration subject. In one embodiment, the dosage of the pharmaceutical composition can be, for example, 0.01 mg to 100 mg per kg of body weight of the subject, 0.1 mg to 50 mg per kg, or 0.3 mg to 10 mg per kg of body weight of the subject.

[0220] The pharmaceutical composition of the present disclosure may be used, for example, in vitro or in vivo. The pharmaceutical composition of the present disclosure may be used, for example, as a research reagent or as a pharmaceutical. In the former case, the pharmaceutical composition of the present disclosure may also be referred to as a test reagent or test kit.

[0221] The subject to which the pharmaceutical composition of the present disclosure is administered is not particularly limited. When the pharmaceutical composition of the present disclosure is used in vitro, the subject to which the pharmaceutical composition is administered can be, for example, a cell, a tissue, an organ, etc. Examples of the cells include cells collected from a living body, cultured cells, etc., and examples of the tissue or organ include tissue (biological tissue) or an organ, etc. collected from a living body. Examples of the cells include immune cells such as T cells, B cells, NK cells, and dendritic cells. When the pharmaceutical composition of the present disclosure is used in vivo, the subject (subject to which the pharmaceutical composition is administered) can be, for example, the subjects exemplified above.

[0222] When administered to a subject, the pharmaceutical composition of the present disclosure can induce an immune response against HTLV-1 (particularly, cellular immunity against HTLV-1, particularly cellular immunity mediated by cytotoxic T cells). Therefore, the pharmaceutical composition of the present disclosure can also be referred to as a vaccine, vaccine composition, or vaccine preparation. The vaccine, vaccine composition, or vaccine preparation of the present disclosure can prevent the onset of HTLV-1-associated diseases (e.g., ATL) by activating cellular immunity and suppressing the proliferation of infected cells. The pharmaceutical composition of the present disclosure can efficiently release the nucleic acid into the cytoplasm by administering it in the form of a lipid complex encapsulating the nucleic acid. According to some aspects of the present disclosure, a pharmaceutical composition for inducing an immune response against HTLV-1 (particularly, cellular immunity against HTLV-1, particularly cellular immunity mediated by cytotoxic T cells) is provided. According to another aspect of the present disclosure, a method for inducing an immune response against HTLV-1 is provided, comprising the step of administering the pharmaceutical composition to a subject in need thereof.

[0223] The pharmaceutical composition of the present disclosure can induce an immune response against HTLV-1 and, therefore, can be useful for the prevention and / or treatment of HTLV-1-associated diseases. That is, according to some aspects of the present disclosure, a pharmaceutical composition for use in the prevention and / or treatment of HTLV-1-associated diseases is provided. According to another aspect of the present disclosure, a method for preventing and / or treating HTLV-1-associated diseases is provided, comprising the step of administering the pharmaceutical composition to a subject in need thereof. According to another aspect of the present disclosure, use of a lipid complex or a pharmaceutical composition for use in the prevention and / or treatment of HTLV-1-associated diseases is provided.

[0224] The subject in need may be a subject infected with HTLV-1, a subject determined to be at high risk of being infected with HTLV-1, a subject who may be infected with HTLV-1, or a healthy person who is not infected with HTLV-1. Also, the subject in need may be a subject determined to be at high risk of developing or recurring an HTLV-1-associated disease, or a subject who has developed an HTLV-1-associated disease.

[0225] In some embodiments, the pharmaceutical composition of the present disclosure can prevent or alleviate at least one symptom caused by HTLV-1 infection in a subject to which the composition is administered. An example of a symptom of HTLV-1 infection is the onset of ATL. The prevention of the symptom can be evaluated subjectively or objectively, and specific examples include self-assessment by the subject to which the composition is administered; evaluation by a physician; quality of life (QOL) evaluation; evaluation of the onset of ATL or delay in the progression of ATL symptoms, or reduction in the severity of ATL symptoms. The objective evaluation may be evaluation using animals or humans.

[0226] In some forms of the pharmaceutical composition, the nucleic acid is mRNA, and the pharmaceutical composition can be used to induce an immune response against HTLV-1 (particularly cellular immunity against HTLV-1, particularly cellular immunity by cytotoxic T cells). In particular, this form of pharmaceutical composition can also be referred to as an mRNA vaccine, mRNA vaccine composition, or mRNA vaccine preparation. In some embodiments, the pharmaceutical composition can be used for the prevention and / or treatment of HTLV-1-associated diseases. In some embodiments, the pharmaceutical composition is a vaccine. In some forms of the pharmaceutical composition, the nucleic acid is mRNA, and the pharmaceutical composition can be used as an mRNA vaccine. In a specific embodiment, the pharmaceutical composition can be used as an mRNA vaccine for the prevention of HTLV-1-associated diseases.

[0227] In some embodiments, the pharmaceutical composition of the present disclosure is used for the prevention and / or treatment of ATL. In certain embodiments, the pharmaceutical composition of the present disclosure is used for the prevention of ATL onset, aggravation, or recurrence. In certain embodiments, the nucleic acid comprises a nucleic acid comprising a polypeptide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof. As described in the Examples below, it has been shown that Gag, a structural protein of HTLV-1, is expressed in cells of ATL patients and that patients without ATL recurrence have cytotoxic T cells against Gag, confirming that an effective immune response against HTLV-1 can be induced by using HTLV-1 Gag as an antigen. The present inventors have found that a cellular immune response of T cells can be induced by using a pharmaceutical composition containing HTLV-1 Gag p15, p19, and p24 as active ingredients. Therefore, the pharmaceutical composition of the present disclosure can induce an immune response against, for example, HTLV-1.

[0228] In some embodiments, the pharmaceutical composition of the present disclosure is a vaccine for use in preventing, for example, the onset of ATL, the aggravation of ATL, or the recurrence of ATL.

[0229] In these embodiments, the subject may be a patient infected with HTLV-1 who has not yet developed ATL, a patient who has developed ATL, or a patient in remission from ATL. The subject is preferably a patient infected with HTLV-1 in whom prevention of the onset of ATL or the recurrence of ATL is desired. The subject may also be a patient who has undergone bone marrow transplantation or hematopoietic stem cell transplantation after developing ATL.

[0230] According to a further aspect of the present disclosure, there is provided a method for preventing the onset, aggravation, or recurrence of ATL, comprising the step of administering a pharmaceutical composition of the present disclosure to a subject in need thereof. This prevention method is characterized by administering to a subject a pharmaceutical composition comprising a lipid complex of the present disclosure, and other configurations and conditions are not particularly limited. This prevention method comprises, as an active ingredient, a nucleic acid comprising a polypeptide encoding at least one selected from Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof, and therefore, when administered to a subject, is capable of inducing an immune response against HTLV-1.

[0231] Since the prevention method of this embodiment can induce, for example, an immune response against HTLV-1, it can also be referred to as, for example, a vaccination method against HTLV-1, a method for inducing protective immunity, a method for stimulating an immune response, or a method for inducing an immune response.

[0232] In this prevention method, the active ingredient is administered to a subject to induce an immune response in the subject, thereby conferring immunity to HTLV-1 in the subject. In this prevention method, for example, an immune response against Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof is induced in the subject, resulting in a cellular immune response by T cells or the like against Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof. In this prevention method, the administration step is carried out once or multiple times.

[0233] In some embodiments, the present disclosure provides the use of a pharmaceutical composition of the present disclosure for use in a method for preventing the onset, aggravation, or recurrence of ATL. The present disclosure provides the use of a lipid conjugate or pharmaceutical composition of the present disclosure for use in the manufacture of a vaccine for use in suppressing the onset, aggravation, or recurrence of ATL.

[0234] The present disclosure will be described in more detail below with reference to Examples, Production Examples, and Test Examples, but the present disclosure is not limited to these Examples. In this specification, "room temperature" generally refers to a temperature of about 10°C to about 35°C.

[0235] Example 1 Preparation of LNP Encapsulating mRNA (1) Preparation of Template DNA To prepare template plasmid DNA for use in in vitro transcription (IVT), a plasmid was synthesized containing a DNA fragment in which the T7 promoter sequence (TAATACGACTCACTATA: SEQ ID NO: 18), 5'UTR sequence (AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA: SEQ ID NO: 19), KOZAK sequence (GCCACC: SEQ ID NO: 20), each antigen sequence (Gag antigen, Tax antigen, HBZ antigen), and 3'UTR sequence (TAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGG: SEQ ID NO: 21) were linked in this order. 1 μg of the plasmid was dissolved in nuclease-free water (210 μL), to which Q5 Hot Start High-Fidelity 2X Master Mix (250 μL, NEB# M0494L), 10 μM sense primer (20 μL), and antisense primer containing 10 μM poly-T (20 μL) were added. After incubation at 95°C for 1 minute, 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 3 minutes were carried out, followed by further incubation at 72°C for 5 minutes, thereby amplifying the template DNA by PCR. After the reaction, the reaction mixture was purified using a NucleoSpin Gel and PCR Clean Up Kit (TaKaRa #U0609A) to obtain template DNAs having the antigen sequences of interest (SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24).

[0236]

[0237]

[0238] <SEQ ID NO: 24> Template DNA containing the sequence of HBZ antigen TAATACGACTCACTATAAGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGCTAGCGGCCTGTTTCGCTGCCTGCCTGTGAGCTGCCCTGAAGACCTGCTGGTGGAGGAACTGGTGGACGGCCTGCTGAGCCTGGAAGAAGAACTGAAGGACAAGGAGGAGGAAGAAGCCGTGCTGGACGGCCTGCTGAGCCTGGAAGAAGAATCTCGCGGACGCCTTCGCCGCGGACCTCCTGGAGAAAAAGCTCCTCCTAGAGGAGAAACACACAGAGATAGACAAAGAAAAGCTGAGGAGAAGCGCAAGCGCAAGAAGGAGCGCGAGAAGGAGGAGGAGAAGCAGATCGCCGAGTACCTGAAGCGCAAAGAAGAAGAAAAAGCTCGCCGCCGCCGCCGCGCTGAAAAAAAAGCTGCTGATGTGGCCCGCCGCAAACAGGAAGAACAGGAACGCCGCGAACGCAAATGGCGCCAGGGCGCTGAAAAAGCTAAGCAGCACAGCGCCCGCAAGGAGAAGATGCAGGAACTGGGCATCGACGGCTACACCCGCCAGCTGGAAGGCGAAGTGGAAAGCCTGGAAGCCGAACGCCGCAAGCTGCTGCAGGAGAAGGAGGACCTGATGGGCGAGGTGAACTACTGGCAGGGCCGCCTGGAAGCCATGTGGCTGCAGTGATAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGG

[0239] (2) Preparation of mRNA Using each of the obtained template DNAs, mRNA was prepared by in vitro transcription (IVT). 300μg / mL template DNA (80μL), 100mM CleanCap AG (32μL, TriLink catalog #N-7113), 100mM ATP (40μL, TriLink catalog #N-1510), 100mM CTP (40μL, TriLink catalog #N-1511), 100mM GTP (40μL, TriLink catalog #N-1512), 100mM N1-methyl-ψ-Uridine-5'-Triphosphate (40μL), UltraPure DNase / RNase-Free Distilled Water (405.6 μL, Thermo Fisher catalog #10977015), T7 Transcription 10x buffer (80 μL), RNase inhibitor (20 μL, NEB catalog #M0314L), yeast inorganic pyrophosphatase (16 μL, NEB catalog #M2403L), and T7 RNA polymerase (6.4 μL, Roche catalog #08140669103) were mixed and incubated at 37°C for 3 hours. RNase-Free DNase I (24 μL, TaKaRa catalog #2270A) was added and incubated at 37°C for 30 minutes. 10x phosphatase buffer (96 μL, NEB catalog #B0289S) and Antarctic phosphatase (48 μL, NEB catalog #M0289L) were then added and incubated at 37°C for 30 minutes. The mixture was mixed with 8 M LiCl solution (484 μL, Sigma-Aldrich catalog #L7026), allowed to stand at -20°C for at least 1 hour, centrifuged (4°C, 15,000 rpm, 30 minutes), and the supernatant was discarded. 75% ethanol was then added, followed by centrifugation (4°C, 15,000 rpm, 5 minutes), and the supernatant was discarded. This procedure was repeated three times.The resulting precipitate was dissolved in nuclease-free water and purified using an RNeasy Maxi kit (Qiagen catalog #75162) according to the accompanying manual. Alternatively, the resulting precipitate was treated with RNase III (NEB, catalog #M0245S) for 10 minutes at room temperature, followed by purification on an Oligo dT column (Sartorius, catalog #311.1218-2). From the above, mRNAs (SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:9) expressing proteins with the respective amino acid sequences (SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:8) were obtained. A cap structure and poly(A) sequence were added to each mRNA. The cap structure was the structure shown below, CleanCap (Cap1) (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0240]

[0241]

[0242] <SEQ ID NO:9> HBZ mRNA AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCUGCUAGCGGCCUGUUUCGCUGCCUGCCUGUGAGCUGCCCUGAAGACCUGCUGGUGGAGGAACUGGUGGACGGCCUGCUGAGCCUGGAAGAAGAACUGAAGGACAAGGAGGAGGAAGAAGCCGUGCUGGACGGCCUGCUGAGCCUGGAAGAAGAAUCUCGCGGACGCCUUCGCCGCGGACCUCCUGGAGAAAAAGCUCCUCCUAGAGGAGAAACACACAGAGAUAGACAAAGAAAAGCUGAGGAGAAGCGCAAGCGCAAGAAGGAGCGCGAGAAGGAGGAGGAGAAGCAGAUCGCCGAGUACCUGAAGCGCAAAGAAGAAGAAAAAGCUCGCCGCCGCCGCCGCGCUGAAAAAAAAGCUGCUGAUGUGGCCCGCCGCAAACAGGAAGAACAGGAACGCCGCGAACGCAAAUGGCGCCAGGGCGCUGAAAAAGCUAAGCAGCACAGCGCCCGCAAGGAGAAGAUGCAGGAACUGGGCAUCGACGGCUACACCCGCCAGCUGGAAGGCGAAGUGGAAAGCCUGGAAGCCGAACGCCGCAAGCUGCUGCAGGAGAAGGAGGACCUGAUGGGCGAGGUGAACUACUGGCAGGGCCGCCUGGAAGCCAUGUGGCUGCAGUGAUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0243] <SEQ ID NO: 1> Amino acid sequence of Gag MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVLPVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAET RGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHT NSPLGDMLRACQTWTPKDKTKVLVVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNSIGGEV

[0244] <SEQ ID NO: 4> Amino acid sequence of Tax MAHFPGFGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0245] <SEQ ID NO: 8> Amino acid sequence of HBZ MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQIAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ

[0246] (3) Preparation of LNP encapsulating mRNA Each mRNA was dissolved in 50 mM sodium citrate (pH 3.5) to prepare an mRNA dilution solution. 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate (TS202), DPPC (Nippon Fine Chemicals), cholesterol (Nippon Fine Chemicals), and MPEG2000-DMG (NOF Corp.) were dissolved in ethanol at a molar ratio of 47 / 11 / 40.5 / 1.5 to prepare a lipid solution. The ratio of mRNA to lipid was set to a weight ratio of 0.05, and the mRNA dilution solution and lipid solution were mixed at a volume ratio of 3:1 at a flow rate to obtain lipid nanoparticles (LNP). The resulting LNP aqueous solution was dialyzed using a Float-A-Lyzer G2 (SPECTRUM, 100K MWCO) to replace the external solution with PBS, followed by a sucrose solution. After concentration, the solution was sterilized by filtration, and the formulation quality of the LNP was evaluated. 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate was synthesized according to Example A-2 of International Publication WO2017 / 222016.

[0247] The mRNA concentration and encapsulation rate were measured using Quant-iT RiboGreen RNA Reagent (Invitrogen, Cat# R11491). Specifically, the mRNA concentration (A) measured after dilution with RNase-Free Water was defined as the mRNA present in the LNP external solution, and the mRNA concentration (B) measured after dilution with 1% Triton X-100 was defined as the total mRNA concentration in the formulation, and the encapsulation rate was calculated as follows: Encapsulation rate (%) = 100 - (A / B) x 100 The average particle size (Z-average) and polydispersity index (PDI) of the LNP were measured using a particle size analyzer (Malvern, Zetasizer Nano ZS). The results of evaluating the formulation quality of the prepared LNP are shown in Table 1.

[0248] The above results demonstrate that lipid nanoparticles (LNPs) were obtained in which the mRNA encoding the Gag protein or its immunogenic fragment, the mRNA encoding the Tax protein or its immunogenic fragment, and the mRNA encoding the HBZ protein or its immunogenic fragment were encapsulated in lipids. The encapsulation rate for each LNP was greater than 90%, demonstrating high mRNA encapsulation rates. The polydispersity index (PDI) was also less than 0.05.

[0249] Example 2: Study of Cellular Immune Responses in Mice (1) Immunization of Mice and Splenocyte Preparation. LNPs containing mRNA for each antigen were inoculated intramuscularly into the thigh of C57BL / 6 mice (female, 8 weeks old, n = 4 per Gag group; n = 3 per Tax or HBZ group) at 10 μg mRNA / mouse on Day 0 and Day 5. On Day 12, the mice were euthanized and the spleens were removed. The spleens were crushed on a cell strainer using a sterile syringe plunger and washed with 10 ml of RPMI to collect the cell suspension. The cell suspension was centrifuged, the supernatant was removed, and the cells were hemolyzed with 1 ml of NH4Cl for 2 minutes. The reaction was then stopped with 9 ml of RPMI and centrifuged. The cell pellet was resuspended in 20 ml of RPMI with 10% FBS.

[0250] (2) Analysis of Cellular Immune Responses The ability to induce cellular immune responses was evaluated using an ELISPOT assay. Peptides used to stimulate cells were designed and pooled using the following method. Based on the amino acid sequence of Gag (GenBank accession number AAA85841.1) (SEQ ID NO: 1), overlapping peptides of 9 amino acids in length (offset: 1 amino acid) were designed. 97 peptides of Gag p15 were cloned into Gag-1 (p15-1 (p15 1-50 )), and Gag-2 (p15-2 (p15 51-97 )). 121 peptides of Gag p19 were pooled as Gag-3 (p19-1 (p19 1-50 )), Gag-4 (p19-2 (p19 51-100 )), and Gag-5 (p19-3 (p19 101-121 )). Gag p24 peptides were pooled as Gag-6 (p24-1 (p24 1-51 ), Gag-7(p24-2(p24 52-103 )), Gag-8 (p24-3 (p24 104-154 )), and Gag-9 (p24-4 (p24 155-206 Similarly, 345 peptides were pooled as Tax-1 (Tax 1-50 ), Tax-2 (Tax 51-100 ), Tax-3 (Tax 101-150 ), Tax-4 (Tax 151-200 ), Tax-5 (Tax 201-250 ), Tax-6 (Tax 251-300 ), Tax-7 (Tax 301-345 ) were pooled. Similarly, 198 peptides were pooled as HBZ-1 (HBZ 1-50 ), HBZ-2 (HBZ 51-100 ), HBZ-3 (HBZ 101-150 ), HBZ-4 (HBZ 151-198 The ELISpot assay was performed using MABTECH ELISpot Flex: IFN-γ, Mouse-ALP according to the following procedure. The assay plate MAIPS4510 (Millipore) was treated with the coating antibody, and the prepared mouse splenocytes were pooled at 10 6Cells were seeded at 1000 cells / well. After 24 hours of stimulation with 1 μM of the above peptide pool, IFN-γ-producing cells were stained with the detection antibody. The stained spots were analyzed using an ImmunoSpot S6 ENTRY Analyzer (CTL), and the number of spots was counted.

[0251] Figures 1 to 3 show the results indicating the number of spots for each peptide pool. Figure 1 shows the results for the Gag peptide pool, Figure 2 shows the results for the peptide pool, and Figure 3 shows the results for the HBZ peptide pool. "No Peptide" indicates the negative control (no peptide stimulation), and "Positive Control" indicates the positive control (stimulation with the mitogens PMA / Ionomycin). In Figure 1, Gag-1 to Gag-2 correspond to p15-1 and p15-2, Gag-3 to Gag-5 correspond to p19-1 to p19-3, and Gag-6 to Gag-9 correspond to p24-1 to p24-4.

[0252] As shown in Figure 1, spots were formed in the peptide pools of Gag p19 (Gag-3 to Gag-5), Gag p15 (Gag-1 to Gag-2), or Gag p24 (Gag-6 to Gag-9). In particular, many spots were formed in the peptide pools of Gag p19-1 (Gag-3), p19-2 (Gag-4), and p19-3 (Gag-5) (especially p19-1 (Gag-3) and p19-2 (Gag-4)). These results demonstrated that IFN-γ was produced in mice immunized with LNPs encapsulating mRNA encoding Gag proteins. The production of IFN-γ suggested the induction of a cellular immune response. As shown in Figure 2, spots were formed in the peptide pool of Tax. In particular, many spots were formed in the Tax-4 or Tax-6 (especially Tax-6) peptide pools. These results demonstrated that IFN-γ was produced in mice immunized with LNPs encapsulating mRNA encoding the Tax protein. Furthermore, the production of IFN-γ suggested the induction of a cellular immune response. As shown in Figure 3, spots were formed in the HBZ peptide pool. In particular, many spots were formed in the HBZ-1 peptide pool. These results demonstrated that IFN-γ was produced in mice immunized with LNPs encapsulating mRNA encoding the HBZ protein. Furthermore, the production of IFN-γ suggested the induction of a cellular immune response. The above results confirmed the induction of cellular immunity (IFN-γ production), suggesting that cellular immunity is induced in animal models with transplanted cancer, resulting in an antitumor effect.Molecular Therapy 2024, 32(3): 704-721.Lymph node macrophages drive innate immune responses to enhance the anti-tumor efficacy of mRNA vaccines; Molecular Therapy 2021, 29(7): 2227-2238. mRNA-encoded, constitutively active STINGV155M is a potent genetic adjuvant of antigen-specific CD8+ T cell response; Nature Biotechnology 2019, 37(10): 1174-1185. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation。

[0253] Example 3: To analyze the efficacy of Gag mRNA vaccines using animal models, an experimental system using a syngeneic mouse model was established. EL4, a T-cell lymphoma line derived from the inbred C57BL / 6 mouse strain, can form tumors when subcutaneously transplanted into wild-type C57BL / 6 mice. An EL4 subline, EL4-Gag, expressing the gag gene, one of the HTLV-1 antigens, was established as follows: First, the gene sequence encoding the full-length Gag protein was amplified by RT-PCR and inserted into the multicloning site of the PiggyBac Transposon Vector System (System Biosciences, PB-CMV-EF1-Puro) to construct a plasmid expressing full-length Gag. Gene transfer into EL4 was performed using lipofection with a Gag expression PiggyBac vector and a transposase expression vector. Subsequently, EL4 (EL4-Gag) stably expressing Gag was established by adding puromycin to the culture medium for selection. Gag expression in EL4-Gag is confirmed by real-time PCR. Next, wild-type C57BL / 6 mice (n=4 / group) are injected intramuscularly with Gag mRNA vaccine at 10 μg mRNA / mouse twice (e.g., on Day 1 and Day 5) into the thigh muscle. As a control, mice are also injected twice with saline using the same protocol. After the second vaccination (e.g., from the day after vaccination to 2 weeks later, Day 6 to Day 19), 1×10 6 EL4-Gag cells are subcutaneously transplanted into mice. Thereafter, the mice are weighed twice a week, and tumor engraftment is confirmed and tumor diameter is measured. Furthermore, tumor volume is calculated using the following formula: Tumor volume = 1 / 2*(Length × Width) 2 ) Weigh the mice, check for the presence or absence of tumors, and calculate the volume if tumors are visible.

[0254] Reference Example 1 (1) Study of HTLV-1 Antigens Antibody responses to HTLV-1 antigens were studied using serum or plasma from HTLV-1-infected individuals, ATL patients, and other individuals. Specifically, luciferase immunoprecipitation (LIPS assay) was performed. Serum or plasma was obtained from HTLV-1-infected individuals, ATL patients, HAM / TSP patients, and HTLV-1-uninfected individuals. All clinical samples were collected after obtaining written informed consent in accordance with the Declaration of Helsinki. The experiments described in the Examples were approved by the Institutional Ethics Committee of Kumamoto University (Accession Nos. G489, G499, and E2214). The antigens used were Tax (SEQ ID NO: 4), Env (SEQ ID NO: 25), Gag p15 (SEQ ID NO: 11), Gag p19 (SEQ ID NO: 13), and Gag p24 (SEQ ID NO: 14). As the antigen, vectors expressing fusion proteins of various proteins and NanoLuc were prepared, and after the preparation, the vectors were introduced into the 293T cell line, and the cell lysate of the cells was used. ７A count of antigen, LIPS buffer, and 1 μl of the serum or plasma were mixed to prepare a total volume of 100 μl. After the preparation, the plate was incubated at room temperature (hereinafter, approximately 24°C) for 30 minutes using a plate mixer. After the incubation, Protein A / G magnetic beads (Thermo Fisher Scientific) were washed 3 to 5 times with LIPS buffer using a magnetic plate. After the washing, the magnetic beads were resuspended in LIPS buffer. After the resuspension, 10 μl of the magnetic beads were added to each well of the plate. After the addition, the plate was incubated at room temperature for 30 minutes using a plate mixer. After the incubation, to concentrate immune complexes consisting of antibodies and NanoLuc-viral antigen fusion proteins in the patient samples, the plate was placed on a magnetic plate, and the pellets in each well were washed five times with 150 to 200 μl of LIPS buffer. After washing, the LIPS buffer used for washing was removed, and 100 μl of Nano-Glo luciferase assay substrate (Promega) was added to each well. After the addition, luminescence was measured using a luminometer (Promega). The results are shown in FIG. 1.

[0255] Amino acid sequence of Tax (SEQ ID NO: 4) MAHFPGFGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0256] Amino acid sequence of Env (SEQ ID NO: 25) GKFLATLILFFQFCPLIFGDYSPSCCTLTIGVSSYHSKPCNPAQPVCSWTLDLLALSADQALQPPCPNLVSYSSYHATYSLYLFPHWIKKPNRNGGGYYSASYSDPCSLKCPYLGCQSWTCPYTGAVSSPYWKFQHDVNFTQEVSRLNINLHFSKCGFPFSLLVDAPGYDPIWFLNTEPSQLPPTAPPLLPHSNLDHILEPSIPWKSKLLTLVQLTLQSTNYTCIVCIDRASLSTWHVLYSPN VSVPSSSSTPLLYPSLALPAPHLTLPFNWTHCFDPQIQAIVSSPCHNSLILPPFSLSPVPTLGSRSRRAVPVAVWLVSALAMGAGVAGGITGSMSLASGKSLLHEVDKDISQLTQAIVKNHK NLLKIAQYAAQNRRGLDLLFWEQGGLCKALQEQCCFLNITNSHVSILQERPPLENRVLTGWGLNWDLGLSQWAREALQTGITLVALLLLVILAGPCILRQLRHLPSRVRYPHYSLINPESL

[0257] Amino acid sequence of Gag p15 (SEQ ID NO: 11) VVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNLHRGGGLTSPPTLQQVLPNQDPASIL

[0258] Amino acid sequence of Gag p19 (SEQ ID NO: 13) MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVL

[0259] Amino acid sequence of Gag p24 (SEQ ID NO: 14) PVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAETRGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHTNSPLGDMLRACQTWTPKDKTKVL

[0260] FIG. 4 is a graph showing the results of the LIPS assay. In FIG. 4, the horizontal axis indicates the type of subject, and the vertical axis indicates luminescence intensity. FIG. 4(A) shows the antibody response to Tax, FIG. 4(B) shows the antibody response to Env, FIG. 4(C) shows the antibody response to Gag p15, FIG. 4(D) shows the antibody response to Gag p19, and FIG. 4(E) shows the antibody response to Gag p24. As shown in FIG. 4, antibody responses to Tax and Env were found to be lower in ATL patients compared to HTLV-1-infected patients. Furthermore, antibody responses to Gag p19 and Gag p24 were found to be higher in ATL patients compared to HTLV-1-infected patients.

[0261] (2) Study of Gag Expression In Reference Example 1(1), the ATL patients had low antibody responses to Tax and high antibody responses to Gag, so gene expression of Tax and Gag was studied. Specifically, quantitative reverse transcription PCR (RT-qPCR) was performed. For HTLV-1 cases, ATL cell lines, MT1 cells, MT4 cells, 55T(+) cells, ED cells, and TLOm1 cells, and an HTLV-1-negative T cell line, Jurkat cells, were used. For ATL cases, MT1 cells and serum collected from ATL patients (ATL-1, ATL-2, ATL-3, ATL-4, and ATL-5) were used. Total RNA was extracted from each cell line using TRIzol Reagent (Invitrogen). After extraction, cDNA was synthesized using SuperScript IV Reverse Transcriptase (Invitrogen). Gag cDNA was prepared using the following Gag cDNA synthesis primers. Tax cDNA and 18Sr RNA were synthesized using random primers. After synthesis, Gag expression was measured using Taqman real-time PCR. Tax expression was measured using SYBR-green real-time PCR. 18Sr RNA was quantified using Taqman Gene Expression Assays (Hs99999901_s1; Applied Biosystems). The amplification conditions were 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, with each cycle consisting of 40 cycles. The results are shown in Figure 5.

[0262] Gag cDNA synthesis primer Primer 1 (SEQ ID NO: 26) 5'- TGCAGGATATGGGCC-3'. Gag primer set Primer 2 (SEQ ID NO: 27) 5'-AGTACCTTTGCTCCTCCCTC-3' Primer 3 (SEQ ID NO: 28) 5'-TAATACCTCGGGTTTCGGCC-3'. Gag probe Probe (SEQ ID NO: 29) 5'-TTCCCTCCATCACCAGCTAGATAGCCT-3'. Tax primer set Primer 4 (SEQ ID NO: 30) 5'-CCGCCGATCCCAAAGAA-3' Primer 5 (SEQ ID NO: 31) 5'-CCTGTCCAAACCCTGGGAA-3'

[0263] Figure 5 is a graph showing the results of RT-qPCR. In Figure 5, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative mRNA expression level. Figure 5(A) shows the expression level of Gag mRNA in various cell lines, Figure 5(B) shows the expression level of Tax mRNA in various cell lines, Figure 5(C) shows the expression level of Gag mRNA in ATL case samples, and Figure 5(D) shows the expression level of Tax mRNA in ATL case samples. As shown in Figure 5, it was found that Gag was expressed even in ATL cells and ATL case samples that did not express Tax, thereby revealing a Tax-independent Gag expression mechanism.

[0264] (3) Examination of Gag Expression in Non-Tax-Expressing Cells Next, we examined whether Gag expression was observed in cells that did not express Tax. Specifically, a proximity ligation assay (PLA) was performed. PLA was performed using the Duolink PLA kit (Sigma-Aldrich) according to the manufacturer's instructions. The ATL cell lines, MT4 cells, ATL-55T(+) cells, and ED cells, and the T cell line, Jurkat cells, were used as a negative control. The cells were concentrated onto glass coverslips using a cytospin (Cytospin 2, Shandon). After concentration, 4% paraformaldehyde was added and fixation was performed at room temperature for 15 minutes. After fixation, 0.2% Triton® X-100 was added and permeabilization was performed at room temperature for 15 minutes. After permeabilization, the cells were blocked with Duolink In Situ PLA Probe-Blocking Solution. After blocking, the cells were stained with antibodies. For staining, mouse anti-HTLV-1 p24 monoclonal antibody (clone 46 / 3.24.4, ZeptoMetrix), rabbit polyclonal antibody against HTLV-1 p24 Gag (Cat No. 5418, ABL), or mouse anti-HTLV-1 p19 Gag monoclonal antibody (Cat No. 0801003, ZeptoMetrix) were used. After staining, the glass coverslips were sealed with DAPI-containing In Situ Mounting Medium (Sigma-Aldrich). After sealing, the cells were observed using a Nikon C2 confocal microscope (Nikon). The results are shown in Figure 6.

[0265] Figure 6 is a photograph showing the results of a proximity ligation assay. Figure 6(A) shows the results for Gag p19 antibody. Figure 6(B) shows the results for Gag p24 antibody. In Figure 6, the upper row shows the results of DAPI staining, the middle row shows the results of Gag p19 or Gag p24 staining, and the lower row shows the results of DAPI and Gag p19 or Gag p24 staining. In Figure 6, the area surrounded by a dashed line indicated by an arrow indicates the expression of Gag p19 or Gag p24. As shown in Figure 6(A), it was found that Gag p19 was expressed not only in MT4 cells that express Tax, but also in ATL-55T(+) cells and ED cells that do not express Tax. Furthermore, as shown in Figure 6(B), Gag p24 was found to be expressed not only in MT4 cells that express Tax, but also in ATL-55T(+) cells and ED cells that do not express Tax. These results demonstrate that Gag p19 and p24 are expressed in ATL cells, regardless of the presence or absence of Tax expression.

[0266] (4) Study of cellular immune responses induced by Gag in ATL patients after hematopoietic stem cell transplantation. Next, we investigated whether Gag induces cellular immune responses in ATL patients who have undergone hematopoietic stem cell transplantation and are maintaining long-term remission. Specifically, we used the ELISPOT assay. Based on the amino acid sequence of Gag (GenBank accession number AAA85841.1, SEQ ID NO: 1), overlapping peptides of 9 amino acids in length (offset: 1 amino acid) were designed. The 97 peptides of Gag p15 were cloned into p15-1 (p15 １－５０ ), and p15-2 (p15 ５１－９７ ) and pooled as p19-1 (p19). １－５０ ), p19-2 (p19 ５１－１００ ), and p19-3 (p19 １０１－１２１) were pooled. Peripheral blood mononuclear cells (PBMCs) were collected from ATL patients who had undergone hematopoietic stem cell transplantation and maintained long-term remission. ELISPOT assays were performed using a human IFN-γ ELISPOT kit (MABTECH). The PBMCs were seeded onto ELISPOT plates. After seeding, 0.25 μmol / L of the pooled peptides and 1 μg / ml of purified NA / LE mouse anti-human CD28 antibody (Cat No: 555725, BD Biosciences) were added. After the addition, stimulation was carried out for 6 hours. Spots of IFN-γ-producing cells were developed using an AP Conjugate Kit (Bio-Rad) and counted using an ImmunoSpot S6 Analyzer (CTL). The results are shown in Figure 7.

[0267] Figure 7 is a photograph showing the results of an ELISPOT assay. In Figure 7, the left panel shows the results of stimulation with Gag p19 peptide. The upper and middle panels on the right panel show the results of stimulation with Gag p15 peptide, and the lower panel on the right panel shows the results of unstimulated stimulation. As shown in Figure 7, spots were formed when stimulated with Gag p19 or Gag p15 peptide. These results demonstrated that IFN-γ was produced by Gag p19 or Gag p15 in PBMCs from ATL patients who had undergone hematopoietic stem cell transplantation and maintained long-term remission. Furthermore, the production of IFN-γ suggested the induction of a cellular immune response.

[0268] (5) Study of Gag-induced cellular immune response in mice Next, we investigated whether a cellular immune response was induced in mice immunized with recombinant Gag protein. Specifically, an ELISPOT assay was used. Gag p15 peptides and Gag p19 peptides were designed and pooled in the same manner as in Reference Example 1 (4). Gag p24 peptides were designed in the same manner as in Reference Example 1 (4). 206 Gag p24 peptides were cloned into p24-1 (p24 １－５１ ), p24-2 (p24 ５２－１０３ ), p24-3 (p24 １０４－１５４), and p24-4 (p24 １５５－２０６ ) and pooled. Mice (C57BL / 6J, Jackson Laboratory Japan) were infected with HTLV-1, and 14 days later, splenocytes were collected from the mice. The ELISPOT assay was performed in the same manner as in Reference Example 1 (4), except that splenocytes collected from mice were used as samples.

[0269] Figure 8 is a photograph showing the results of the ELISPOT assay. In Figure 8, the top row (A) shows the results for splenocytes from mouse 1, the middle row (B) shows the results for splenocytes from mouse 2, and the bottom row (C) shows the results for splenocytes from mouse 3. In Figure 8, from left to right, column 2 (Gag p15-1 and 2) shows the results for stimulation with Gag p15, column 3 (Gag p19-3 to 5) shows the results for stimulation with Gag p19, column 4 (Gag p24-6 to 9) shows the results for stimulation with Gag p24, column 1 (10) shows the positive control, and column 1 (11) shows the negative control. As shown in Figure 8, spots were formed when stimulated with Gag p19, Gag p15, or Gag p24 peptides. In particular, many spots were formed when stimulated with Gag p19-3 peptide. These results demonstrated that IFN-γ was produced in mice immunized with Gag proteins, either Gag p15, Gag p19, or Gag p24, suggesting that a cellular immune response was induced.

[0270] The lipid complexes or pharmaceutical compositions of the present disclosure can induce an immune response against HTLV-1. Therefore, the lipid complexes of the present disclosure and pharmaceutical compositions containing the same have potential applications in, for example, the prevention and / or treatment of HTLV-1-associated diseases.

Claims

1. a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of the human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein; a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic Tax protein; and a nucleic acid comprising a polynucleotide encoding an immunogenic fragment of an HTLV-1 antigenic HBZ protein; A lipid complex in which at least one type of nucleic acid selected from the group consisting of: is encapsulated in a lipid.

2. The nucleic acid is a nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof; a nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p19 (Gag p19) or an immunogenic fragment thereof; a nucleic acid comprising a polynucleotide encoding the HTLV-1 antigenic Gag protein p24 (Gag p24) or an immunogenic fragment thereof; a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic Tax protein or an immunogenic fragment thereof; and a nucleic acid comprising a polynucleotide encoding an HTLV-1 antigenic HBZ protein or an immunogenic fragment thereof; The lipid complex according to claim 1, which is at least one selected from the group consisting of:

3. The lipid complex of claim 1, wherein the lipid comprises a cationic lipid and at least one lipid selected from the group consisting of a neutral lipid, a polyethylene glycol-modified lipid, and a sterol.

4. The nucleic acid comprises a nucleic acid comprising a polynucleotide encoding at least one selected from the group consisting of Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and Gag p24 or an immunogenic fragment thereof. The lipid complex of claim 1.

5. The nucleic acid (i) at least one amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 8, 11-14; (ii) an amino acid sequence having at least 80% homology to at least one amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 8, 11 to 14; (iii) an amino acid sequence in which 1 to 31 amino acids in the amino acid sequence of SEQ ID NO: 1, 4, 7, 8, 11 to 14 have been modified by at least one of deletion, substitution, insertion, and addition; (iv) an amino acid sequence comprising at least one amino acid sequence selected from SEQ ID NO: 11 or 12, SEQ ID NO: 13, or SEQ ID NO: 14, and having at least 80% homology to SEQ ID NO: 1; (v) an amino acid sequence comprising at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1, the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1, or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1; (vi) an amino acid sequence comprising at least one of the amino acid sequence of positions 1 to 59 of SEQ ID NO: 1, the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1, or the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1, and having at least 80% homology to SEQ ID NO: 1; (vii) an amino acid sequence comprising at least one of the amino acid sequence of 151 to 208 of SEQ ID NO: 4 or the amino acid sequence of 251 to 308 of SEQ ID NO: 4; (viii) an amino acid sequence comprising at least one of the amino acid sequence of 151 to 208 of SEQ ID NO: 4 or the amino acid sequence of 251 to 308 of SEQ ID NO: 4, and having at least 80% homology to SEQ ID NO: 4; (ix) the amino acid sequence of positions 1 to 58 of SEQ ID NO: 8, or (x) an amino acid sequence comprising the 1st to 58th amino acids of SEQ ID NO: 8 and having at least 80% homology to SEQ ID NO: 8; The lipid complex of claim 1, which is a nucleic acid comprising a polynucleotide encoding:

6. The lipid complex of claim 1, wherein the nucleic acid is a nucleic acid comprising a polynucleotide encoding an antigenic Gag protein.

7. The lipid complex of claim 1, wherein the lipid complex is a lipid nanoparticle (LNP).

8. The lipid complex of claim 1, wherein the nucleic acid is mRNA.

9. The nucleic acid is (i) a polynucleotide consisting of at least one base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 9, 10, 15 to 17; (ii) a polynucleotide consisting of the base sequence of (i) above, in which 1 to 94 bases have been modified by at least one of deletion, insertion, substitution, and addition; (iii) a polynucleotide consisting of a nucleotide sequence having 80% or more identity to the nucleotide sequence of (i); (iv) a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a base sequence complementary to the base sequence of (i); (v) a polynucleotide consisting of at least one of the nucleotide sequences of bases 1 to 177 of SEQ ID NO: 3, bases 154 to 327 of SEQ ID NO: 3, or bases 304 to 390 of SEQ ID NO: 3; (vi) a polynucleotide comprising at least one of the nucleotide sequence of 1 to 177 of SEQ ID NO: 3, the nucleotide sequence of 154 to 327 of SEQ ID NO: 3, or the nucleotide sequence of 304 to 390 of SEQ ID NO: 3, and having an identity of 80% or more to the nucleotide sequence of SEQ ID NO: 3; (vii) a polynucleotide consisting of at least one of the nucleotide sequences of bases 451 to 624 of SEQ ID NO: 6 or the nucleotide sequence of bases 751 to 924 of SEQ ID NO: 6; (viii) a polynucleotide comprising at least one of the nucleotide sequence of 451 to 624 of SEQ ID NO: 6 or the nucleotide sequence of 751 to 924 of SEQ ID NO: 6, and having an identity of 80% or more to the nucleotide sequence of SEQ ID NO: 6; (ix) a polynucleotide consisting of the 1st to 174th base sequence of SEQ ID NO: 10; (x) a polynucleotide comprising a base sequence comprising bases 1 to 174 of SEQ ID NO: 10 and having an identity of 80% or more to the base sequence of SEQ ID NO: 10; The lipid complex according to claim 1, wherein the nucleic acid comprises at least one selected from the group consisting of:

10. The cationic lipid is represented by the following formula (I): 【Transformation 8】 [In the formula, L １ and L ２ each independently represents an alkylene group having 3 to 10 carbon atoms; R １ and R ２ each independently represents an alkyl group having 4 to 22 carbon atoms or an alkenyl group having 4 to 22 carbon atoms; X １ represents a single bond or —CO—O—, and ring P represents any one of the following formulae (P-1) to (P-6). 【Chemistry 9】 [In the formula, R ３ represents an alkyl group having 1 to 3 carbon atoms. The lipid complex according to claim 1, comprising a compound represented by the formula: or a pharmaceutically acceptable salt thereof.

11. The cationic lipid has the following formula (II): 【Chemistry 10】 2-{9-oxo-9-[(3-pentyloctyl)oxy]nonyl}dodecyl 1-methylpiperidine-4-carboxylate or a pharmaceutically acceptable salt thereof. The lipid complex according to claim 1, comprising:

12. A pharmaceutical composition comprising the lipid complex of any one of claims 1 to 11.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is a vaccine.

14. The pharmaceutical composition according to claim 12, for use in the prevention or treatment of an HTLV-1 associated disease.

15. Use of the lipid complex of any one of claims 1 to 11 for the manufacture of a pharmaceutical composition.

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