HTLV-1 nucleic acid lipid particle vaccine

Abstract

Provided is a vaccine for the treatment and / or prevention of infection by human T-cell leukemia virus type 1 (HTLV-1).　A lipid particle of the present invention has encapsulated therein a nucleic acid that enables expression of the Tax antigen or the gp46 antigen of HTLV-1. The particle includes a lipid including a cationic lipid represented by general formula (Ia) or a pharmaceutically acceptable salt thereof. In the formula: R1 and R2 independently represent a C1-C3 alkyl group; L1 represents a C17-C19 alkenyl group optionally having one or more C2-C4 alkanoyloxy groups; L2 represents a C10-C19 alkyl group optionally having one or more C2-C4 alkanoyloxy groups or represents a C10-C19 alkenyl group optionally having one or more C2-C4 alkanoyloxy groups; and p represents 3 or 4.

Description

[Technical field]

[0001] The present invention relates to a nucleic acid lipid particle vaccine encapsulating HTLV-1 mRNA. [Background technology]

[0002] Human T-cell leukemia virus type 1 (HTLV-1) is a highly oncogenic retrovirus. There are 10 to 20 million HTLV-1-infected people worldwide, and 1 million in Japan (Non-Patent Document 1). 3 to 5% of HTLV-1-infected people develop adult T-cell leukemia (ATL) and have a poor prognosis. At present, there is no vaccine to prevent HTLV-1 infection and no drug to prevent ATL onset. In addition, no standard treatment that contributes to a satisfactory therapeutic effect for ATL has been established. For example, the long-term survival rate of patients with multi-drug chemotherapy, which is one of the standard treatments, is less than 10%.

[0003] In recent years, allogeneic hematopoietic stem cell transplantation has been performed for ATL patients, and some patients have achieved high therapeutic effects. The antitumor effect of allogeneic hematopoietic stem cell transplantation is thought to be due to the effects of pre-transplant treatment using large amounts of anticancer drugs and whole-body radiation, as well as the contribution of the antitumor effect (graft versus leukemia effect; GVL effect) by the donor's immune cells after transplantation (Non-Patent Document 2). In particular, it has been reported that cytotoxic T lymphocytes (CTLs) specific to the transcriptional activator Tax, which is essential for HTLV-1 replication, increase (Non-Patent Document 3). However, the mortality rate due to graft versus host disease (GVHD), a side effect of allogeneic hematopoietic stem cell transplantation, is high.

[0004] Tax has the ability to activate NF-kB and is a protein essential for HTLV-1 replication (Non-Patent Document 4). Excessive NF-kB activation via Tax has also been observed in HTLV-1-infected cells and ATL cells, and it has been revealed that NF-kB activation by Tax correlates with the canceration of infected cells (Non-Patent Document 5, Non-Patent Document 6). In addition, Non-Patent Document 7 identifies amino acids that are important for NF-kB activation by Tax, and the mechanism of action of NF-kB activation by Tax has been analyzed using Tax mutants with point mutations inserted into those amino acids (Non-Patent Document 8, Non-Patent Document 9). However, there are no examples of effective vaccines that use Tax or Tax mutants to prevent or treat HTLV-1.

[0005] The results of HTLV-1 infection experiments using immunodeficient mice transplanted with human peripheral blood mononuclear cells (hPBMCs) have revealed that HTLV-1 infection is neutralized by anti-gp46 antibodies that target gp46, an envelope protein of HTLV-1 (Non-Patent Document 10). In addition, a vertical infection model using rats suggests that anti-gp46 antibodies with neutralizing activity may prevent vertical infection. However, there are no examples of effective vaccines that use gp46 to prevent or treat HTLV-1.

[0006] Known LNP formulations (LNP-mRNA) in which mRNA encoding an antigen is encapsulated in lipid nanoparticles (LNP) include the SARS-CoV-2 vaccine (Non-Patent Document 11) and the influenza vaccine (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2015 / 164674 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Front Microbiol.(2012)3:388. [Non-Patent Document 2] Blood (2012) 119(10):2409-2416. [Non-Patent Document 3] Cancer Research (2010)70(15):6181-6192. [Non-Patent Document 4] Lancet Infect Dis(2007)7:266-281. [Non-Patent Document 5] Front Microbiol.(2012)3:406. [Non-Patent Document 6] Viruses(2011)3(6):714-749. [Non-Patent Document 7] Gene Dev.(1990)4:1875. [Non-Patent Document 8] J.Biochem.(2011)150(6):679-686. [Non-Patent Document 9] J. Biol Chem. (2006)281(19):13075-13082. [Non-Patent Document 10] Viruses (2016) 8(2):41. [Non-Patent Document 11] Science (2021) 371:1152. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a vaccine for preventing and / or treating infection with Human T-cell Leukemia Virus type 1 (HTLV-1). [Means for solving the problem]

[0010] The present inventors have found that nucleic acid lipid particles (LNP-mRNA) encapsulating HTLV-1 mRNA can be applied to the treatment and prevention of onset of adult T-cell leukemia, and have completed the present invention. Specifically, they have produced LNP-mRNA-HTLV-1 encapsulating mRNA expressing HTLV-1 Tax and gp46, and have found that in mice and monkeys administered with LNP-mRNA-HTLV-1, antibody production and CTL against HTLV-1 Tax and gp46, as well as neutralizing activity that inhibits the proliferation of HTLV-1-infected cells, are induced.

[0011] The gist of the present invention is as follows. (1) A lipid particle encapsulating a nucleic acid capable of expressing the gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1), wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharma- ceutical acceptable salt thereof. [ka] During the ceremony, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 may have one or more C2-C4 alkanoyloxy groups; 17 -C 19 represents an alkenyl group; L 2 may have one or more C2-C4 alkanoyloxy groups; 10 -C 19 C which may have one or more alkyl groups or C2-C4 alkanoyloxy groups 10 -C 19 represents an alkenyl group; p is 3 or 4. (2) R in general formula (Ia) 1 and R 2 and are both methyl groups. (3) The particle according to (1) or (2), wherein p in general formula (Ia) is 3. (4) L in general formula (Ia) 1 C may have one or more acetoxy groups 17 -C 19 The particle according to any one of (1) to (3), wherein the group is an alkenyl group. (5) L in general formula (Ia) 2 C may have one or more acetoxy groups 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 10 -C 19 The particle according to any one of (1) to (4), wherein the group is an alkenyl group. (6) L in general formula (Ia) 2 C may have one or more acetoxy groups 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 17 -C 19 The particle according to any one of (1) to (4), wherein the group is an alkenyl group. (7) L in general formula (Ia) 1 The particles according to any one of (1) to (6), wherein is a (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or a (8Z,11Z)-heptadecadienyl group. (8) L in general formula (Ia) 2 is a decyl group, a cis-7-decenyl group, a dodecyl group, or a (R)-11-acetyloxy-cis-8-heptadecenyl group. (9) The cationic lipid has the following structural formula: [ka] The particle according to (1), (10) The cationic lipid has the following structural formula: [ka] The particle according to (1), (11) The cationic lipid has the following structural formula: [ka] The particle according to (1), (12) The particle according to (9) or (10), wherein the lipid further comprises an amphiphilic lipid, a sterol, and a PEG lipid. (13) The particle according to (11), wherein the lipid further comprises an amphiphilic lipid, a sterol, and a PEG lipid. (14) The particles according to (12), wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. (15) The particles according to (13), wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. (16) The particles according to (12) or (14), wherein the sterol is cholesterol. (17) The particles according to (13) or (15), wherein the sterol is cholesterol. (18) The particle according to any one of (12), (14), and (16), wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine. (19) The particle according to any one of (13), (15), and (17), wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine. (20) The particles according to any one of (12) to (19), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid. (21) The particles according to (20), wherein the amphiphilic lipid is 10 to 25%. (22) The particle according to any one of (12), (14), (16), and (18), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 15% amphipathic lipid, 35 to 50% sterols, 40 to 55% cationic lipid, and 1 to 3% PEG lipid. (23) The particles according to (22), comprising 10 to 15% amphiphilic lipid, 35 to 45% sterols, 40 to 50% cationic lipid, and 1 to 2.5% PEG lipid. (24) The particles according to (23), wherein the PEG lipid content is 1 to 2%. (25) The particle according to any one of (13), (15), (17), and (19), wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% PEG lipid. (26) The particles according to (25), comprising 10 to 45% sterols, 42.5 to 65% cationic lipid, and 1 to 2.5% PEG lipid. (27) The particles according to (26), wherein the PEG lipid content is 1 to 2%. (28) The particles according to any one of (20) to (27), wherein the ratio of the total lipid weight to the nucleic acid weight is 15 to 30. (29) The particles according to (28), wherein the ratio of the total lipid weight to the nucleic acid weight is 15 to 25. (30) The particles according to (29), wherein the ratio of the total lipid weight to the nucleic acid weight is 17.5 to 22.5.

[0012] (31) The particle according to any one of (1) to (30), wherein the gp46 antigen of human T-cell leukemia virus type 1 (HTLV-1) is a fusion protein with an oligomerization domain. (32) The particle according to (31), wherein the oligomerization domain is fibritin. (33) The particle according to (31) or (32), wherein the gp46 antigen of human T-cell leukemia virus type 1 (HTLV-1) consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:14. (34) The particle according to any one of (1) to (33), wherein the Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) has at least one mutation selected from the group consisting of T130A, L131S, L319R and L320S with respect to the amino acid sequence of SEQ ID NO: 11, and when compared with an amino acid sequence other than the mutated amino acids, the amino acid sequence has at least 95% identity with the amino acid sequence of SEQ ID NO: 11. (35) A particle described in (34), in which the Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) has the mutations T130A, L131S, L319R and L320S with respect to the amino acid sequence of SEQ ID NO: 11, and when compared with the amino acid sequence other than the mutated amino acids, the amino acid sequence has at least 95% identity with the amino acid sequence of SEQ ID NO: 11. (36) The particle according to any one of (1) to (35), wherein the Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) is a fusion protein with a signal peptide. (37) The particle according to (36), wherein the signal peptide is a peptide consisting of the 1st to 18th amino acids of the amino acid sequence of SEQ ID NO:13. (38) A particle described in any of (31) to (37), wherein the nucleic acid capable of expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) is an mRNA including a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of gp46 antigen or Tax antigen, a 3' untranslated region (3'-UTR), and a polyA tail (polyA). (39) The particle described in (38), wherein the nucleic acid sequence capable of expressing the gp46 antigen of human T-cell leukemia virus type 1 (HTLV-1) consists of a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 17 or 18. (40) The particle described in (38), wherein the nucleic acid sequence capable of expressing the Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) consists of a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 20.

[0013] (41) The particle according to any one of (1) to (40), wherein the nucleic acid contains at least one modified nucleotide. (42) The particle according to (41), wherein the modified nucleotide comprises at least one pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine nucleotide optionally substituted at the 1-position. (43) The particle according to (41), wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine. (44) The particle according to (41), wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine. (45) The particles according to any one of (1) to (44), having an average particle size of 30 to 300 nm.

[0014] (46) Use of the particles according to any one of (1) to (45) for producing a composition for preventing and / or treating infection with human T-cell leukemia virus type 1 (HTLV-1). (47) A composition comprising the particles according to any one of (1) to (45). (48) The composition according to (47) for expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) in vivo or in vitro. (49) The composition according to (47) or (48) for use as a medicine. (50) The composition according to (49) for inducing an immune response against human T-cell leukemia virus type 1 (HTLV-1). (51) The composition according to (49) or (50) for preventing and / or treating infection with human T-cell leukemia virus type 1 (HTLV-1). (52) The composition according to (49) or (50) for preventing and / or treating the onset of a disease caused by HTLV-1 selected from the group consisting of adult T-cell leukemia-lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU) in an HTLV-1-infected individual. (53) A method for expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) in vitro, comprising introducing the composition according to any one of (47) to (50) into a cell. (54) A method for expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) in vivo, comprising administering the composition according to any one of (47) to (51) to a mammal. (55) A method for inducing an immune response against human T-cell leukemia virus type 1 (HTLV-1), comprising administering to a mammal the composition according to (49) or (50). (56) A method for preventing and / or treating infection with human T-cell leukemia virus type 1 (HTLV-1), comprising administering to a mammal the composition according to any one of (49) to (52). (57) A method for preventing and / or treating the onset of a disease caused by HTLV-1 selected from the group consisting of adult T-cell leukemia-lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU), comprising administering to a mammal any of the compositions according to (49) to (52). (58) A peptide comprising a Tax antigen of human T-cell leukemia virus type 1 (HTLV-1), which has a mutation that reduces or eliminates carcinogenicity, fused with a signal peptide. (59) The peptide of (58), wherein the Tax antigen has at least one mutation selected from the group consisting of T130A, L131S, L319R and L320S with respect to the amino acid sequence of SEQ ID NO: 11, and when compared with the amino acid sequence excluding the mutated amino acids, the peptide has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11. (60) The peptide of (58) or (59), wherein the signal peptide is a peptide consisting of the 1st to 18th amino acids of the amino acid sequence of SEQ ID NO: 13. (61) A particle described in any of (31) to (37), wherein the nucleic acid capable of expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), and the translation region and 3' untranslated region (3'-UTR) of the gp46 antigen or Tax antigen. (62) A particle described in (61), wherein the cap structure (Cap), 5' untranslated region (5'-UTR), translation region and 3' untranslated region (3'-UTR) of gp46 antigen or Tax antigen are a nucleotide sequence having at least 90% identity to the sequence from base 1 to base 1141 of SEQ ID NO: 17 or the sequence from base 1 to base 1222 of SEQ ID NO: 18. (63) A particle described in (61), wherein the cap structure (Cap), 5' untranslated region (5'-UTR), translation region and 3' untranslated region (3'-UTR) of gp46 antigen or Tax antigen are a nucleotide sequence having at least 90% identity to the sequence from the 1st to the 1315th positions of SEQ ID NO: 20. This specification includes the disclosures of Japanese Patent Application No. 2021-084942, which is the priority basis of this application. Effect of the Invention

[0015] The present invention makes it possible to prevent and / or treat infection with human T-cell leukemia virus type 1 (HTLV-1). The present invention also makes it possible to prevent and / or treat diseases caused by infection with HTLV-1. The particles of the present invention have excellent properties in terms of metabolic stability, in vitro activity, in vivo activity, rapidity of onset of efficacy, duration of efficacy, physical stability, drug interactions, safety, etc., and are useful as medicines for treating or preventing the above-mentioned diseases. [Brief description of the drawings]

[0016] [Figure 1]This figure shows the HIV-1 LTR transcription activity (A) and HTLV-1 LTR transcription activity (B) of Tax wild-type, Tax mutant, and secreted Tax mutant. pHIV-1 LTR indicates the HIV-1 LTR reporter plasmid-administered group, pHTLV-1 LTR indicates the HTLV-1 LTR reporter plasmid-administered group, and Empty indicates the negative control group, pcDNA3.1+ vector. Experiments were performed in triplicate, and the vertical bars indicate the average value, with the error bars indicating SD. The white circles on the bar graphs indicate individual data from triplicates. Tax WT: Tax wild-type, Tax Mut: Tax mutant, Sec-Tax Mut: secreted Tax mutant. [Diagram 2] 2 shows the Tax-specific CTL induction level and blood anti-Tax antibody titer in C3H mice administered the lipid particles of Example 12. Fig. 2A shows the Tax-specific CTL induction level, and Fig. 2B shows the blood anti-Tax antibody titer. [Diagram 3] This is a diagram showing anti-gp46 antibody titers and anti-Tax antibody titers in monkey blood. Figure 3A shows anti-gp46 specific antibody titers in blood, and Figure 3B shows anti-Tax antibody titers in blood. 4 monkeys per group. 0W, 2W, 4W, and 6W respectively show data before administration, 2 weeks after the first administration, 4 weeks after (2 weeks after the second administration), and 6 weeks after (2 weeks after the third administration). Bars show average values, and white circles show individual data. [Figure 4] Figure 1 shows gp46-specific and Tax-specific cellular immune responses. Four mice per group. Vertical bars indicate mean values, error bars indicate SD. White circles on bar graphs indicate individual data. [Diagram 5] This is a diagram showing anti-HTLV-1 neutralizing activity in monkey blood. The assay negative control shows the result of no antibody treatment, and the assay positive control shows the result of treatment with a known HTLV-1 neutralizing antibody. There were 4 monkeys in each group. #736, #741, #743, and #737 indicate the individual monkey IDs of the negative control group, and #745, #742, #740, and #735 indicate the individual monkey IDs of the mixed preparation groups of Examples 5 and 6. The arrows indicate syncytial formation. [Figure 6]FIG. 1 shows anti-gp46 antibody titers in the blood of C57BL / 6 mice. [Figure 7] 7A and 7B show gp46- and Tax-specific cellular immune responses in C57BL / 6 mice, with FIG 7A showing IFN-γ production levels and FIG 7B showing IL-2 production levels. [Figure 8] 8A and 8B show the expression levels of gp46 and Tax proteins in CHO-S cells treated with the lipid particles of Examples 9 to 12. Fig. 8A shows the expression levels in the cell lysate, and Fig. 8B shows the expression levels in the culture supernatant. [Figure 9] FIG. 1 shows the nucleotide sequence of template plasmid DNA for HTLV-I SU gp46 IVT (SEQ ID NO:1). [Figure 10] FIG. 2 shows the nucleotide sequences of the sense primer (SEQ ID NO: 2) and the antisense primer (SEQ ID NO: 3). [Figure 11] FIG. 4 shows the nucleotide sequence of template DNA for in vitro transcription (IVT) of SU gp46 (SEQ ID NO:4). [Figure 12] FIG. 5 shows the nucleotide sequence of template plasmid DNA for HTLV-I gp46-Fib IVT. [Figure 13] FIG. 6 shows the nucleotide sequence of template DNA for in vitro transcription (IVT) of gp46-Fib (SEQ ID NO: 6). [Figure 14] FIG. 7 shows the nucleotide sequence of template plasmid DNA for HTLV-I dgp62-Fib IVT (SEQ ID NO:7). [Figure 15] FIG. 8 shows the nucleotide sequence of template DNA for in vitro transcription (IVT) of dgp62-Fib (SEQ ID NO: 8). [Figure 16] FIG. 9 shows the nucleotide sequence of template plasmid DNA for HTLV-I sec Tax mutant IVT. [Figure 17]FIG. 1 shows the nucleotide sequence (SEQ ID NO: 10) of template DNA for in vitro transcription (IVT) of sec Tax mutant. [Figure 18] FIG. 1 shows the amino acid sequence of Tax wild-type (SEQ ID NO:11). [Figure 19] FIG. 12 shows the amino acid sequence of Tax mutant (T130A / L131S / L319R / L320S). [Figure 20] FIG. 1 shows the amino acid sequence (SEQ ID NO: 13) of the secreted Tax mutant (T130A / L131S / L319R / L320S). [Figure 21] FIG. 1 shows the amino acid sequence of gp46 (SEQ ID NO:14). [Figure 22] FIG. 1 shows the amino acid sequence of gp46-Fib (SEQ ID NO: 15). [Figure 23] FIG. 1 shows the amino acid sequence of dgp62-Fib (SEQ ID NO: 16). [Figure 24] FIG. 17 shows the nucleotide sequence of SU gp46 mRNA. [Diagram 25] FIG. 1 shows the nucleotide sequence of gp46-Fib mRNA (SEQ ID NO: 18). [Figure 26] FIG. 1 shows the nucleotide sequence of dgp62-Fib mRNA (SEQ ID NO: 19). [Figure 27] FIG. 2 shows the nucleotide sequence of sec Tax mutant mRNA (SEQ ID NO: 20). [Figure 28] 1 shows the expression levels of gp46 protein in CHO-S cells treated in Examples 30 to 34. The dotted line indicates the OD value of the negative control Buffer. [Figure 29] FIG. 2 shows the nucleotide sequence of the template DNA of gp46-Fib, polyA95 (SEQ ID NO: 21). [Diagram 30] FIG. 2 shows the nucleotide sequence of the template DNA of gp46-Fib, polyA80 (SEQ ID NO: 22). [Diagram 31]FIG. 2 shows the nucleotide sequence polyA60 (SEQ ID NO: 23) of the template DNA of gp46-Fib. [Diagram 32] FIG. 2 shows the nucleotide sequence of the template DNA of gp46-Fib, polyA40 (SEQ ID NO: 24). [Diagram 33] FIG. 2 shows the nucleotide sequence of the template DNA of gp46-Fib, polyA20 (SEQ ID NO: 25). [Diagram 34] FIG. 2 shows the nucleotide sequence of polyA95 (SEQ ID NO: 26) of the template DNA of the sec Tax mutant. [Diagram 35] FIG. 2 shows the nucleotide sequence of polyA80 (SEQ ID NO: 27) of the template DNA of the sec Tax mutant. [Diagram 36] FIG. 2 shows the nucleotide sequence of polyA60 (SEQ ID NO: 28) of the template DNA of the sec Tax mutant. [Figure 37] FIG. 2 shows the nucleotide sequence of polyA40 (SEQ ID NO: 29) of the template DNA of the sec Tax mutant. [Figure 38] FIG. 1 shows the nucleotide sequence of polyA20 (SEQ ID NO: 30) of the template DNA of the sec Tax mutant. [Figure 39] FIG. 1 shows the gp46-Fib mRNA sequence polyA95 (SEQ ID NO: 31). [Diagram 40] FIG. 1 shows the gp46-Fib mRNA sequence polyA80 (SEQ ID NO: 32). [Diagram 41] FIG. 1 shows the gp46-Fib mRNA sequence polyA60 (SEQ ID NO: 33). [Diagram 42] FIG. 1 shows the gp46-Fib mRNA sequence polyA40 (SEQ ID NO: 34). [Diagram 43] FIG. 1 shows the gp46-Fib mRNA sequence polyA20 (SEQ ID NO: 35). [Diagram 44] FIG. 1 shows the mRNA sequence polyA95 (SEQ ID NO: 36) of the sec Tax mutant. [Diagram 45] FIG. 1 shows the mRNA sequence polyA80 (SEQ ID NO: 37) of the sec Tax mutant. [Diagram 46] FIG. 1 shows the mRNA sequence polyA60 (SEQ ID NO: 38) of the sec Tax mutant. [Figure 47] FIG. 1 shows the mRNA sequence polyA40 (SEQ ID NO: 39) of the sec Tax mutant. [Figure 48] FIG. 1 shows the mRNA sequence polyA20 (SEQ ID NO: 40) of the sec Tax mutant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, the embodiment of the present invention will be described in more detail.

[0018] The present invention provides lipid particles encapsulating a nucleic acid capable of expressing HTLV-1 gp46 antigen or Tax antigen, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharma- ceutical acceptable salt thereof.

[0019] [ka]

[0020] During the ceremony, R 1 and R 2 each independently represents a C1-C3 alkyl group; L 1 may have one or more C2-C4 alkanoyloxy groups; 17 -C 19 represents an alkenyl group; L 2 may have one or more C2-C4 alkanoyloxy groups; 10 -C 19 C which may have one or more alkyl groups or C2-C4 alkanoyloxy groups 10 -C 19 represents an alkenyl group; p is 3 or 4.

[0021] R in general formula (Ia) 1 and R 2 each independently represents a C1-C3 alkyl group, and preferably, both are a methyl group.

[0022] In the general formula (Ia), p is 3 or 4, and is preferably 3.

[0023] L in general formula (Ia) 1 may have one or more C2-C4 alkanoyloxy groups; 17 -C 19 It represents an alkenyl group, and preferably represents an alkenyl group which may have one or more acetoxy groups. 17 -C 19 It is an alkenyl group. 1 Specific examples of such an alkyl group include an (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, and a (8Z,11Z)-heptadecadienyl group.

[0024] L in general formula (Ia) 2 may have one or more C2-C4 alkanoyloxy groups; 10 -C 19 C which may have one or more alkyl groups or C2-C4 alkanoyloxy groups 10 -C 19 It represents an alkenyl group, and preferably represents an alkenyl group which may have one or more acetoxy groups. 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 10 -C 19 Alternatively, L in formula (Ia) is an alkenyl group. 2 C may have one or more acetoxy groups 10 -C 12 C which may have one or more alkyl groups or acetoxy groups 17 -C 19It is also preferred that L is an alkenyl group. 2 Specific examples of the alkyl group include a decyl group, a cis-7-decenyl group, a dodecyl group, and an (R)-11-acetyloxy-cis-8-heptadecenyl group.

[0025] Specific examples of cationic lipids that are components constituting the particles of the present invention include those having the following structural formula: [ka] [ka] [ka] Examples of the compound include (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate, (9Z,12Z)-3-dimethylaminopropyl(9Z,12Z)-octacos-19,22-dien-11-yl carbonate, and (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate, each of which is represented by the formula:

[0026] The cationic lipid represented by general formula (Ia) may be one type of compound or a combination of two or more types of compounds.

[0027] A method for producing cationic lipids of general formula (Ia) is described in WO 2015 / 005253.

[0028] The lipids of the present invention may further include amphipathic lipids, sterols and PEG lipids.

[0029] The amphipathic lipid is a lipid having affinity for both polar and non-polar solvents, and specific examples thereof include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and combinations thereof. The amphipathic lipid used in the particles of the present invention is preferably distearoylphosphatidylcholine and / or dioleoylphosphatidylethanolamine, and more preferably distearoylphosphatidylcholine.

[0030] The sterols are sterols having a hydroxyl group, and a specific example thereof is cholesterol.

[0031] The PEG lipid is a PEG-modified lipid, specifically, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, combinations thereof, etc., are exemplified, but 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol is preferred. The average molecular weight of the PEG lipid is not particularly limited, but is, for example, 1000 to 5000, preferably 1500 to 3000, more preferably 1800 to 2200.

[0032] The lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but may be, for example, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid in molar amounts. It is preferable that the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid. It is more preferable that the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 22.5% amphipathic lipid, 15 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid. In the lipid composition, the ratio of PEG lipid is more preferably 1-3% by molar amount, even more preferably 1-2%, even more preferably 1.2-2%, even more preferably 1.25-2%, even more preferably 1.3-2%, and particularly preferably 1.5-2%. In addition, in the lipid composition, the ratio of total lipid weight to nucleic acid weight is not particularly limited, but may be 15-30, preferably 15-25, more preferably 15-22.5, and even more preferably 17.5-22.5.

[0033] When 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate or (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate is used as the cationic lipid, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, but for example, in molar amounts, the amphipathic lipid is 5 to 25%, the sterols are 10 to 55%, the cationic lipid is 40 to 65%, and the PEG lipid is 1 to 5%, and it is preferable that the amphipathic lipid is 15% or less, the sterols are 20 to 55%, the cationic lipid is 40 to 65%, and the PEG lipid is 1 to 5%. It is more preferable that the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 15% amphipathic lipid, 35 to 50% sterols, 40 to 55% cationic lipid, and 1 to 3% PEG lipid; it is even more preferable that the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 15% amphipathic lipid, 35 to 45% sterols, 40 to 50% cationic lipid, and 1 to 2.5% PEG lipid; it is even more preferable that the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 15% amphipathic lipid, 35 to 45% sterols, 40 to 50% cationic lipid, and 1 to 2% PEG lipid. In the lipid composition, the PEG lipid is more preferably 1.2 to 2%, even more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and even more preferably 1.5 to 2%. In the lipid composition, the ratio of the total lipid weight to the nucleic acid weight is not particularly limited, but may be 15 to 30, preferably 15 to 25, more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0034] When (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate is used as the cationic lipid, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is not particularly limited, and for example, the lipid composition is preferably, in molar amounts, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid, and more preferably, the lipid composition is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid, and more preferably, the lipid composition is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% PEG lipid. It is more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 10 to 25% amphipathic lipids, 10 to 45% sterols, 42.5 to 65% cationic lipids, and 1 to 2.5% PEG lipids. It is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 15 to 22.5% amphipathic lipids, 15 to 40% sterols, 45 to 65% cationic lipids, and 1 to 2% PEG lipids. It is even more preferable that the lipid composition of amphipathic lipids, sterols, cationic lipids, and PEG lipids is, in molar amounts, 17.5 to 22.5% amphipathic lipids, 15 to 40% sterols, 45 to 65% cationic lipids, and 1 to 2% PEG lipids. In the lipid composition, the PEG lipid is more preferably 1.2 to 2%, even more preferably 1.25 to 2%, even more preferably 1.3 to 2%, and even more preferably 1.5 to 2%. In the lipid composition, the ratio of the total lipid weight to the nucleic acid weight is not particularly limited, but is preferably 15 to 30, more preferably 15 to 25, even more preferably 15 to 22.5, and even more preferably 17.5 to 22.5.

[0035] Specific lipid combinations in the present invention include distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or dioleoylphosphatidylethanolamine as amphipathic lipids, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacos-19,22-dien-11-yl carbonate, or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as cationic lipids, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol or N-[methoxy] Poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine may be used in combination. Also preferred is a lipid combination using distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine as amphiphilic lipid, cholesterol as sterols, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as cationic lipid, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as PEG lipid. More preferred specific lipid combinations in the present invention include distearoylphosphatidylcholine as the amphipathic lipid, cholesterol as the sterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate as the cationic lipid, and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol as the PEG lipid.

[0036] In the present invention, the nucleic acid encapsulated in the lipid particles is capable of expressing the gp46 antigen or Tax antigen of HTLV-1.

[0037] The amino acid sequence of the HTLV-1 gp46 antigen is shown in SEQ ID NO: 14. The nucleic acid to be encapsulated in the lipid particles may encode the HTLV-1 gp46 antigen, which consists of an amino acid sequence having at least 95%, preferably 96%, more preferably 97%, and more preferably 98% identity to the amino acid sequence of SEQ ID NO: 14.

[0038] The gp46 antigen may be a fusion protein with an oligomerization domain. The oligomerization domain is a protein that multimerizes a fused protein, and an example of this is fibritin derived from T4 bacteriophage. A foldon domain, which is a trimerization domain of fibritin, may also be used. The amino acid sequence of the fusion protein of gp46 of HTLV-1 and fibritin is shown in SEQ ID NO: 15. The amino acid sequence of 313th to 339th amino acids in the amino acid sequence of SEQ ID NO: 15 is the amino acid sequence of fibritin. The oligomerization domain may be located at the C-terminus or N-terminus of the gp46 antigen.

[0039] The amino acid sequence of the wild-type Tax antigen of HTLV-1 is shown in SEQ ID NO: 11. The nucleic acid to be encapsulated in the lipid particles may be one that encodes the Tax antigen of HTLV-1, which is composed of an amino acid sequence having at least 95%, preferably 96%, more preferably 97% identity with the amino acid sequence of SEQ ID NO: 11. The wild-type Tax antigen has HTLV-1 LTR transcription activity and is carcinogenic, so that it is necessary to reduce or eliminate the toxicity of carcinogenicity to the living body. For example, a Tax antigen having a mutation that reduces or eliminates carcinogenicity may be used. In addition, since the carcinogenicity of Tax to cells decreases when it is secreted outside the cells, it may be a fusion protein with a signal peptide. Examples of mutations that reduce or eliminate carcinogenicity include T130A, L131S, L319R, and L320S. T130A and L131S are HIV-1 LTR and HTLV-1 transcription activity-inactivating mutations, and L319R and L320S are HTLV-1 transcription activity-deleting mutations. It is sufficient that the mutant Tax antigen has at least one of these mutations, and preferably has two, more preferably three, and particularly preferably four mutations. The amino acid sequence of the mutant Tax antigen having the above four mutations is shown in SEQ ID NO: 12. An example of a signal peptide for secreting the Tax antigen outside the cell is an IgE secretory signal peptide. The signal peptide is preferably fused to the N-terminus of the Tax antigen. The amino acid sequence of the fusion protein of the signal peptide and the mutant Tax antigen having the above four mutations is shown in SEQ ID NO: 13. The 1st to 18th amino acids of the amino acid sequence of SEQ ID NO: 13 are the amino acid sequence of the IgE secretory signal peptide.

[0040] The present invention encompasses a peptide that is a secretory Tax mutant in which an IgE secretion signal is added to a Tax mutant in which a signal peptide is fused to a Tax antigen having at least one, preferably two, more preferably three, and particularly preferably four of the above four amino acid mutations. The peptide is a Tax antigen of human T-cell leukemia virus type 1 (HTLV-1), in which a signal peptide is fused to a Tax antigen having a mutation that reduces or eliminates carcinogenicity. Examples of mutations that reduce or eliminate carcinogenicity include T130A, L131S, L319R, and L320S. That is, examples of the above peptide include a peptide in which a Tax antigen has at least one mutation selected from the group consisting of T130A, L131S, L319R, and L320S with respect to the amino acid sequence of SEQ ID NO: 11, and when the amino acid sequence other than the mutant amino acid is compared, the peptide has an amino acid sequence that has at least 95%, preferably 96%, and more preferably 97% identity with the amino acid sequence of SEQ ID NO: 11. Furthermore, an example of a signal peptide for secreting the Tax antigen outside the cell is an IgE secretory signal peptide. The signal peptide is preferably fused to the N-terminus of the Tax antigen. The amino acid sequence of the IgE secretory signal peptide is the 1st to 18th amino acids of the amino acid sequence of SEQ ID NO: 13. That is, an example of the above peptide is a peptide in which the signal peptide consists of the 1st to 18th amino acids of the amino acid sequence of SEQ ID NO: 13.

[0041] In the present invention, the nucleic acid encapsulated in the lipid particle may be capable of expressing a protein in which the gp46 antigen and the Tax antigen of HTLV-1 are linked by a linker. The linker is composed of an amino acid sequence containing a sequence that is cleaved by a protease, and examples thereof include linkers containing an amino acid sequence that is recognized and cleaved by the protease Furin. The protease cleavage sequence may be any sequence that is cleaved by the Furin protein, and examples thereof include a sequence represented by RXK / RR (R represents arginine, K represents lysine, and X represents any amino acid) (J. Biol. Chem. 1992, 267, 16396; J. Biol. Chem. 1991, 266, 12127).

[0042] The identity of an amino acid sequence is a numerical representation of the percentage of amino acid identity relative to the full length sequence, with amino acids that are completely identical to the corresponding amino acids being considered as the same amino acid. The identity of a sequence in the present invention is calculated using sequence analysis software GENETYX-SV / RC (manufactured by Genetyx Corporation), and this algorithm is commonly used in the technical field. The amino acids encoded by the nucleic acid encapsulated in the lipid particles of the present invention may contain amino acid mutations (substitutions), deletions, insertions and / or additions, so long as they maintain a certain level of identity with SEQ ID NOs: 11 to 16.

[0043] The amino acids encoded by the nucleic acid to be encapsulated in the lipid particles of the present invention retain the above-mentioned sequence identity, and may be substituted, deleted, inserted and / or added at several positions (preferably 5 positions or less, more preferably 3, 2 or 1 position) in the amino acid sequences of SEQ ID NOs: 11 to 16, with several amino acids per position (preferably 10 positions or less, more preferably 7 positions or less, even more preferably 5, 4, 3, 2 or 1 position).

[0044] The nucleic acid capable of expressing the gp46 antigen or Tax antigen of HTLV-1 may be an mRNA containing a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of gp46 or Tax, a 3' untranslated region (3'-UTR) and a polyA tail (polyA). The KOZAK sequence may also be present on the 5' side of the translation region of gp46 or Tax. The cap structure (Cap) is present at the 5' end of many eukaryotic mRNAs and is a site having a 7-methylguanosine structure. Examples of the cap structure include cap0, cap1, cap2, and cap structures when ARCA (Anti-Reverse Cap Analog) is used, and the cap structure is as shown in the following structural formula.

[0045] [ka]

[0046] (In the formula, Base represents any unmodified or modified nucleic acid base, and RNA represents any polynucleotide.)

[0047] [ka]

[0048] [ka]

[0049] [ka]

[0050] The cap structure of the mRNA of the present invention is preferably cap0 or cap1, and more preferably cap1. The sequence of the 5' untranslated region (5'-UTR) can be, for example, a sequence containing the 5' untranslated region of the human β-globin gene. For example, the sequence of the 5' untranslated region of the human β-globin gene is the sequence of base numbers 15 to 64 in the sequence of SEQ ID NO: 17. The sequence of the gp46 translation region is a sequence that can express all or a part of the amino acid sequence of the gp46 antigen, and may contain an initiation codon and / or a stop codon, for example, the sequence of base numbers 71 to 1009 in the sequence of SEQ ID NO: 17. In addition, the sequence of the gp46 translation region and the fibritin sequence may be linked, for example, the sequence of base numbers 71 to 1090 in SEQ ID NO: 18. The sequence of the translation region of dgp62 is a sequence capable of expressing all or part of the amino acid sequence of the dgp62 antigen, and may contain a start codon and / or a stop codon, for example, the sequence of nucleotide numbers 71 to 1396 in the sequence of SEQ ID NO: 19. The sequence of the translation region of dgp62 may also be linked to the sequence of fibritin, for example, the sequence of nucleotide numbers 71 to 1480 in the sequence of SEQ ID NO: 19. The sequence of the translation region of Tax is a sequence capable of expressing all or part of the amino acid sequence of the Tax antigen, and may contain a start codon and / or a stop codon. For example, it has four mutations, T130A, L131S, L319R, and L320S. Examples of the sequence include a sequence of the translation region of a mutant Tax antigen having the above four mutations linked to a sequence encoding an IgE secretion signal peptide for secreting the Tax antigen outside the cell, and is the sequence of nucleotide numbers 71 to 1183 in the sequence of SEQ ID NO: 20. The sequence of the 3' untranslated region (3'-UTR) may be, for example, the 3' untranslated region of the human β-globin gene. For example, it is the sequence of base numbers 1010 to 1141 in the sequence of SEQ ID NO: 17. The sequence of the polyA tail (polyA) may be, for example, the sequence of base numbers 1142 to 1241 in the sequence of SEQ ID NO: 17.The sequences of the cap structure (Cap), 5' untranslated region (5'-UTR), gp46 or Tax translation region, 3' untranslated region (3'-UTR) and polyA tail (polyA) may be modified, and the sequence of the nucleic acid capable of expressing the gp46 antigen may consist of a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 17. The sequence of the nucleic acid capable of expressing the Tax antigen may consist of a nucleotide sequence having at least 90%, preferably 95%, more preferably 97% identity with the sequence of SEQ ID NO: 20.

[0051] The length of the poly A tail is not limited, but is, for example, 10 to 250 bases long, preferably 15 to 120 bases long, more preferably 15 to 115 bases long, and particularly preferably 20 to 110 bases long.

[0052] The mRNA of the present invention may be an mRNA having a sequence including a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of gp46 or Tax, and a 3' untranslated region (3'-UTR), and the portion consisting of the cap structure (Cap), the 5' untranslated region (5'-UTR), the translation region of gp46 antigen or Tax antigen, and the 3' untranslated region (3'-UTR) has a nucleotide sequence that has at least 90%, preferably 95%, and more preferably 97% identity to the sequence from base 1 to base 1141 of SEQ ID NO: 17, the sequence from base 1 to base 1222 of SEQ ID NO: 18, or the sequence from base 1 to base 1315 of SEQ ID NO: 20.

[0053] The nucleic acid to be encapsulated in the lipid particles may be in any form as long as it is capable of expressing the gp46 antigen or Tax antigen of HTLV-1. Examples of such nucleic acids include single-stranded DNA, single-stranded RNA (e.g., mRNA), a single-stranded polynucleotide consisting of a mixture of DNA and RNA, double-stranded DNA, double-stranded RNA, a hybrid polynucleotide of DNA-RNA, and a double-stranded polynucleotide consisting of two types of polynucleotide consisting of a mixture of DNA and RNA, and preferably, mRNA.

[0054] The nucleotides constituting the nucleic acid to be encapsulated in the lipid particles may be natural or modified nucleotides, but it is preferable that the nucleic acid contains at least one modified nucleotide.

[0055] The modified nucleotide may be one in which any of the base, sugar, and phosphodiester bond has been modified. The modification site may be one or more.

[0056] Examples of base modifications include 5-methylation, 5-fluoroation, and N4-methylation of cytosine, 5-methylation (thymine) and 5-fluoroation of uracil, N6-methylation of adenine, and N2-methylation of guanine.

[0057] An example of a sugar modification is 2'-O-methylation of D-ribofuranose.

[0058] An example of a modification of a phosphodiester bond is a phosphorothioate bond.

[0059] The modified nucleotide is preferably one in which the base portion is modified, for example, a pyrimidine nucleotide substituted at the 5-position, or a pseudouridine which may be substituted at the 1-position, specifically, 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, 1-alkylpseudouridine can be exemplified. The 1-alkylpseudouridine can be 1-(C1-C6 alkyl)pseudouridine, preferably 1-methylpseudouridine or 1-ethylpseudouridine. More preferred examples of the modified nucleotide include 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine. Particularly preferred examples of the modified nucleotide include a combination of 5-methylcytidine and 5-methyluridine, or a combination of 5-methylcytidine and 1-methylpseudouridine.

[0060] The nucleic acid capable of expressing the gp46 antigen or Tax antigen of HTLV-1 of the present invention can be produced by in vitro transcription reaction from DNA having a desired base sequence. Enzymes, buffers, and nucleoside-5'-triphosphate mixtures (adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), cytidine-5'-triphosphate (CTP), and uridine-5'-triphosphate (UTP)) required for in vitro transcription are commercially available (AmpliScribe T7 High Yield Transcription Kit (Epicentre), mMESSAGE mMACHINE T7 Ultra Kit (Life technologies), etc.). The DNA used to produce single-stranded RNA is cloned DNA, for example, a plasmid DNA or a DNA fragment. Plasmid DNA or DNA fragments may be commercially available or may be produced by methods generally known in the art (e.g., the methods described in Sambrook, J. et al., Molecular Cloning a Laboratory Manual second edition (1989); Rashtchian, A., Current Opinion in Biotechnology, 1995, 6(1), 30-36; Gibson DG et al., Science, 2008, 319(5867), 1215-1220, etc.).

[0061] To obtain mRNA with improved stability and / or safety, some or all of the unmodified nucleotides in the mRNA can also be replaced with modified nucleotides by replacing some or all of the unmodified nucleoside-5'-triphosphates with modified nucleoside-5'-triphosphates in an in vitro transcription reaction (Kormann, M., Nature Biotechnology, 2011, 29, 154-157.).

[0062] In order to obtain mRNA with improved stability and / or safety, a cap structure (the above-mentioned Cap0 structure) can be introduced into the 5'-end of mRNA by a method using a capping enzyme after an in vitro transcription reaction. Cap0 can also be converted to Cap1 by a method of allowing 2'-O-methyltransferase to act on mRNA having Cap0. Commercially available capping enzymes and 2'-O-methyltransferases can be used (e.g., Vaccinia Capping System, M2080; mRNA Cap 2'-O-Methyltransferase, M0366, both manufactured by New England Biolab). When using commercially available products, mRNA having a cap structure can be produced according to the protocol attached to the product.

[0063] The cap structure at the 5' end of mRNA can also be introduced by a method other than using an enzyme. For example, by adding ARCA or CleanCap (registered trademark) to an in vitro transcription reaction, a cap analog structure possessed by ARCA or a Cap1 structure derived from CleanCap can be introduced into mRNA. Commercially available products can be used for ARCA and CleanCap (ARCA, N-7003; CleanCap Reagent AG, N-7113, both manufactured by TriLink BioTechnologies). When using commercially available products, mRNA having a cap structure can be produced according to the protocol attached to the product.

[0064] In the present invention, the nucleic acid to be encapsulated in the lipid particles may be purified by methods such as desalting, HPLC (reverse phase, gel filtration, ion exchange, affinity), PAGE, ultrafiltration, etc. By removing impurities through the purification process, the production of inflammatory cytokines in a living body to which the nucleic acid is administered can be reduced.

[0065] The nucleic acid-encapsulated lipid particles of the present invention can be produced by a method such as a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, a dehydration-rehydration method, a surfactant dialysis method, a hydration method, or a freeze-thaw method. For example, the nucleic acid-encapsulated lipid particles can be produced by the method described in International Publication No. 2015 / 005253. The nucleic acid-encapsulated lipid particles of the present invention can also be produced by mixing a nucleic acid solution and a lipid solution in a microchannel. For example, the nucleic acid-encapsulated lipid particles can be produced using NanoAssemblr (registered trademark) from Precision Nanosystems, Inc., according to the method described in the attached protocol.

[0066] The particles of the present invention may have an average particle diameter of 30 nm to 300 nm, preferably 30 to 200 nm, and more preferably 30 to 100 nm. The average particle diameter can be obtained by measuring the volume average particle diameter based on the principle of dynamic light scattering using an instrument such as Zeta Potential / Particle Sizer NICOMP (registered trademark) 380ZLS (PARTICLE SIZING SYSTEMS).

[0067] The particles of the present invention can be used to manufacture a composition for preventing and / or treating diseases caused by HTLV-1 infection. Diseases caused by HTLV-1 infection include adult T-cell leukemia (ATL), HTLV-1-associated myelopathy (HAM), HTLV-1-associated uveitis (HU), etc. The lipid particles encapsulating a nucleic acid capable of expressing the gp46 antigen of HTLV-1 and the lipid particles encapsulating a nucleic acid capable of expressing the Tax antigen of HTLV-1 may be formulated in the same lipid particle or as separate lipid particles, but are preferably prepared as separate lipid particles.

[0068] The particles of the present invention can be used to express the gp46 antigen and the Tax antigen of HTLV-1 in vivo or in vitro. That is, lipid particles encapsulating a nucleic acid capable of expressing the gp46 antigen of HTLV-1 and lipid particles encapsulating a nucleic acid capable of expressing the Tax antigen of HTLV-1 may be administered to a subject. Thus, the present invention provides a method for expressing the gp46 antigen and the Tax antigen of HTLV-1 in vitro, which comprises introducing a composition containing the particles into a cell. The present invention also provides a method for expressing the gp46 antigen and the Tax antigen of HTLV-1 in vivo, which comprises administering a composition containing the particles to a mammal. By expressing the gp46 antigen and the Tax antigen of HTLV-1 in vivo, an immune response against HTLV-1 can be induced. As a result, HTLV-1 infection can be prevented and / or treated. Thus, the present invention provides a method for inducing an immune response against HTLV-1, which comprises administering a composition containing the particles to a mammal. The present invention also provides a method for preventing and / or treating HTLV-1 infection, comprising administering a composition containing the above particles to a mammal.The present invention also includes a kit comprising both lipid particles encapsulating a nucleic acid capable of expressing the gp46 antigen of HTLV-1 and lipid particles encapsulating a nucleic acid capable of expressing the Tax antigen of HTLV-1.

[0069] The particles of the present invention can be used as medicines and as experimental reagents. The particles of the present invention are usually added to a carrier such as water, a buffer solution, or physiological saline, and the resulting mixture (composition) can be introduced into cells (in vitro) or administered to a mammal (in vivo). When administered to a mammal, the carrier should be a pharma- ceutically acceptable carrier (e.g., physiological saline). The particles of the present invention may also be formulated into creams, pastes, ointments, gels, lotions, and other dosage forms using fats, fatty oils, lanolin, petrolatum, paraffin, wax, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, and other base materials.

[0070] The particles of the present invention can be administered orally or parenterally, for example, via intramuscular, intravenous, rectal, transdermal, transmucosal, subcutaneous, or intradermal administration, to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, pigs, monkeys, cats, dogs, horses, goats, sheep, and cows.

[0071] When the particles of the present invention are administered to humans, for example, a single dose of about 0.001 to 1 mg, preferably 0.01 to 0.2 mg, of mRNA weight per adult may be administered once or several times by intramuscular injection, subcutaneous injection, intradermal injection, intravenous drip injection, or intravenous injection, although the dose and number of administrations may be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0072] When used as an experimental reagent, the particles of the present invention can be introduced into cells in which it is desired to express HTLV-1 gp46 antigen and Tax antigen (for example, HEK293 cells and their derived cells (HEK293T cells, FreeStyle 293 cells, and Expi293 cells), CHO cells, C2C12 mouse myoblasts, and immortalized mouse dendritic cells (MutuDC1940)), and the HTLV-1 gp46 antigen and Tax antigen can be expressed in vitro. The expression of HTLV-1 gp46 antigen and Tax antigen can be analyzed by detecting HTLV-1 gp46 antigen and Tax antigen proteins in a sample by Western blotting, or by detecting peptide fragments specific to HTLV-1 gp46 antigen and Tax antigen by mass spectrometry.

[0073] In the present invention, "treatment" refers to recovery, remission, alleviation, and / or delay in the worsening of clinical symptoms of a disease caused by a virus, bacteria, or the like, or a disease caused by such infection (e.g., precancerous lesion, cancer, etc.) in a patient who has developed such a disease.

[0074] In the present invention, "prevention" means reducing the incidence of disease caused by infectious diseases such as viruses or bacteria. Prevention includes reducing the risk of progression of diseases caused by infectious diseases such as viruses or bacteria, or reducing the severity of such diseases. The particles of the present invention are effective in preventing and / or treating the above-mentioned diseases because they induce a protective immune response.

[0075] The particles of the present invention are also expected to be used as a preventive drug and a therapeutic drug for diseases caused by HTLV-1 in HTLV-1-infected individuals, such as adult T-cell leukemia-lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU). EXAMPLES

[0076] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention.

[0077] The following abbreviations may be used in the examples. SU gp46: represents gp46, which is the extracellular domain (surface unit) of the HTLV-1 envelope protein. gp46-Fib: a fusion protein between gp46 and the C-terminal region of Fibritin. dgp62-Fib: a fusion protein between a protein in which the transmembrane and intracellular regions have been deleted from gp62 and the C-terminal region of Fibritin. sec Tax mutant: This refers to a fusion protein of a mutated Tax protein and a secretion signal protein.

[0078] [Example 1] Preparation of SU gp46 mRNA-001 (1) Preparation of template DNA for in vitro transcription (IVT) of SU gp46 To prepare template DNA for in vitro transcription (IVT), SU gp46 DNA was amplified by PCR and purified. A DNA fragment (SEQ ID NO: 1) containing the sequence of the T7 promoter, human β-globin 5'-UTR, KOZAK sequence, SU gp46, human β-globin 3'-UTR, and PolyA sequence was introduced into a plasmid. 8 ng of the plasmid was dissolved in nuclease-free water (547.2 μL), to which 10× Buffer for KOD-Plus- Ver.2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 10 μM antisense primer (24 μL, SEQ ID NO: 3), and KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # KOD-211) were added. The mixture was incubated at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 90 seconds, and then further incubated at 68°C for 1 minute to amplify the DNA. After the reaction, the template DNA (sequence number 4) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0079] (2) Preparation of SU gp46 mRNA-001 by in vitro transcription 365.1 μg / mL template DNA obtained in Example 1-(1) (4.38 μL), 100 mM CleanCap AG (8 μL, TriLink catalog # N-7113), 100 mM ATP (8 μL, Hongene catalog # R1331), 100 mM GTP (8 μL, Hongene catalog # R2331), 100 mM CTP (8 μL, Hongene catalog # R3331), 100 mM N1-methylpseudoUTP(8 μL, Hongene catalog # R5-027), Nuclease-free water(67.62 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer(32 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase(16 μL, Promega catalog # P137X) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (8 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8 M LiCl solution (80 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -30°C. After centrifugation (4°C, 5200×g, 35 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5200×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (500 μL) and purified using the RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual. The resulting solution (750 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (85 μL) and enzyme (32 μL) were mixed and incubated at 37°C for 30 minutes, followed by incubation at 75°C for 2 minutes.The resulting solution was purified using an RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual to obtain the target mRNA. The same experimental procedure was performed twice in total, and the resulting mRNA solutions were combined to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 17.

[0080] [Example 2] Preparation of gp46-Fib mRNA-002 (1) Preparation of template DNA for in vitro transcription (IVT) of gp46-Fib To prepare template DNA for in vitro transcription (IVT), gp46-Fib DNA was amplified by PCR and purified. A DNA fragment (SEQ ID NO: 5) containing the sequence of T7 promoter, human β-globin 5'-UTR, KOZAK sequence, gp46-Fib, human β-globin 3'-UTR, and PolyA sequence was introduced into the plasmid. 8 ng of the plasmid was dissolved in nuclease-free water (547.2 μL), to which 10× Buffer for KOD-Plus- Ver.2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 10 μM antisense primer (24 μL, SEQ ID NO: 3), and KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # KOD-211) were added. The mixture was incubated at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 90 seconds, and then further incubated at 68°C for 1 minute to amplify the DNA. After the reaction, the template DNA (sequence number 6) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0081] (2) Preparation of gp46-Fib mRNA-002 by in vitro transcription 345.3 μg / mL template DNA obtained in Example 2-(1) (4.63 μL), 100 mM CleanCap AG (8 μL, TriLink catalog # N-7113), 100 mM ATP (8 μL, Hongene catalog # R1331), 100 mM GTP (8 μL, Hongene catalog # R2331), 100 mM CTP (8 μL, Hongene catalog # R3331), 100 mM N1-methylpseudoUTP(8 μL, Hongene catalog # R5-027), Nuclease-free water(67.37 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer(32 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase(16 μL, Promega catalog # P137X) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (8 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8 M LiCl solution (80 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -30°C. After centrifugation (4°C, 5200×g, 35 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5200×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (500 μL) and purified using the RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual. The resulting solution (750 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (85 μL) and enzyme (32 μL) were mixed and incubated at 37°C for 30 minutes, followed by incubation at 75°C for 2 minutes.The resulting solution was purified using an RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual to obtain the target mRNA. The same experimental procedure was performed twice in total, and the resulting mRNA solutions were combined to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 18.

[0082] [Example 3] Preparation of dgp62-Fib mRNA-003 (1) Preparation of template DNA for in vitro transcription (IVT) of dgp62-Fib To prepare template DNA for in vitro transcription (IVT), dgp62-Fib DNA was amplified by PCR and purified. A DNA fragment (SEQ ID NO: 7) containing the sequence of T7 promoter, human β-globin 5'-UTR, KOZAK sequence, dgp62-Fib, human β-globin 3'-UTR, and PolyA sequence was introduced into the plasmid. 8 ng of the plasmid was dissolved in nuclease-free water (547.2 μL), to which 10× Buffer for KOD-Plus- Ver.2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 10 μM antisense primer (24 μL, SEQ ID NO: 3), and KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # KOD-211) were added. The mixture was incubated at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 90 seconds, and then further incubated at 68°C for 1 minute to amplify the DNA. After the reaction, the template DNA (sequence number 8) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0083] (2) Preparation of dgp62-Fib mRNA-003 by in vitro transcription 366.7 μg / mL template DNA obtained in Example 3-(1) (4.36 μL), 100 mM CleanCap AG (8 μL, TriLink catalog # N-7113), 100 mM ATP (8 μL, Hongene catalog # R1331), 100 mM GTP (8 μL, Hongene catalog # R2331), 100 mM CTP (8 μL, Hongene catalog # R3331), 100 mM N1-methylpseudoUTP(8 μL, Hongene catalog # R5-027), Nuclease-free water(67.64 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer(32 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase(16 μL, Promega catalog # P137X) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (8 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8 M LiCl solution (80 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -30°C. After centrifugation (4°C, 5200×g, 35 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5200×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (500 μL) and purified using the RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual. The resulting solution (750 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (85 μL) and enzyme (32 μL) were mixed and incubated at 37°C for 30 minutes, followed by incubation at 75°C for 2 minutes.The resulting solution was purified using an RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual to obtain the target mRNA. The same experimental procedure was performed twice in total, and the resulting mRNA solutions were combined to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 19.

[0084] [Example 4] Preparation of sec Tax mutant mRNA-004 (1) Preparation of template DNA for in vitro transcription (IVT) of sec Tax mutants To prepare template DNA for in vitro transcription (IVT), sec Tax DNA was amplified by PCR and purified. A DNA fragment (SEQ ID NO: 9) containing the sequence of T7 promoter, human β-globin 5'-UTR, KOZAK sequence, sec Tax, human β-globin 3'-UTR, and PolyA sequence was introduced into the plasmid. 8 ng of the plasmid was dissolved in nuclease-free water (547.2 μL), to which 10× Buffer for KOD-Plus- Ver.2 (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 2 mM dNTP mix (80 μL, Toyobo Co., Ltd. catalog # KOD-211), 25 mM MgSO4 (48 μL, Toyobo Co., Ltd. catalog # KOD-211), 50 μM sense primer (4.8 μL, SEQ ID NO: 2), 10 μM antisense primer (24 μL, SEQ ID NO: 3), and KOD Plus polymerase (16 μL, Toyobo Co., Ltd. catalog # KOD-211) were added. The mixture was incubated at 98°C for 1 minute, followed by 20 cycles of 98°C for 5 seconds, 55°C for 15 seconds, and 68°C for 90 seconds, and then further incubated at 68°C for 1 minute to amplify the DNA. After the reaction, the template DNA (sequence number 10) was purified using Wizard SV Gel and PCR Clean-Up System (Promega catalog # A9281).

[0085] (2) Preparation of sec Tax mutant mRNA-004 by in vitro transcription 401.1 μg / mL template DNA obtained in Example 4-(1) (3.99 μL), 100 mM CleanCap AG (8 μL, TriLink catalog # N-7113), 100 mM ATP (8 μL, Hongene catalog # R1331), 100 mM GTP (8 μL, Hongene catalog # R2331), 100 mM CTP (8 μL, Hongene catalog # R3331), 100 mM N1-methylpseudoUTP(8 μL, Hongene catalog # R5-027), Nuclease-free water(68.01 μL, Thermo Fisher catalog # AM9937), T7 Transcription 5× buffer(32 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase(16 μL, Promega catalog # P137X) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (8 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8 M LiCl solution (80 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -30°C. After centrifugation (4°C, 5200×g, 35 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5200×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (500 μL) and purified using the RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual. The resulting solution (750 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (85 μL) and enzyme (32 μL) were mixed and incubated at 37°C for 30 minutes, followed by incubation at 75°C for 2 minutes.The resulting solution was purified using an RNeasy Midi kit (Qiagen catalog # 75144) according to the attached manual to obtain the target mRNA. The same experimental procedure was performed twice in total, and the resulting mRNA solutions were combined to obtain the target mRNA. The obtained mRNA has the sequence of SEQ ID NO: 20.

[0086] [Example 5] Preparation of SU gp46 mRNA-005 (1) Preparation of template DNA for in vitro transcription (IVT) of SU gp46 The experiment was carried out in the same manner as in Example 1-(1).

[0087] (2) Preparation of SU gp46 mRNA-005 by in vitro transcription 339 μg / mL template DNA obtained in Example 5-(1) (89 μL), 100 mM CleanCap AG (150 μL, TriLink catalog # T-7113), 100 mM ATP (150 μL, Hongene catalog # R1331), 100 mM GTP (150 μL, Hongene catalog # R2331), 100 mM CTP (150 μL, Hongene catalog # R3331), 100 mM N1-methyl-pseudouridine 5'-triphosphate (150 μL, Hongene catalog # R5-027), Nuclease-free water (1261 μL, Qiagen catalog # 129114), T7 Transcription 5× buffer (600 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (300 μL, Promega catalog # P137X) was added and incubated at 37°C for 2 hours. RQ1 RNase-Free DNase (30 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8M LiCl solution (1500 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -20°C. After centrifugation (4°C, 5250×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5250×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The resulting eluate (12 mL, 12.0 mg in terms of UV) was mixed with nuclease-free water (120 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (1,400 μL), and enzyme (480 μL) and incubated at 37°C for 30 minutes.After incubation at 75°C for 2 minutes, 8M LiCl solution (7000μL, Sigma-Aldrich catalog # L7026) was added and the mixture was left to stand overnight at -20°C. After centrifugation (4°C, 5250×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5250×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (2mL) (9.9mg in UV equivalent). The obtained mRNA was purified by reverse phase chromatography (Column: PLRP-S, 4000 Å, 10 μm, 10 x 200 mm (Agilent), Buffer A: 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0), Buffer B: 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), Gradient B%: 27.5% to 35% (20 min), Flow rate: 5 mL / min, Temperature: 80 °C). The target fraction was collected and desalted by ultrafiltration (Amicon Ultra-15 (MWCO: 30 kDa)) (3.4 mg in UV equivalent).

[0088] The obtained mRNA has the sequence of SEQ ID NO: 17. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be of the desired length.

[0089] [Example 6] Preparation of sec Tax mutant mRNA-006 (1) Preparation of template DNA for in vitro transcription (IVT) of sec Tax mutants The same procedure as in Example 4-(1) was followed.

[0090] (2) Preparation of sec Tax mutant mRNA-006 by in vitro transcription 336 μg / mL template DNA obtained in Example 6-(1) (89 μL), 100 mM CleanCap AG (150 μL, TriLink catalog # T-7113), 100 mM ATP (150 μL, Hongene catalog # R1331), 100 mM GTP (150 μL, Hongene catalog # R2331), 100 mM CTP (150 μL, Hongene catalog # R3331), 100 mM N1-methyl-pseudouridine 5'-triphosphate (150 μL, Hongene catalog # R5-027), Nuclease-free water (1261 μL, Qiagen catalog # 129114), T7 Transcription 5× buffer (600 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (300 μL, Promega catalog # P137X) was added and incubated at 37°C for 2 hours. RQ1 RNase-Free DNase (30 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8M LiCl solution (1500 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -20°C. After centrifugation (4°C, 5250×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5250×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The resulting eluate (12 mL, 12.6 mg in terms of UV) was mixed with nuclease-free water (100 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (1,400 μL), and enzyme (500 μL) and incubated at 37°C for 30 minutes.After incubation at 75°C for 2 minutes, 8M LiCl solution (7000μL, Sigma-Aldrich catalog # L7026) was added and the mixture was left to stand overnight at -20°C. After centrifugation (4°C, 5250×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5250×g, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (2mL) (10.9mg in UV equivalent). The obtained mRNA was purified by reverse phase chromatography (Column: PLRP-S, 4000 Å, 10 μm, 10 x 200 mm (Agilent), Buffer A: 5% acetonitrile, 400 mM triethylamine acetate (pH 7.0), Buffer B: 25% acetonitrile, 400 mM triethylamine acetate (pH 7.0), Gradient B%: 27.5% to 35% (20 min), Flow rate: 5 mL / min, Temperature: 80 °C). The target fraction was collected and desalted by ultrafiltration (Amicon Ultra-15 (MWCO: 30 kDa)) (3.7 mg in UV equivalent).

[0091] The obtained mRNA has the sequence of SEQ ID NO: 20. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be of the desired length.

[0092] [Example 7] Preparation of gp46-Fib mRNA-007 (1) Preparation of template DNA for in vitro transcription (IVT) of gp46-Fib The same procedure as in Example 2-(1) was followed.

[0093] (2) Preparation of gp46-Fib mRNA-007 by in vitro transcription The following mixture was prepared: 397 μg / mL template DNA (63 μL) obtained in Example 7-(1), 100 mM CleanCap AG (50 μL, TriLink catalog # T-7113), 100 mM ATP (50 μL, Hongene catalog # R1331), 100 mM GTP (50 μL, Hongene catalog # R2331), 100 mM CTP (50 μL, Hongene catalog # R3331), 100 mM N1-methyl-pseudouridine 5'-triphosphate (50 μL, Hongene catalog # R5-027), Nuclease-free water (387 μL, Qiagen catalog # 129114), T7 Transcription 5× buffer (200 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (100 μL, Promega catalog # The mixture was mixed with RNase-Free DNase (25 μL, Promega catalog # M6101) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (25 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8M LiCl solution (500 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -20°C. After centrifugation (4°C, 5250×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5250×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The resulting eluate (3 mL, 3.6 mg in terms of UV) was mixed with nuclease-free water (332 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (450 μL) and enzyme (718 μL) and incubated at 37°C for 30 minutes.After incubation at 75°C for 2 minutes, the mixture was purified using an RNeasy Maxi kit according to the attached manual to obtain the desired mRNA (4 mL, 2.8 mg in UV equivalent).

[0094] The obtained mRNA has the sequence of SEQ ID NO: 18. It was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be of the desired length.

[0095] [Example 8] Preparation of sec Tax mutant mRNA-008 (1) Preparation of template DNA for in vitro transcription (IVT) of sec Tax mutants The same procedure as in Example 4-(1) was followed.

[0096] (2) Preparation of sec Tax mutant mRNA-008 by in vitro transcription The following mixture was prepared: 391 μg / mL template DNA (64 μL) obtained in Example 8-(1), 100 mM CleanCap AG (50 μL, TriLink catalog # T-7113), 100 mM ATP (50 μL, Hongene catalog # R1331), 100 mM GTP (50 μL, Hongene catalog # R2331), 100 mM CTP (50 μL, Hongene catalog # R3331), 100 mM N1-methyl-pseudouridine 5'-triphosphate (50 μL, Hongene catalog # R5-027), Nuclease-free water (386 μL, Qiagen catalog # 129114), T7 Transcription 5× buffer (200 μL, Promega catalog # P140X), Enzyme mix, T7 RNA Polymerase (100 μL, Promega catalog # The mixture was mixed with RNase-Free DNase (25 μL, Promega catalog # M6101) and incubated at 37°C for 4 hours. RQ1 RNase-Free DNase (25 μL, Promega catalog # M6101) was added and incubated at 37°C for 15 minutes. 8M LiCl solution (500 μL, Sigma-Aldrich catalog # L7026) was added and left to stand overnight at -20°C. After centrifugation (4°C, 5250×g, 30 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 5250×g, 10 minutes), after which the supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water and purified using the RNeasy Maxi kit (Qiagen catalog # 75162) according to the attached manual. The resulting eluate (3 mL, 3.5 mg in terms of UV radiation) was mixed with nuclease-free water (359 μL), rApid Alkaline Phosphatase (Roche catalog # 04 898 141 001) buffer (450 μL), and enzyme (691 μL) and incubated at 37°C for 30 minutes.After incubation at 75°C for 2 minutes, the mixture was purified using an RNeasy Maxi kit according to the attached manual to obtain the desired mRNA (4 mL, 2.8 mg in UV equivalent).

[0097] The obtained mRNA has the sequence of SEQ ID NO: 20. It was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be of the desired length.

[0098] [Examples 9 to 24] Preparation of mRNA-encapsulated nucleic acid lipid particles using the mRNA described in Examples 1 to 8 (1) Preparation of mRNA-encapsulated nucleic acid-lipid particles Distearoylphosphatidylcholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (Cholesterol: hereinafter referred to as Chol, Sigma-Aldrich, Inc.), (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (a compound described in Example 23 of WO2015 / 005253) (hereinafter referred to as LP1) or (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate (a compound described in Example 28 of WO2015 / 005253) (hereinafter referred to as LP2), and polyethylene glycol having a molecular weight of about 2000, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene Glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION, SUNBRIGHT GM-020) was dissolved in ethanol in the molar ratio shown in Table 1 to a total lipid concentration of 5 mM.

[0099] On the other hand, the mRNA obtained in Examples 1 to 8 was diluted with citrate buffer (20 mM citrate buffer, pH 4.0).

[0100] The lipid solution and mRNA solution were mixed in a microchannel using a NanoAssemblr BenchTop (Precision Nanosystems Inc.) so that the total lipid weight ratio to mRNA was the value shown in Table 1 and the volume ratio was 1:3, to obtain a crude dispersion of nucleic acid-lipid particles. The nucleic acid-lipid particle dispersion was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) for 12 to 18 hours against approximately 25 to 50 times the amount of buffer solution to remove ethanol, to obtain a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles.

[0101] LP1 was synthesized according to the method described in Example 23 of WO2015 / 005253, and LP2 was synthesized according to the method described in Example 28 of WO2015 / 005253.

[0102] (2) Characterization of mRNA-encapsulated nucleic acid-lipid particles The characteristics of the dispersion containing the nucleic acid-lipid particles prepared in (1) were evaluated. The methods for evaluating each characteristic are explained below.

[0103] (2-1) Encapsulation rate of mRNA The mRNA encapsulation rate was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) in accordance with the attached instructions. That is, the amount of mRNA in the dispersion of the nucleic acid-lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated by the following formula. {([amount of mRNA in the presence of surfactant]-[amount of mRNA in the absence of surfactant]) / [amount of mRNA in the presence of surfactant]} x 100(%)

[0104] (2-2) Ratio of mRNA to lipid The amount of mRNA in the nucleic acid-lipid particle dispersion was measured by one of the following methods. The nucleic acid-lipid particle dispersion was diluted with 1.0% Triton X-100 and measured by reverse phase chromatography (System: Agilent 1260 series, Column: Bioshell A400 Protein C4 (10 cm × 4.6 mm, 3.4 μm) (SUPELCO), Buffer A: 0.1 M triethylamine acetate (pH 7.0), Buffer B: acetonitrile, (B%): 5-50% (0-15 min), Flow Rate: 1 mL / min, Temperature: 70 °C, Detection: 260 nm).

[0105] The nucleic acid-lipid particle dispersion was diluted and dissolved in 90% methanol, and the amount of mRNA in the nucleic acid-lipid particles was measured using an ultraviolet-visible spectrophotometer (PerkinElmer, LAMBDA (trademark) 465). The mRNA concentration was calculated using the following formula. {[Absorbance at 260 nm]-[Absorbance at 350 nm]} x 40 x dilution factor (μg / mL)

[0106] The amount of each lipid in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH C18 (130 Å, 3.5 μm, 3.0 mm × 150 mm,) (Waters catalog # 186005263), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75-100% (0-6 min), 100% (6-15 min), Flow Rate: 0.45 mL / min, Temperature: 50 °C, Detection: Corona CAD (Charged Aerosol Detector)).

[0107] The ratio of the total lipid amount to the mRNA was calculated by the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt)

[0108] (2-3) Average particle diameter The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume average particle size, and ± indicates deviation.

[0109] The results of the characteristic evaluation are shown in Table 2.

[0110] [Table 1]

[0111] [Table 2]

[0112] From the above results, it was revealed that these nucleic acid-lipid particles had 95% or more of the mRNA encapsulated within the lipid particles, and had an average particle size of approximately 100 nm to approximately 140 nm.

[0113] (Test Example 1) Plasmid construction for LTR reporter assay DNAs encoding HTLV-1 Tax wild type (Uniprot Accession Number: P03409), Tax mutant, and secretory Tax mutant with an IgE secretion signal added to the Tax mutant were synthesized by Eurofin, the contractor. Each DNA fragment was inserted into the NheI / NotI site of the pcDNA3.1+ vector (Thermo Scientific Inc.) and cloned. DNAs encoding HIV-1 LTR and HTLV-1 LTR were synthesized by Eurofin, the contractor. Each DNA fragment was inserted into the KpnI / XhoI site of the pGL4.17 vector (Promega) and cloned.

[0114] HIV-1 LTR and HTLV-1 LTR reporter assays A total of 500 ng of plasmids, including 350 ng of Tax wild type, Tax mutant, or secreted Tax mutant expressing plasmid, 100 ng of HIV-1 LTR reporter plasmid or HTLV-1 LTR reporter plasmid, and 50 ng of pRG-RK plasmid (Promega) for transfection efficiency correction, was transfected into HEK293T cells using TransIT-LT1 gene transfer reagent (Takara). 48 hours after transfection, the expression levels of firefly luciferase and Renilla luciferase were measured using Dual-Glo (registered trademark) Luciferase Assay System (Promega), and the ratio of the expression level of firefly luciferase / the expression level of Renilla luciferase was calculated to evaluate the transcriptional activity of HIV-1 LTR and HTLV-1 LTR.

[0115] (Test Example 2) Administration of Example 12 to C3H mice C3H / HeJJcl mice were obtained from CLEA Japan and acclimatized. Example 12 was administered at 5 μg mRNA / 20 μL / mouse to the disinfected triceps surae muscle of anesthetized mice at two-week intervals. The first administration was to the right leg, and the second administration was to the left leg. Buffer was used to adjust the concentration of lipid particles and for the negative control group.

[0116] Preparation of serum and spleen cells Two weeks after the first administration of lipid particles and one week after the second administration, blood was collected from anesthetized mice. The blood was left to stand at room temperature for more than one hour and centrifuged at 3000 RPM for 10 minutes to separate and collect serum. Spleens were also collected from mice that were exsanguinated under anesthesia. The spleens were mashed using the mesh of a Cell Strainer (FALCON) and the gasket of a syringe (TERUMO), adjusted to single cell suspension with RPMI 1640 (nacalai tesque), and then centrifuged to collect cells. The supernatant was discarded, and red blood cells were lysed using DB Pharm Lyse Lysing buffer (BECTON DICKINSON). After centrifugation, the cell suspension was resuspended in RPMI 1640 and passed through mini Cell Strainers (Hitec Corporation, Cat. HT-AMS-14002). After centrifugation, the supernatant was discarded, the cells were resuspended, and the cell concentration was measured. After centrifugation, the supernatant was discarded and the cell concentration was adjusted with cell culture medium (RPMI 1640 supplemented with 10% Fetal Bovine Serum (inactivated and filtered, HyClone), 1% Penicillin-Streptomycin Mixed Solution (Nacalai Tesque), 1% Sodium Pyruvate (Gibco), 1% MEM Non-Essential Amino Acids (Gibco), 1% HEPES Buffer Solution (Gibco), and 1% StemSure Monothioglycerol Solution (FUJIFILM)). Centrifugation was performed at 1500 RPM for 5 minutes at 4°C.

[0117] Tax-specific CTL induction level 3 x 10 prepared mouse spleen cells 6The cells were resuspended in 1X Dulbecco's Phosphate Buffered Saline (Gibco), centrifuged, and the supernatant discarded. This process was repeated twice. Then, TruStain FcX Antibody (BioLegend) diluted 20-fold with 1X PBS was added to the cells and allowed to stand at room temperature for 5 minutes. 5 μL of H-2D K HTLV-1 Tax38-46 Tetramer-ARLHTHALL-PE (MBL) was added to each sample and incubated at 37°C, 5% CO2 for 15 minutes. APC / Fire™ 750 anti-mouse CD3 Antibody (BioLegend) and Anti-CD8 (Mouse) mAb-FITC (MBL) were added to the sample on ice at a final concentration of 100 times dilution, and the sample was left in the dark for 30 minutes. After centrifugation, the supernatant was discarded and the cells were washed twice with 1X PBS. Finally, the cells were resuspended in 400 μL PBS, and the cells labeled with FACSVerse (BD) were detected and analyzed with FlowJo (verification). The centrifugation was performed at 1500 RPM for 5 minutes at 4°C.

[0118] Anti-Tax antibody titer in mouse blood Tax (MYBiosource) recombinant protein was added to a 96-well flat-bottom plate for use as a solid-phase antigen and left to stand overnight at 4°C. A standard curve dilution series was prepared by diluting Mouse IgG (SouthernBiotech) 3-fold from a maximum concentration of 0.25 μg / mL in 7 steps. Each well was washed, blocking solution was added, and the plate was left to stand at room temperature for 1 hour. Serum samples were prepared by diluting 4-fold from a maximum concentration of 100-fold using blocking solution in 7 steps. The blocked plate was washed, and the diluted samples were added to the plate and left to stand at room temperature for 1 hour. Detection antibodies, Goat Anti-Mouse IgG and Human ads-HRP (SouthernBiotech), were diluted 3000-fold in blocking solution and added to the washed wells. After 1 hour, the plate was washed, TMB Microwell Peroxidase Substrate System (seracare) was added, and the plate was left to stand for 2-3 minutes. The reaction was stopped using TMB Stop Solution (KPL, Cat. 51500-0021). The corrected absorbance was calculated by subtracting the absorbance at 540 nm from the absorbance at 450 nm using a plate reader, and the anti-gp46 antibody titer and anti-Tax antibody titer were calculated. The blocking solution was 1X Dulbecco's Phosphate Buffered Saline (gibco) supplemented with 1% Bovine Serum Albumin (Sigma) and 0.05% Tween (BIO-RAD), and washing was performed three times with 1X Dulbecco's Phosphate Buffered Saline ((10X) gibco) supplemented with 0.05% Tween.

[0119] (Test Examples 3 to 5) Administration to cynomolgus monkeys The lipid particles of Example 13 and Example 14 were administered to the upper arm of the monkey once every two weeks, a total of four times. The first administration was to the right upper arm, and then administration was alternated between the left and right. The lipid particle dosage was a mixture of equal amounts of 25 μg mRNA of Example 13 and Example 14, and 50 μg mRNA / 200 μL / body was administered per administration.

[0120] (Test Example 3) Anti-gp46 and Tax antibody titers in monkey blood Recombinant gp46 protein (RayBiotech) and recombinant Tax protein (MY Biosource) were added to a 96-well plate and immobilized overnight at 4°C. After that, the plate was washed (three times with PBS containing 0.05% Tween 20) using a plate washer, and blocking was performed by adding blocking solution (1% BSA, PBS containing 0.05% Tween 20). A monkey plasma sample dilution series was prepared in seven stages of 4-fold dilution from the highest concentration of 100-fold diluted serum using blocking solution. A standard curve dilution series of monkey IgG concentration was prepared in eight stages of 3-fold dilution from the highest concentration of 0.25 μg / mL of monkey IgG solution (manufacturer) using blocking solution. The sample dilution solution and standard curve dilution solution were added, left at room temperature for 1 hour, and then washed with a plate washer. The detection antibody was HRP-labeled anti-monkey IgG antibody (Sigma-Aldrich) diluted 4000-fold with blocking solution, added to the plate, and left at room temperature for 1 hour. After washing with a plate washer, TMB Microwell Peroxidase Substrate System (SERACARE Life Sciences) was added and left to stand for 10 minutes. TMB Stop Solution (SERACARE Life Sciences) was used as a reaction stop solution. The absorbance at a wavelength of 450 nm (control wavelength 540 nm) was measured using a plate reader, and the corrected absorbance (Delta) obtained by subtracting the absorbance measured at 540 nm from the absorbance measured at 450 nm was used for analysis. A calibration curve was created using Nonlinear Regression: 4 Parameters from the monkey IgG concentration and Delta of the standard curve. The anti-gp46 and Tax antibody concentrations of the sample were calculated from the calibration curve, the dilution ratio of the measurement sample, and Delta.

[0121] (Test Example 4) gp46- and Tax-specific cellular immune responses in monkeys. PBMCs isolated from monkey peripheral blood using Ficoll (Cytiva) were diluted at 2 × 10 in RPMI Complete medium (10% FBS [Sigma-Aldrich], 1% PS [Penicilin-Streptomycin Mixed Solution, Nacalai Tesque], 1 mM Sodium Pyruvate [Thermo Fisher Scientific], 10 mM HEPES [Thermo Fisher Scientific], 1 × StemSure [Fujifilm Wako Pure Chemical Industries, Ltd.], 1 × MEM Non-Essential Amino Acids Solution [Thermo Fisher Scientific]). 6 The cells were prepared at a final concentration of 0.1% (v / v) of Tax epitope peptide pool (Eurofins) or 1 μg / mL of gp46 recombinant protein (RayBiotech) was added at 100 μL / well in RPMI Complete medium, and the cells were cultured for 48 hours at 37°C and 5% CO2. Antigen-specific IFN-γ producing cells were cultured in Monkey IFN-γ ELISpot plate (MABTech) at 100 μL / well. PLUS The antigen-specific IFN-γ producing cells were counted using an ELISPOT analyzer (CTL).

[0122] (Test Example 5) Anti-HTLV-1 neutralizing activity in monkey blood Using a spin column-based Antibody Purification Kit (Cosmo Bio), total IgG was purified from monkey plasma collected and prepared 2 weeks after the fourth administration. The purified IgG was added to the HTLV-1-infected YT#1 cell line, and then the HTLV-1-uninfected CEM cells were mixed with the YT#1 cells at a constant ratio of 1:1. Patient serum was added to the co-culture system, and the rate of syncytium formation after overnight culture was measured and examined under a microscope.

[0123] (Test Example 6) Mouse blood gp46-specific total IgG antibody titer To use gp46 (Ray Biotech Inc.) recombinant protein as a solid-phase antigen, it was added to a 96-well flat-bottom plate and left to stand overnight at 4°C. A standard curve dilution series was prepared by diluting Mouse IgG (SouthernBiotech) 3-fold from the highest concentration of 0.25 μg / mL in 7 steps. Each well was washed, blocking solution was added, and the plate was left to stand at room temperature for 1 hour. Serum samples were prepared by diluting 4-fold from the highest concentration of 100-fold using blocking solution in 7 steps. The blocked plate was washed, and the diluted samples were added to the plate and left to stand at room temperature for 1 hour. Detection antibodies, Goat Anti-Mouse IgG and Human ads-HRP (SouthernBiotech), were diluted 3000-fold in blocking solution and added to the washed wells. After 1 hour, the plate was washed, TMB Microwell Peroxidase Substrate System (seracare) was added, and the plate was left to stand for 2-3 minutes. The reaction was stopped using TMB Stop Solution (KPL, Cat. 51500-0021). The corrected absorbance, calculated by subtracting the absorbance at 540 nm from the absorbance at 450 nm, was used for analysis using a plate reader to calculate the anti-gp46 antibody titer and anti-Tax antibody titer. The blocking solution was 1X Dulbecco's Phosphate Buffered Saline (gibco) supplemented with 1% Bovine Serum Albumin (Sigma) and 0.05% Tween (BIO-RAD), and washing was performed three times with 1X Dulbecco's Phosphate Buffered Saline ((10X)gibco) supplemented with 0.05% Tween.

[0124] (Test Example 7) gp46- and Tax-specific cellular immune responses in mice. Prepared mouse spleen cells 10 6The cells were seeded at 100 cells / well in a 96-well U-bottom plate (FALCON) and stimulated with gp46 and Tax pooled peptides (Eurofins) at a final concentration of 10 μg / mL at 37°C and 5% CO2. After 24 hours, the culture supernatant was collected and IFN-γ (R&D Systems) and IL-2 (R&D Systems) production was measured according to the kit protocol.

[0125] (Test Example 8) Protein Expression Analysis Examples 9, 10, 11, and 12 were added to CHO-S cells (Thermo Fisher Scientific) so that the mRNA concentration during culture was 3 μg / mL. 72 hours after addition, the culture supernatant and cell pellet were collected. The cell pellet was mixed well with M-PER (trademark) Tissue Protein Extraction Reagent (Thermo Scientific) and left to stand at room temperature for 10 minutes. The mixture was mixed again with a vortex and centrifuged (3000 RPM, 4°C, 10 minutes) to collect the cell lysate. The gp46 protein in the culture supernatant and cell lysate was detected using Western blotting.

[0126] (Results of Test Example 1) Selection of Tax antigen designs based on the transcriptional activity of HIV-1 LTR and HTLV-1 LTR The HIV-1 LTR and HTLV-1 LTR transcriptional activities of wild-type Tax, mutant Tax, and secreted Tax mutant were evaluated (Fig. 1). Compared with wild-type Tax, the mutant Tax showed reduced HIV-1 LTR and HTLV-1 LTR transcriptional activities. The secreted Tax mutant also showed HIV-1 LTR and HTLV-1 LTR transcriptional activities equivalent to those of the negative control. These results suggest that the secreted Tax mutant, which has the lowest HIV-1 LTR and HTLV-1 LTR transcriptional activities, is a vaccine antigen candidate with the lowest carcinogenicity.

[0127] (Results of Test Example 2) CTL induction level and blood anti-Tax antibody titer in Example 12 The CTL induction level was evaluated in C3H mice administered Example 12 (FIG. 2). As a result, Example 12 induced Tax-specific CTL and anti-Tax antibody responses in the blood.

[0128] (Results of Test Example 3) Anti-gp46 and Tax antibody titers in monkey blood The blood anti-gp46 antibody response and blood anti-Tax antibody response induced by a lipid particle formulation in which equal amounts of Example 13 and Example 14 were mixed were evaluated ( FIG. 3 ). As a result, the blood anti-gp46 antibody titer and anti-Tax antibody titer were higher after two administrations (4W) and three administrations (6W) compared to before administration (0W).

[0129] (Results of Test Example 4) gp46- and Tax-specific IFN-γ-producing cell levels in monkey PBMCs The blood anti-gp46 antibody responses and blood anti-Tax antibody responses induced by the mixed preparations of Examples 13 and 14 were evaluated ( FIG. 4 ). As a result of examining PBMCs collected and prepared 7 weeks after the fourth administration, the mixed preparation groups of Examples 5 and 6 were found to induce gp46-specific and Tax-specific IFN-γ-producing cells.

[0130] (Results of Test Example 5) Anti-HTLV-1 neutralizing activity in monkey blood The anti-HTLV-1 neutralizing activity in the blood induced by the mixed preparations of Example 13 and Example 14 was evaluated (Figure 5). As a result of examining total IgG in the blood collected and purified two weeks after the fourth administration, anti-HTLV-1 neutralizing activity in the blood was observed in three out of four animals in the lipid particle administration group, compared to the negative control group.

[0131] (Results of Test Example 6) Anti-gp46 antibody titer in mouse blood The blood anti-gp46 antibody response induced by administration of the lipid particles of Example 9, Example 10, Example 11, or Example 12 was evaluated ( FIG. 6 ). As a result, high blood anti-gp46 antibody responses were observed in the Example 9 group, Example 10 group, and Example 11 group, compared to the negative control group.

[0132] (Results of Test Example 7) gp46-specific and Tax-specific cellular immune responses in C57BL / 6 mice. The gp46-specific and IL-2-specific cellular immunity induced by administration of the lipid particles of Example 9, Example 10, Example 11, or Example 12 was evaluated using the IFN-γ production level and the IL-2 production level as indicators ( FIG. 7 ). As a result, a high gp46-specific cellular immune response was observed in the Example 9 group, the Example 10 group, and the Example 11 group, compared to the negative control group. In addition, a high Tax-specific cellular immune response was observed in the Example 12 group, compared to the negative control group.

[0133] (Results of Test Example 8) Expression levels of antigen proteins by lipid particles of Examples 9 to 12 The expression levels of antigen proteins expressed by each lipid particle in the culture supernatant and cell lysate of CHO-S cells treated in Examples 9 to 12 were evaluated (FIG. 8). As a result, expression of antigen proteins bound by anti-gp46 antibodies expressed by each lipid particle was confirmed. The gp46 protein is 39 kDa.

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[0143] [Industrial Applicability]

[0144] The present invention can be used for the prevention and / or treatment of infection by HTLV-1. [Sequence List Free Text]

[0145] SEQ ID NO:1: Nucleotide sequence of template plasmid DNA for SU gp46 IVT SEQ ID NO:2: Nucleotide sequence of the sense primer SEQ ID NO: 3: Nucleotide sequence of the antisense primer SEQ ID NO: 4: Nucleotide sequence of template DNA for in vitro transcription (IVT) of SU gp46 SEQ ID NO: 5: Nucleotide sequence of template plasmid DNA for HTLV-I gp46-Fib IVT SEQ ID NO: 6: Nucleotide sequence of template DNA for in vitro transcription (IVT) of gp46-Fib SEQ ID NO: 7: Nucleotide sequence of template plasmid DNA for HTLV-I dgp62-Fib IVT SEQ ID NO: 8: Nucleotide sequence of template DNA for in vitro transcription (IVT) of dgp62-Fib SEQ ID NO: 9: Nucleotide sequence of template plasmid DNA for HTLV-I sec Tax mutant IVT SEQ ID NO: 10: Nucleotide sequence of template DNA for in vitro transcription (IVT) of sec Tax mutants SEQ ID NO: 11: Amino acid sequence of Tax wild type SEQ ID NO: 12: Amino acid sequence of Tax mutant (T130A / L131S / L319R / L320S) SEQ ID NO: 13: Amino acid sequence of the secreted Tax mutant (T130A / L131S / L319R / L320S) SEQ ID NO: 14: Amino acid sequence of gp46 SEQ ID NO: 15: Amino acid sequence of gp46-Fib SEQ ID NO: 16: Amino acid sequence of dgp62-Fib SEQ ID NO: 17: SU gp46 mRNA SEQ ID NO: 18: gp46-Fib mRNA SEQ ID NO: 19: dgp62-Fib mRNA SEQ ID NO: 20: sec Tax mutant mRNA SEQ ID NO: 21: Nucleotide sequence of template DNA for gp46-Fib polyA95 SEQ ID NO: 22: Nucleotide sequence of template DNA of gp46-Fib polyA80 SEQ ID NO: 23: Nucleotide sequence of template DNA of gp46-Fib polyA60 SEQ ID NO: 24: Nucleotide sequence of template DNA of gp46-Fib polyA40 SEQ ID NO: 25: Nucleotide sequence of template DNA of gp46-Fib polyA20 SEQ ID NO: 26: Nucleotide sequence of template DNA of sec Tax mutant polyA95 SEQ ID NO: 27: Nucleotide sequence of template DNA of sec Tax mutant polyA80 SEQ ID NO: 28: Nucleotide sequence of template DNA of sec Tax mutant polyA60 SEQ ID NO: 29: Nucleotide sequence of template DNA of sec Tax mutant polyA40 SEQ ID NO: 30: Nucleotide sequence of template DNA of sec Tax mutant polyA20 SEQ ID NO: 31: gp46-Fib mRNA sequence polyA95 SEQ ID NO: 32: gp46-Fib mRNA sequence polyA80 SEQ ID NO: 33: gp46-Fib mRNA sequence polyA60 SEQ ID NO: 34: gp46-Fib mRNA sequence polyA40 SEQ ID NO: 35: gp46-Fib mRNA sequence polyA20 SEQ ID NO: 36: mRNA sequence of sec Tax mutant polyA95 SEQ ID NO: 37: mRNA sequence of sec Tax mutant polyA80 SEQ ID NO: 38: mRNA sequence of sec Tax mutant polyA60 SEQ ID NO: 39: mRNA sequence of sec Tax mutant polyA40 SEQ ID NO: 40: mRNA sequence of sec Tax mutant polyA20 All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A lipid particle encapsulating a nucleic acid capable of expressing a Tax antigen of human T-cell leukemia virus type 1 (HTLV-1), The Tax antigen is With respect to the amino acid sequence of SEQ ID NO: 11, the amino acid sequence has at least one mutation selected from the group consisting of T130A, L131S, L319R and L320S; When the amino acid sequence other than the mutated amino acid is compared, the amino acid sequence has at least 95% identity with the amino acid sequence of SEQ ID NO: 11, The above particles, wherein the lipid comprises a cationic lipid represented by general formula (Ia) or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 During the ceremony, R １ and R ２ is independently １ -C ３ represents an alkyl group; L １ is C ２ -C ４ C which may have one or more alkanoyloxy groups １７ -C １９ represents an alkenyl group; L ２ is C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ Alkyl group, or C ２ -C ４ C which may have one or more alkanoyloxy groups １０ -C １９ represents an alkenyl group; p is 3 or 4.

2. The particle described in claim 1, wherein the Tax antigen is a fusion protein with a signal peptide.

3. The particle described in claim 2, wherein the signal peptide is an IgE secretory signal peptide.

4. A particle described in claim 3, wherein the IgE secretory signal peptide is a peptide consisting of the amino acid sequence from the 1st to the 18th amino acids of the amino acid sequence of SEQ ID NO:

13.

5. A particle as described in claim 1, further comprising a nucleic acid capable of expressing a gp46 antigen in the same lipid particle.

6. A lipid particle composition comprising a lipid particle containing a nucleic acid capable of expressing a gp46 antigen and the lipid particle described in claim 1 as a separate particle.

7. The cationic lipid has the following structural formula: 【Chemistry 2】 The particle according to claim 1 , wherein the particle is represented by the formula:

8. The cationic lipid has the following structural formula: 【Chemistry 3】 The particle according to claim 1 , wherein the particle is represented by the formula:

9. The cationic lipid has the following structural formula: 【Chemistry 4】 The particle according to claim 1 , wherein the particle is represented by the formula:

10. The particle of claim 7 , wherein the lipid further comprises an amphipathic lipid, a sterol, and a PEG lipid.

11. The particle described in claim 8, wherein the lipid further comprises an amphipathic lipid, a sterol and a PEG lipid.

12. 10. The particle of claim 9, wherein the lipid further comprises an amphipathic lipid, a sterol, and a PEG lipid.

13. 11. The particle according to claim 10, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

14. The particle of claim 11, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

15. 13. The particle according to claim 12, wherein the amphiphilic lipid is at least one selected from the group consisting of distearoylphosphatidylcholine, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine.

16. 11. The particle according to claim 10, wherein the sterol is cholesterol.

17. 12. The particle according to claim 11, wherein the sterol is cholesterol.

18. 13. The particle according to claim 12, wherein the sterol is cholesterol.

19. 11. The particle of claim 10, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

20. The particle according to claim 11, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

21. 13. The particle of claim 12, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.

22. The particle according to any one of claims 10 to 21, wherein the lipid composition of the amphipathic lipid, the sterols, the cationic lipid, and the PEG lipid is, in molar amounts, 5 to 25% amphipathic lipid, 10 to 55% sterols, 40 to 65% cationic lipid, and 1 to 5% PEG lipid.

23. 23. The particle according to claim 22, wherein the amphiphilic lipid is 10 to 25%.

24. The particle according to any one of claims 10, 11, 13, 14, 16, 17, 19, or 20, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 5 to 15% amphipathic lipid, 35 to 50% sterols, 40 to 55% cationic lipid, and 1 to 3% PEG lipid.

25. 25. The particle according to claim 24, comprising 10-15% amphiphilic lipid, 35-45% sterols, 40-50% cationic lipid, and 1-2.5% PEG lipid.

26. 26. The particle of claim 25, wherein the PEG lipid is 1-2%.

27. 22. The particle according to any one of claims 12, 15, 18, or 21, wherein the lipid composition of amphipathic lipid, sterols, cationic lipid, and PEG lipid is, in molar amounts, 10 to 25% amphipathic lipid, 10 to 50% sterols, 40 to 65% cationic lipid, and 1 to 3% PEG lipid.

28. 28. The particle according to claim 27, comprising 10-45% sterols, 42.5-65% cationic lipid, and 1-2.5% PEG lipid.

29. 29. The particle of claim 28, wherein the PEG lipid is 1-2%.

30. 23. The particle according to claim 22, wherein the ratio of total lipid weight to nucleic acid weight is 15 to 30.

31. 31. The particle according to claim 30, wherein the ratio of total lipid weight to nucleic acid weight is 15 to 25.

32. 32. The particle according to claim 31, wherein the ratio of total lipid weight to nucleic acid weight is 17.5 to 22.

5.

33. The particle according to claim 5, wherein the gp46 antigen of human T-cell leukemia virus type 1 (HTLV-1) is a fusion protein with an oligomerization domain.

34. 34. The particle of claim 33, wherein the oligomerization domain is fibritin.

35. The particle according to claim 34, wherein the gp46 antigen of human T-cell leukemia virus type 1 (HTLV-1) consists of an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

15.

36. The particle described in claim 1, wherein the Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) has the mutations T130A, L131S, L319R and L320S with respect to the amino acid sequence of SEQ ID NO:11, and when compared with the amino acid sequence excluding the mutated amino acids, the particle consists of an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO:

11.

37. The particle described in claim 33, wherein the nucleic acid capable of expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) is an mRNA comprising a cap structure (Cap), a 5' untranslated region (5'-UTR), a translation region of gp46 antigen or Tax antigen, a 3' untranslated region (3'-UTR) and a poly A tail (poly A).

38. The particle described in claim 37, wherein the sequence of the nucleic acid capable of expressing the gp46 antigen of human T-cell leukemia virus type 1 (HTLV-1) consists of a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 17 or 18.

39. The particle described in claim 37, wherein the nucleic acid sequence capable of expressing the Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) consists of a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO:

20.

40. The particle of claim 1 , wherein the nucleic acid comprises at least one modified nucleotide.

41. 41. The particle of claim 40, wherein the modified nucleotide comprises at least one pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine nucleotide optionally substituted at the 1-position.

42. The particle according to claim 40, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

43. The particle according to claim 40, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.

44. 2. The particles according to claim 1, having an average particle size of 30 to 300 nm.

45. Use of the particles according to claim 1 for the manufacture of a composition for preventing and / or treating infection with human T-cell leukemia virus type 1 (HTLV-1).

46. A composition comprising the particles of claim 1.

47. The composition according to claim 46, for expressing gp46 antigen or Tax antigen of human T-cell leukemia virus type 1 (HTLV-1) in vivo or in vitro.

48. 48. The composition according to claim 46 or 47, for use as a medicine.

49. The composition according to claim 48, for inducing an immune response against human T-cell leukemia virus type 1 (HTLV-1).

50. The composition according to claim 48 for preventing and / or treating infection with human T-cell leukemia virus type 1 (HTLV-1).

51. The composition described in claim 49 for preventing and / or treating infection with human T-cell leukemia virus type 1 (HTLV-1).

52. The composition according to claim 48, for preventing and / or treating the onset of a disease caused by HTLV-1 selected from the group consisting of adult T-cell leukemia-lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU) in an HTLV-1-infected individual.

53. The composition described in claim 49 for preventing and / or treating the onset of a disease caused by HTLV-1 selected from the group consisting of adult T-cell leukemia / lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU) in an HTLV-1-infected individual.

54. A Tax antigen of human T-cell leukemia virus type 1 (HTLV-1), comprising: A peptide in which a Tax antigen and a signal peptide are fused, The Tax antigen is a peptide having an amino acid sequence that has at least one mutation selected from the group consisting of L131S, L319R and L320S with respect to the amino acid sequence of SEQ ID NO:11, and that has at least 95% identity with the amino acid sequence of SEQ ID NO:11 when compared with the amino acid sequence other than the mutated amino acids.

55. A Tax antigen described in claim 54, further having a T130A mutation.

56. A Tax antigen described in claim 54 or 55, wherein the signal peptide is an IgE secretory signal peptide, preferably a peptide consisting of the 1st to 18th amino acids of the amino acid sequence of SEQ ID NO:

13.

57. A nucleic acid expressing the Tax antigen described in claim 54.

Images