WO2020022272A1 - インフルエンザワクチンを含む組成物 - Google Patents
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- WO2020022272A1 WO2020022272A1 PCT/JP2019/028674 JP2019028674W WO2020022272A1 WO 2020022272 A1 WO2020022272 A1 WO 2020022272A1 JP 2019028674 W JP2019028674 W JP 2019028674W WO 2020022272 A1 WO2020022272 A1 WO 2020022272A1
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- A61K39/145—Orthomyxoviridae, e.g. influenza virus
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16171—Demonstrated in vivo effect
Definitions
- the present invention relates to a composition comprising a vaccine adjuvant and a universal influenza vaccine antigen.
- Component vaccines consisting of virus-derived proteins or their partial peptides are superior in safety to live vaccines and whole-particle inactivated vaccines.
- component vaccines tend to have low efficacy of immunostimulation.
- An adjuvant is an additive that enhances a humoral immune response and / or a cellular immune response to an antigen, and Alum, saponin, and the like have been used as vaccine adjuvants.
- TLR Toll-like receptor
- TLR7 and TLR8 small molecules that mimic single-stranded RNA of a virus that is a natural ligand are known as Activators.
- synthetic compounds such as pyrimidine compounds (see Patent Documents 1 and 2) and imidazoquinoline compounds (see Patent Document 3) have been reported.
- TLR7 and / or TLR8 Activation of TLR7 and / or TLR8 by its agonist activates dendritic cells (DC) via TLR and myeloid differentiation factor 88 (MyD88) -dependent signaling pathways.
- DC dendritic cells
- MyD88 myeloid differentiation factor 88
- T-cell co-stimulatory molecules co-stimulatory molecules CD80, CD86, CD40
- inflammatory cytokines including type I interferon (especially IFN ⁇ ), TNF ⁇ , IL-6 or IL-12 are produced. .
- TLR7 and / or TLR8 agonist is known to have an activity of activating T cells and B cells in addition to activating DC, and further stimulating NK cells to promote IFN ⁇ production. It is expected to have vaccine adjuvant activity. Indeed, vaccine adjuvant activity of TLR7 and / or TLR8 agonists such as resiquimod or imiquimod has been reported (see Non-Patent Document 2).
- Examples of the complex between the TLR7 and / or 8 agonist and other substances include a vaccine adjuvant in which a fatty acid and an imidazoquinoline compound are covalently linked (see Patent Documents 4, 5, 6, and Non-Patent Document 4), A conjugate compound of a phospholipid and an adenine compound (see Patent Literatures 7, 8, and 9), a conjugate compound of a fatty acid and an adenine compound (Non-Patent Literature 5), a fatty acid glyceride or a polyethylene glycol of a phospholipid and an adenine compound A conjugate compound (see Patent Document 10), a conjugate compound of squalene and an adenine compound (see Patent Document 11), and a conjugate compound of squalene and a pyrimidine compound (see Patent Document 12) are known.
- influenza hemagglutinin also called influenza HA split vaccine
- the HA antibody binds to a portion of the hemagglutinin that is exposed from the viral membrane, called the head region, which is the region having the most structural change among the virus strains.
- the head region which is the region having the most structural change among the virus strains.
- stem cell antibody's ability to protect infection not only depends on its antigen binding property and antibody amount, but also strongly depends on the subclass of the induced IgG antibody (see Non-Patent Document 3).
- stem IgG2 antibodies are functional and cross-reactive, indicating that a vaccine that strongly induces stem IgG2 antibodies is effective in protecting a wide range of influenza infections.
- no method has been found to effectively enhance the ability to induce stem IgG2 antibodies.
- the problem to be solved by the present invention is to find an influenza HA split vaccine that effectively produces an antibody that binds to the HA stem region of an influenza virus that is unlikely to cause antigen mutation, and a method for improving the inducing ability of an IgG2 antibody. It is to provide.
- the present inventors have conducted intensive studies and found that the stem region of hemagglutinin is exposed to the outside by subjecting the current influenza HA split vaccine to acidic treatment, and the influenza HA split vaccine after the acid treatment has an HA stem region. It has been found that it can function as a universal influenza vaccine antigen by producing a cross-reactive antibody that binds to LAH (long alpha alpha helix). Furthermore, it has been found that by adding a compound that enhances the physiological activity of TLR7 to the influenza HA split vaccine after the acid treatment, the compound functions as a vaccine adjuvant, and its IgG2 antibody-inducing ability is improved. Was completed.
- the present invention relates to the following.
- composition comprising the following (1) and (2); (1) a universal influenza vaccine antigen; and (2) a vaccine adjuvant.
- composition according to Item 1 wherein the universal influenza vaccine antigen is an influenza HA split vaccine antigen capable of producing an antibody that binds to LAH in the HA stem region.
- composition according to Item 2 wherein the HA stem region of the influenza HA split vaccine antigen is in a form exposed to the outside.
- the universal influenza vaccine antigen is a vaccine antigen containing two or more influenza HA split vaccine antigens produced by subjecting an influenza HA split vaccine, each of which is a single HA subtype, to an acidic treatment.
- Item 8. The composition according to any one of Items 1 to 7.
- the vaccine adjuvant is a substance that enhances the physiological activity of one or more TLRs selected from the group consisting of TLR7, TLR8, and TLR9.
- Composition [Item 12] The composition according to any one of Items 1 to 11, wherein the vaccine adjuvant is a substance that enhances at least the physiological activity of TLR7.
- the vaccine adjuvant is a substance that enhances at least the physiological activity of TLR9.
- the substance that enhances the biological activity of TLR7 is a low molecular weight compound that enhances the biological activity of TLR7, or a low molecular weight compound that enhances the biological activity of TLR7 is chemically bound to a lipid via a spacer.
- Item 13 The composition according to Item 11 or 12, which is a conjugate compound.
- the low molecular weight compound that enhances the physiological activity of TLR7 or the low molecular weight compound that enhances the physiological activity of TLR7 contained in the conjugate compound has a molecular weight of 200 to 600, and has an adenine skeleton and a pyrimidine. Item 16.
- composition according to Item 15 having a skeleton, an imidazoquinoline skeleton, an imidazopyridine skeleton, or a quinazoline skeleton.
- the low molecular weight compound that enhances the physiological activity of TLR7 or the low molecular weight compound that enhances the physiological activity of TLR7 contained in the conjugate compound has a pyrimidine skeleton, an adenine skeleton, or an imidazoquinoline skeleton.
- the conjugate compound wherein the compound that enhances the biological activity of TLR7 is a low molecular weight compound having an adenine skeleton and a lipid derived from squalene or squalane chemically bonded via a spacer, or a pharmaceutically acceptable compound thereof Item 19.
- a conjugate compound or a pharmaceutical thereof, wherein the compound that enhances the biological activity of TLR7 is a low molecular weight compound having a pyrimidine skeleton and a squalene or squalane-derived lipid chemically bonded via a spacer.
- Item 19 The composition according to any one of Items 15 to 18, which is a physiologically acceptable salt.
- the compound that enhances the physiological activity of TLR7 is represented by the following formula (1): [In the formula (1), X represents methylene, R 1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms which may be substituted with a hydroxy group; R 2 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R 3 represents an alkyl group having 1 to 3 carbon atoms, R 4 represents a hydrogen atom, a halogen atom, a hydroxy group, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms, Y 1 represents a single bond or methylene, Y 2 represents a single bond or —C (O) —, L represents a straight-chain alkylene having 2 or 3 carbon atoms, R 5 and R 6 independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, or R 5 together with R 6 is a substituted or unsub
- the conjugate compound is (4E, 8E, 12E, 16E, 20E) -N- ⁇ 2-[ ⁇ 4-[(2-amino-4- ⁇ [(3S) -1-hydroxyhexane-3-yl] amino ⁇ -6-methyl Pyrimidin-5-yl) methyl] benzyl ⁇ (methyl) amino] ethyl ⁇ -4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide ; (4E, 8E, 12E, 16E, 20E) -1- (4- ⁇ 4-[(2-amino-4- ⁇ [(2S) -1-hydroxypentan-2-yl] amino ⁇ -6-methylpyrimidine -5-yl) methyl] -3-methoxybenzyl ⁇ piperazin-1-yl) -4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24
- [Item 25] A method for preventing influenza, comprising administering a prophylactically effective amount of the composition according to any of Items 1 to 23 to a warm-blooded animal in need of prevention.
- a prophylactically effective amount of a universal influenza vaccine antigen and a prophylactically effective amount of a vaccine adjuvant eg, a substance that enhances the biological activity of TLR7
- a vaccine adjuvant eg, a substance that enhances the biological activity of TLR7
- a kit for preventing influenza comprising a universal influenza vaccine antigen and a vaccine adjuvant (for example, a substance that enhances the biological activity of TLR7).
- a vaccine adjuvant for example, a substance that enhances the biological activity of TLR7.
- a vaccine adjuvant for example, a substance that enhances the biological activity of TLR7 for use in preventing influenza in combination with a universal influenza vaccine antigen.
- a universal influenza vaccine antigen for use in the prevention of influenza in combination with a vaccine adjuvant eg, a substance that enhances the biological activity of TLR7.
- [Item 30] Use of a vaccine adjuvant (for example, a substance which enhances the biological activity of TLR7) in the manufacture of a composition for preventing influenza containing a universal influenza vaccine antigen.
- a vaccine adjuvant for example, a substance which enhances the biological activity of TLR7
- a universal influenza vaccine antigen in the manufacture of a composition for preventing influenza comprising a vaccine adjuvant (for example, a substance that enhances the biological activity of TLR7).
- a method for producing a composition comprising an influenza HA split vaccine antigen and a vaccine adjuvant (for example, a substance that enhances the biological activity of TLR7), comprising the following steps: a) a step of producing an influenza HA split vaccine antigen that produces an antibody that binds to LAH in the HA stem region by subjecting an influenza HA split vaccine that has not been subjected to formalin treatment to an acidic treatment; and b) the step (a). Mixing the obtained vaccine antigen with a vaccine adjuvant.
- a vaccine adjuvant for example, a substance that enhances the biological activity of TLR7
- a method for producing a composition comprising an influenza HA split vaccine antigen and a vaccine adjuvant (for example, a substance that enhances the biological activity of TLR7), comprising the following steps: a) a step of subjecting the influenza HA split vaccine to acidic treatment; b) subsequently performing formalin treatment to produce an influenza HA split vaccine antigen that produces an antibody that binds to LAH in the HA stem region; and c) combining the vaccine antigen obtained in step b) with a vaccine adjuvant. Mixing.
- a vaccine adjuvant for example, a substance that enhances the biological activity of TLR7
- a method for producing a kit containing an influenza HA split vaccine antigen and a vaccine adjuvant comprising the following steps: a) a step of producing an influenza HA split vaccine antigen that produces an antibody that binds to LAH in the HA stem region by subjecting an influenza HA split vaccine that has not been subjected to formalin treatment to an acidic treatment; and b) the step (a). A step of combining the obtained vaccine and a vaccine adjuvant into a kit.
- a vaccine adjuvant for example, a substance that enhances the biological activity of TLR7
- a method for producing a kit comprising an influenza HA split vaccine antigen and a vaccine adjuvant comprising the following steps: a) a step of subjecting the influenza HA split vaccine to acidic treatment; b) a step of producing an influenza HA split vaccine antigen that produces an antibody that binds to LAH in the HA stem region by subsequent formalin treatment; and c) combining the vaccine obtained in step b) with a vaccine adjuvant.
- a vaccine adjuvant for example, a substance that enhances the biological activity of TLR7
- a universal influenza vaccine antigen is provided, and the vaccine activity can be further improved by adding a vaccine adjuvant.
- FIG. 3 is a diagram showing the improvement of cross-protection ability of an mouse against which an H3N2-type membrane fusion HA split vaccine has been inoculated with an antigen mutant strain. It is a figure which shows the rise of LAH antibody titer in the serum of the mouse
- FIG. 3 is a diagram showing the improvement of cross-protection ability of an mouse against which an H3N2-type membrane fusion HA split vaccine has been inoculated with an antigen mutant strain. It is a figure which shows the rise of LAH antibody titer in the serum of the mouse
- FIG. 3 is a diagram showing the improvement of cross-protection ability of an mouse against which an H1N1 membrane fusion HA split vaccine has been inoculated with an antigen mutant strain.
- FIG. 4 shows that LAH-binding monoclonal antibodies bind more strongly to membrane-fused HA split vaccines than current HA split vaccines.
- FIG. 2 is a view showing the cross-reactive IgG2c-inducing activity of a membrane-fused HA split vaccine to which A-1, A-2, or CpG has been added.
- FIG. 2 is a view showing the cross-reactive IgG1 inducing activity of a membrane fusion HA split vaccine to which A-1, A-2, or CpG has been added.
- FIG. 4 shows the cross-reactive IgG2c-inducing activity of a membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added.
- FIG. 2 is a view showing the cross-reactive IgG1 inducing activity of a membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added.
- FIG. 4 is a view showing a cross-reactive IgG2c / IgG1 ratio of a membrane fusion HA split vaccine to which A-1, A-3, or AddaVax (TM) is added.
- FIG. 2 is a view showing the cross-protection ability of an mouse against which an H3N2 membrane-fused HA split vaccine supplemented with A-1, A-3, or AddaVax (TM) has been inoculated against an antigen mutant.
- FIG. 4 is a graph showing the inhibitory effect of H3N2 membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added, on weight loss in mice due to infection with an antigenic mutant.
- FIG. 4 shows that LAH-binding monoclonal antibodies strongly bind to membrane-fused HA split vaccine that has been subjected to formalin treatment after acid treatment. It is a figure which shows the rise of the LAH antibody titer in the serum of the mouse
- FIG. 4 shows the cross-reactive IgG2c-inducing activity of a membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added.
- FIG. 2 is a view showing the cross-reactive IgG1 inducing activity of a membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added.
- FIG. 4 is a view showing a cross-reactive IgG2c / IgG1 ratio of a membrane fusion HA split vaccine to which A-1, A-3, or AddaVax (TM) is added.
- FIG. 4 is a diagram showing the cross-protection ability of mice vaccinated with the H1N1 membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added, against antigen mutants.
- FIG. 3 is a graph showing the inhibitory effect of H1N1 membrane-fused HA split vaccine to which A-1, A-3, or AddaVax (TM) has been added on weight loss in mice due to antigen mutant infection.
- a vaccine adjuvant is a substance having an immunopotentiating action or a preparation containing the same, and means a substance that improves the immunogenicity of an antigen when added to the antigen.
- the vaccine adjuvants herein include alum, emulsions (eg, Freund's adjuvant, MF59 (registered trademark), AddaVax (TM), AS03, etc.), substances that enhance the physiological activity of NOD receptors, RIG-I receptors Substances that enhance physiological activity, substances that enhance the biological activity of C-type lectin receptor, substances that enhance the biological activity of cytoplasmic DNA receptor, substances that enhance the biological activity of STING, and substances that enhance the biological activity of TLR Etc. are included.
- Preferred examples of the vaccine adjuvant in the present specification include substances that enhance the physiological activity of TLR.
- the substance that enhances the physiological activity of TLR means a TLR agonist having TLR receptor agonist activity, and is not particularly limited as long as it is a substance that enhances the function of TLR receptor.
- Examples of the substance that enhances the physiological activity of TLR in the present specification include monophosphoryl lipid A and its derivatives, adenine compounds, pyrimidine compounds, imidazoquinoline compounds, benzapine compounds, imidazopyridine compounds, quinazoline compounds, guanine compounds, dihydropteridine compounds , CpG ODN and the like.
- the substance that enhances the biological activity of TLR in the present specification preferably includes a substance that enhances the biological activity of one or more TLRs selected from the group consisting of TLR7, TLR8, and TLR9, more preferably And a substance that enhances at least the physiological activity of TLR7.
- the substance that enhances the physiological activity of TLR7 means a TLR7 agonist having TLR7 receptor agonist activity, and is not particularly limited as long as it is a substance that enhances the function of TLR7 receptor.
- the substance that enhances the biological activity of TLR8 means a TLR8 agonist having TLR8 receptor agonist activity, and is not particularly limited as long as it is a substance that enhances the function of TLR8 receptor.
- the substance that enhances the biological activity of TLR9 means a TLR9 agonist having TLR9 receptor agonist activity, and is not particularly limited as long as it is a substance that enhances the function of TLR9 receptor.
- Examples of the substance that enhances the biological activity of TLR7 in the present specification include a substance that enhances the biological activity of TLR7 or a substance that enhances the biological activity of TLR7 and TLR8.
- TLR7-selective means that TLR receptor agonist activity other than TLR7 is weaker than TLR7 receptor agonist activity.
- TLR7 receptor agonist activity is TLR8 receptor activity using single-stranded RNA as an endogenous ligand.
- the case where the activity is stronger than the body agonist activity more specifically, the case where the TLR7 receptor agonist activity (EC50 value) is 10 times or more the TLR8 receptor agonist activity can be exemplified.
- Examples of the substance that enhances the biological activity of TLR7 in the present specification include a low molecular weight compound that enhances the biological activity of TLR7, and a low molecular weight compound that enhances the biological activity of TLR7 and a lipid are chemically bonded via a spacer. Conjugate compounds that are bound to are included.
- the low molecular weight compound that enhances the biological activity of TLR7 in the present specification include those having a molecular weight of 200 to 600, preferably 250 to 500, more preferably 300 to 500.
- a compound having an adenine skeleton, a pyrimidine skeleton, an imidazoquinoline skeleton, an imidazopyridine skeleton, a quinazoline skeleton, a guanine skeleton, or a dihydropteridine skeleton is used.
- a compound having an adenine skeleton, a pyrimidine skeleton, or an imidazoquinoline skeleton is used.
- Examples of the compound having an adenine skeleton include a compound having a 4-amino-8-oxo-purine (8-oxoadenine) skeleton, for example, an aromatic carbon ring having 5 to 6 members at the 9-position, and 5 to 6 members. Substituted with an alkyl group which may be substituted with a 4-membered aromatic heterocyclic ring or a 4- to 7-membered aliphatic nitrogen-containing heterocyclic ring (for example, a linear alkyl group having 1 to 6 carbon atoms). Examples include compounds having an oxo-purine skeleton.
- GSK-2245035 [6-amino-2- ⁇ [(1S) -1-methylbutyl] oxy ⁇ -9- [5- (1-piperidinyl) pentyl] -7,9-dihydro-8H-purine -8-one], PF-4171455 [4-amino-1-benzyl-6-trifluoromethyl-1,3-dihydroimidazo [4,5-c] pyridin-2-one] and the like.
- the compounds having an adenine skeleton preferably include the following compounds or pharmaceutically acceptable salts thereof: 6-amino-9-( ⁇ 6- [2- (dimethylamino) ethoxy] pyridin-3-yl ⁇ methyl) -2-ethoxy-7,9-dihydro-8H-purin-8-one; 6-amino-2- (butylamino) -9-( ⁇ 6- [2- (dimethylamino) ethoxy] pyridin-3-yl ⁇ methyl) -7,9-dihydro-8H-purin-8-one; N- (4- ⁇ [6-amino-2- (butylamino) -8-oxo-7,8-dihydro-9H-purin-9-yl] methyl ⁇ benzoyl) glycine; Methyl (3- ⁇ [[3- (6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl) propyl]
- Examples of the compound having a pyrimidine skeleton include a compound having a 2,4-diaminopyrimidine skeleton.
- the 6-position is appropriately substituted with an alkyl group or the like
- the 5-position is a 5- to 6-membered aromatic carbon ring.
- 2,4 substituted with an alkyl group which may be substituted with a 5- to 6-membered aromatic heterocycle or a 4- to 7-membered aliphatic nitrogen-containing heterocycle (for example, a linear alkyl group having 1 to 6 carbon atoms) -Diaminopyrimidine.
- Specific examples include the compounds described in WO00 / 12487, WO2010 / 133885, WO2013 / 172479 or WO2012 / 136834.
- Examples of the compound having an imidazoquinoline skeleton include imiquimod, resiquimod or 852A [N- [4- (4-amino-2-ethyl-1H-imidazo [4,5-c] quinoline-1). -Yl) butyl] methanesulfonamide] and other compounds having a 4-amino-1H-imidazo [4,5-c] quinoline skeleton, for example, a C1-6 alkyl group or C1-6 alkoxy at the 1-position. 4-amino-1H [4,5-c] quinoline substituted with a C1-6 alkyl group or a C1-6 alkoxy group.
- the compound having an imidazoquinoline skeleton is preferably Imiquimod.
- Examples of the compound having an imidazopyridine skeleton include a compound having a 4-amino-1,3-dihydro-2H-imidazo [4,5-c] pyridin-2-one skeleton.
- a compound having a 6- or 7-position Is substituted with a C1-6 alkyl group or a C1-6 alkoxy group which may be substituted with a halogen atom, and the 1-position is a 5- to 6-membered aromatic carbocycle, a 5- to 6-membered aromatic heterocycle or 4-amino-1,3-dihydro-2H-imidazo [substituted with an alkyl group which may be substituted with a 7-membered aliphatic nitrogen-containing heterocyclic ring (eg, a linear alkyl group having 1 to 4 carbon atoms)] 4,5-c] pyridin-2-one.
- a 7-membered aliphatic nitrogen-containing heterocyclic ring eg, a linear alkyl
- Examples of the compound having a quinazoline skeleton include a derivative having a 2,4-diaminoquinazoline skeleton, and an alkyl group optionally substituted at the 4-position with a hydroxyl group or a halogen atom (for example, a linear or branched alkyl group).
- Examples of the compound having a guanine skeleton include a compound having a 2-amino-6-oxopurine skeleton, and specifically, loxoribine.
- Examples of the compound having a dihydropteridine skeleton include compounds having a 4-amino-7,8-dihydropteridin-6 (5H) -one skeleton, and specifically, besatrimod [4-amino-2-butoxy-8- [[3-[(pyrrolidin-1-yl) methyl] phenyl] methyl] -7,8-dihydropteridin-6 (5H) -one].
- low molecular weight compounds that are TLR7 agonists include Isatoribine, ANA-773, and the compounds described in WO2010 / 077613.
- Examples of the lipid in the conjugate compound in which a low molecular weight compound that promotes the biological activity of TLR7 and the lipid are chemically bonded via a spacer herein include saturated or unsaturated fatty acids having less than 100 carbon atoms.
- a saturated or unsaturated fatty acid having 18 to 30 carbon atoms is used, and more preferably, a fatty acid derived from squalene or squalane is used.
- Fatty acids derived from squalene include (4E, 8E, 12E, 16E, 20E) -4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24- Hexanoic acid ((4E, 8E, 12E, 16E, 20E) -4,8,12,17,21,25-Hexamethylhexacosa-4,8,12,16,20,24-hexaenoic acid) and the like.
- Examples of lipids derived from squalane include 4,8,12,17,21,25-hexamethylhexacosanoic acid (4,8,12,17,21,25-hexamethylhexacosanoic acid).
- Examples of the low molecular weight compound moiety that enhances the biological activity of TLR7 in the conjugate compound in which the low molecular weight compound that enhances the biological activity of TLR7 and the lipid of the present invention are chemically bonded via a spacer include the above-mentioned compounds. Low molecular weight compounds that enhance the biological activity of TLR7.
- Examples of the conjugate compound in which a low molecular weight compound that enhances the biological activity of TLR7 and a lipid are chemically bonded via a spacer in the present specification include a vaccine in which a fatty acid and an imidazoquinoline compound are covalently linked.
- Adjuvants (Patent Literatures 4, 5, 6 and Non-Patent Literature 4), Conjugate Compounds of Phospholipids and Adenine Compounds (Patent Literatures 7, 8, 9), Conjugate Compounds of Fatty Acids and Adenine Compounds (Non-Patent Literature 5) , A conjugate compound of a fatty acid glyceride and an adenine compound via polyethylene glycol (Patent Document 10), a conjugate compound of a squalene compound and an adenine compound (Patent Document 11), a conjugate compound of a squalene compound and a pyrimidine compound (Patent Document 12) And the like.
- conjugate compound in which a low-molecular weight compound that promotes the biological activity of TLR7 and a lipid are chemically bonded via a spacer for example, a low-molecular weight compound having an adenine skeleton and squalene or squalane are preferably used.
- Conjugate compounds in which derived lipids are chemically bonded via a spacer low molecular weight compounds having a pyrimidine skeleton and squalene or squalane-derived lipids are chemically bonded through a spacer Conjugate compounds and pharmaceutically acceptable salts thereof are included.
- conjugate compound in which a low molecular weight compound having an adenine skeleton and a lipid derived from squalene or squalane are chemically bonded via a spacer include a compound described in Patent Document 11 and the like.
- Preferred examples of the conjugate compound in which a low-molecular weight compound having a pyrimidine skeleton and a lipid derived from squalene or squalane are chemically bonded via a spacer include a compound described in Patent Document 12. More preferably, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof is used.
- X represents methylene
- R 1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms which may be substituted with a hydroxy group
- R 2 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
- R 3 represents an alkyl group having 1 to 3 carbon atoms
- R 4 represents a hydrogen atom, a halogen atom, a hydroxy group, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms
- Y 1 represents a single bond or methylene
- Y 2 represents a single bond or —C (O) —
- L represents a straight-chain alkylene having 2 or 3 carbon atoms
- R 5 and R 6 independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, or R 5 together with R 6 is a substituted or unsubstituted 5- to 8-membered nitrogen-containing nitrogen-containing compound A saturated heterocycl
- Halogen atom includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom or a chlorine atom.
- straight alkylene having 2 or 3 carbon atoms examples include, but are not limited to, ethylene and n-propylene.
- alkyl group having 1 to 3 carbon atoms a linear or branched alkyl group having 1 to 3 carbon atoms can be mentioned. Examples include, but are not limited to, methyl, ethyl, propyl, and isopropyl.
- alkoxy group having 1 to 3 carbon atoms a straight or branched alkoxy group having 1 to 3 carbon atoms can be mentioned. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy.
- the “5- to 8-membered nitrogen-containing saturated heterocycle” includes at least 2 nitrogen atoms selected from 2 or 3 nitrogen atoms, 0 or 1 oxygen atom and 0 or 1 sulfur atom.
- a 5- to 8-membered nitrogen-containing saturated heterocycle containing 1 to 3 heteroatoms in the ring is mentioned. Specific examples include pyrrolidine, piperidine, perhydroazepine, imidazolidine, piperazine, morpholine, thiomorpholine, perhydro-1,4-diazepine and the like.
- the substituent when the nitrogen-containing saturated hetero ring is substituted preferably a methyl group, an ethyl group, a propyl group, a hydroxymethyl group, a hydroxyethyl group, a carbonyl group, a hydroxy group or a halogen atom, more preferably a hydroxy group Or a halogen atom, and may be substituted with the same or different 1 to 4 substituents.
- conjugate compound in which a low molecular weight compound having a pyrimidine skeleton and a lipid derived from squalene or squalane are chemically bound via a spacer
- examples of the pharmaceutically acceptable salt include an acid addition salt and a base addition salt.
- examples of the acid addition salt include an acid addition salt with an inorganic or organic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, citric acid, and maleic acid).
- examples of the base addition salt include an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a calcium salt, and an ammonium salt.
- the universal influenza vaccine antigen means an influenza vaccine antigen that induces an antibody capable of broadly protecting influenza (including a mutant virus) (for example, a cross-protective antibody).
- Examples of the universal influenza vaccine antigen include a particle antigen containing an influenza virus-derived protein described in Patent Document 14 and an influenza HA antigen shown in Non-Patent Documents 6, 7, and 8.
- an influenza HA split vaccine antigen that produces an antibody that binds to the HA stem region LAH. More preferably, "an influenza HA split vaccine antigen that has an HA stem region in an externally exposed form and produces an antibody that binds to the HA stem region LAH" (hereinafter, referred to as the influenza HA split vaccine of the present invention), Can be produced, for example, by a method including a step of performing an acid treatment described below.
- influenza HA split vaccine of the present invention induces an antibody that binds to LAH in the stem region of hemagglutinin, which is unlikely to cause antigen mutation, so that the antigen site to which the antibody induced by the conventional influenza HA split vaccine binds (this is the hemagglutinin (In the bulb region) may also be effective against mutated influenza viruses.
- influenza HA split vaccine of the present invention binds more strongly to the LAH-binding monoclonal antibody than the current HA split vaccine.
- it preferably binds to LAH binding monoclonal antibody 1.05 times or more, preferably 1.1 times or more, more preferably 1.5 times or more, and still more preferably 2 times or more stronger than the current HA split vaccine.
- binding 1.05 times, 1.1 times, 1.5 times, or 2 times or more stronger than the current HA split vaccine means that, for example, the reciprocal of the antibody concentration when the absorbance obtained by regression is 0.7 indicates that the current HA split is 1.05 times, 1.1 times, 1.5 times, or 2 times or more of the reciprocal of the antibody concentration of the vaccine.
- the influenza HA split vaccine of the present invention has a higher binding property to the LAH-binding monoclonal antibody as compared with the current HA split vaccine, and the upper limit is not particularly limited, but is, for example, 1.05 to 200 times, 1.1 to 150 times. , 1.5 to 100 times, and 2 to 50 times.
- the range of binding of the influenza HA split vaccine of the present invention to the LAH-binding monoclonal antibody as compared to the current HA split vaccine is 1.05, 1.1, 1.5, 2, 3, 4, and 5 with a lower limit selected from 200. , 150, 100, 50, 30, and 20 in combination with an upper limit.
- the method for measuring the binding property of the influenza HA split vaccine to the LAH-binding monoclonal antibody is not particularly limited, and can be performed by a general method known to those skilled in the art. .
- the LAH-binding monoclonal antibody means a monoclonal antibody that binds to LAH, and its production method is not particularly limited, and can be produced by a general method known to those skilled in the art.
- the LAH binding monoclonal antibody can bind to a peptide corresponding to at least a portion of LAH of the influenza virus from which the influenza HA split vaccine is derived. Means something.
- the current HA split vaccine means a whole particle vaccine which has been treated with ether to remove a lipid component that becomes a pyrogen, and can be produced by, for example, the method of Example 1 of the present application.
- the current HA split vaccine may be an influenza HA split vaccine produced without applying an acid treatment to the influenza HA split vaccine of the present invention produced by a method having the following acidic treatment step.
- the production method includes a step of subjecting the influenza HA split vaccine to acidic treatment.
- the influenza HA split vaccine is obtained by treating the whole-particle vaccine with ether to remove lipid components, which are pyrogens.
- the HA protein on the surface of virus particles required for immunization is recovered by density gradient centrifugation. HA protein is the main component for production.
- Influenza A virus has two types of spike proteins, HA and NA (neuraminidase), and plays a role in causing the virus to cause infection.
- HA binds to the cells to be infected and takes up the virus into the cells.
- HA frequently causes antigenic mutations.
- NA plays a role in breaking HA from infected cells and releasing the replicated virus from the cells.
- the HA protein changes into a structure called a membrane fusion type.
- the membrane-fused HA protein has a shape in which the stem region is exposed to the outside from the viral membrane instead of the globular region, with a large change in the antigen stem three-dimensional structure.
- fused HA was used as a vaccine, an antibody that binds to LAH in the stem region was induced, and it was found in vivo that this antibody has a protective effect against antigen-mutated viruses.
- the acidic treatment is not particularly limited, but is, for example, pH 3.0 to 6.5, preferably 4.0 to 6.0, and more preferably 4.4 to 5.8.
- the acid used for performing the acid treatment is not particularly limited, but for example, phosphoric acid, citric acid, maleic acid and the like can be used.
- the timing of applying the acid treatment to the influenza HA split vaccine is preferably before the formalin treatment.
- the HA fraction used for the current influenza HA split vaccine is subjected to acidic treatment.
- the formalin treatment followed by formalin treatment it is possible to obtain an influenza HA split vaccine antigen that has a strong effect of producing a cross-reactive antibody, and thus is more preferable as a universal influenza vaccine antigen. That is, in the present application, it is preferred that the virus fraction is treated with ether or the like, and the HA fraction from which the lipophilic solvent is removed is subjected to an acidic treatment and then to a formalin treatment.
- influenza HA split vaccine before the acidic treatment is preferably a split vaccine that has not been subjected to formalin treatment.
- Commercially available influenza HA vaccine (brand name) is described in the standard for biological products (March 30, 2004 Notification of Ministry of Health, Labor and Welfare No. 155, last revision: November 30, 2018 Notification of Ministry of Health, Labor and Welfare No. 409) As described above, a step of decomposing a virus with ether or the like, removing a lipophilic solvent, and then treating with formaldehyde or a substance having an equivalent action has been already performed.
- a commercially available influenza HA vaccine (trade name) is an influenza HA split vaccine, it is preferably not used for the production of the influenza HA split vaccine of the present invention since it has been treated with formaldehyde or the like.
- the concentration of formalin in the formalin-treated solution when formalin is applied to the acid-treated influenza HA split vaccine is, for example, 0.0005 v / v% to 10 v / v%, preferably 0.001 v / v% to 1 v / v%, More preferably, 0.003 v / v% to 0.5 v / v%, and still more preferably, 0.005 v / v% to 0.1 v / v%.
- the grade of formalin used is preferably a grade usable for medical use.
- HA of influenza A virus is divided into 18 subtypes (H1 to H18) and NA is divided into 9 subtypes (N1 to N9). Subtypes can be targeted.
- the method for producing an influenza HA split vaccine of the present invention can produce not only type A but also a type B vaccine having HA, the type A and type B vaccines fall under the category of the influenza HA split vaccine of the present invention. It is.
- Preferred examples of the influenza HA split vaccine of the present invention include those derived from H3N2 influenza virus and H1N1 influenza virus.
- influenza HA split vaccine that is a single HA subtype refers to one subtype of influenza A virus selected from 18 subtypes (H1 to H18) or influenza B virus. Influenza HA split vaccine. If a single HA subtype, the NA subtype may be the same or different. Preferred HA subtypes include H1, H3 and B types.
- an influenza HA split vaccine consisting of a single HA subtype is subjected to acid treatment, and the obtained influenza HA split vaccines are treated in a plurality (two or more).
- a plurality two or more
- it can be manufactured by mixing.
- it can be produced by first subjecting an influenza HA split vaccine obtained by mixing two or more HA subtypes to acidic treatment.
- one to three subtypes selected from the group consisting of H1, H3 and B types are included.
- influenza HA split vaccine of the present invention produces antibodies that bind to LAH with few mutations, cross-protection against influenza viruses known as antigenic mutants is possible within the same HA subtype. Can be Further, cross-reactivity may be exhibited between HA subtypes having similar amino acid sequences of LAH (eg, H3 type and H7 type).
- the influenza HA split vaccine obtained by the above-mentioned production method has a protective effect on an antigen-mutated virus strain.
- the current HA split vaccine is prepared from H3N2 influenza virus particles (A / Fujian / 411/02 (H3N2)) and subjected to acidic treatment, not only A / Fujian / 411/02 (H3N2), but also , A / Guizhou / 54/89 (H3N2), A / OMS / 5389/88 (H3N2), A / Beijing / 32/92 (H3N2), A / England / 427/88 (H3N2), A / Africa / 33 / 94 (H3N2), A / Leningrad / 360/86 (H3N2), A / Mississippi / 1/85 (H3N2), A / Philippines / 2/82 (H3N2), A / Shangdong / 9/93 (H3N2), A / Shanghai / 16
- the current HA split vaccine is prepared from H1N1 influenza virus particles (A / Puerto Rico / 8/34 (H1N1)) and subjected to acidic treatment, not only A / Puerto Rico / 8/34 (H1N1)
- the influenza HA split vaccine of the present invention has an enhanced ability to induce a stem IgG2 antibody by combining a compound that enhances the biological activity of TLR7, and exhibits excellent vaccine activity.
- Another embodiment of the combination of the universal influenza vaccine antigen and the vaccine adjuvant includes a combination of an influenza HA split vaccine antigen that produces an antibody that binds to the HA stem region LAH, and a substance that enhances the biological activity of TLR.
- the influenza HA split vaccine antigen which has an HA stem region exposed to the outside and produces an antibody that binds to the HA stem region LAH, and the biological activity of TLR7 Combination of a low molecular weight compound that enhances or a substance that enhances the biological activity of TLR7 selected from conjugate compounds in which a lipid and a low molecular weight compound that enhances the biological activity of TLR7 are chemically bonded via a spacer Is mentioned.
- an influenza HA split vaccine antigen that has an HA stem region exposed to the outside and produces an antibody that binds to the HA stem region LAH, and a molecular weight of 200 to 600 A conjugate comprising a lipid and a low molecular weight compound that enhances the biological activity of TLR7 having an adenine skeleton, a pyrimidine skeleton, an imidazoquinoline skeleton, an imidazopyridine skeleton, or a quinazoline skeleton, and a lipid chemically bound via a spacer Combinations with compounds are mentioned.
- the HA stem region has a shape that is exposed to the outside, thereby enhancing the antigenicity of the HA stem region LAH.
- an influenza HA split vaccine antigen capable of producing a binding antibody, and a conjugate compound in which a low molecular weight compound having a pyrimidine skeleton and a lipid derived from squalene or squalane are chemically bound via a spacer Is mentioned.
- the HA stem region has a shape that is exposed to the outside, thereby enhancing the antigenicity of the HA stem region LAH.
- a combination of an influenza HA split vaccine antigen capable of producing a binding antibody and a compound that enhances the biological activity of TLR7 represented by the following formula (1): Equation (1): [In the formula (1), X represents methylene, R 1 represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms which may be substituted with a hydroxy group; R 2 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R 3 represents an alkyl group having 1 to 3 carbon atoms, R 4 represents a hydrogen atom, a halogen atom, a hydroxy group, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms, Y 1 represents a single bond or methylene, Y 2 represents a single
- the HA stem region has a shape that is exposed to the outside, thereby enhancing the antigenicity of the HA stem region LAH.
- a combination of an influenza HA split vaccine antigen capable of producing a binding antibody and a compound that enhances the biological activity of TLR7 selected from the following: (4E, 8E, 12E, 16E, 20E) -N- ⁇ 2-[ ⁇ 4-[(2-amino-4- ⁇ [(3S) -1-hydroxyhexane-3-yl] amino ⁇ -6-methyl Pyrimidin-5-yl) methyl] benzyl ⁇ (methyl) amino] ethyl ⁇ -4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide ; (4E, 8E, 12E, 16E, 20E) -1- (4- ⁇ 4-[(2-amino-4- ⁇ [(2S) -1-hydroxypentan-2-yl] amino ⁇ -6-methylpyrimidine -5-yl) methyl] -3-methoxybenzyl ⁇ piperazin-1-yl) -4,8,
- the HA stem region has a shape that is exposed to the outside, thereby enhancing the antigenicity of the HA stem region LAH.
- a combination of an influenza HA split vaccine antigen capable of producing a binding antibody and a compound that enhances the biological activity of TLR7 selected from the following: (4E, 8E, 12E, 16E, 20E) -N- ⁇ 2-[ ⁇ 4-[(2-amino-4- ⁇ [(3S) -1-hydroxyhexane-3-yl] amino ⁇ -6-methyl Pyrimidin-5-yl) methyl] benzyl ⁇ (methyl) amino] ethyl ⁇ -4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide ; 4-[(2-amino-4- ⁇ [(2S) -1-hydroxypentan-2-yl] amino ⁇ -6-methylpyrimidin-5
- an influenza HA split vaccine antigen capable of producing an antibody that binds to LAH in the HA stem region produced by subjecting an influenza HA split vaccine to acidic treatment.
- a prophylactically effective amount is the amount of a universal influenza vaccine antigen and / or vaccine adjuvant required to confer a benefit in preventing influenza on a subject.
- the universal influenza vaccine antigen and the vaccine adjuvant may be contained together in one composition, or may be prepared as separate compositions, respectively.
- the universal influenza vaccine antigen and vaccine adjuvant are included in one composition so that they can be administered simultaneously.
- the administration route of the universal influenza vaccine antigen composition and the vaccine adjuvant composition may be the same or different.
- the vaccine antigen and the vaccine adjuvant can be administered simultaneously or staggered, ie, the vaccine antigen composition and the vaccine adjuvant composition can be administered simultaneously, separately (eg, prior to administration of the vaccine antigen composition or The vaccine adjuvant composition is administered after administration).
- the universal influenza vaccine antigen composition and vaccine adjuvant composition may be provided as a kit containing them.
- a composition comprising a universal influenza vaccine antigen and / or vaccine adjuvant may further comprise one or more pharmaceutically acceptable diluents or carriers, for example, tablets, capsules, granules, powders, troches.
- Oral preparations such as syrups, emulsions and suspensions, or parenteral preparations such as external preparations, suppositories, injections, eye drops, nasal preparations, pulmonary preparations, etc., can be prepared by ordinary methods.
- Preferred examples of the formulation include a liquid preparation for injection or nasal drop, and a lyophilized preparation prepared by freeze-drying the liquid preparation.
- liquid for injection examples include emulsions and liposomes containing aqueous solutions and oily compositions, universal influenza vaccine antigens and / or vaccine adjuvants (eg, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof). ) Is dissolved or dispersed in water, or an aqueous solution preparation or suspension, or a universal influenza vaccine antigen and / or vaccine adjuvant (eg, a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof) Or an oily suspension in which is dissolved or dispersed in oil.
- universal influenza vaccine antigens and / or vaccine adjuvants eg, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof
- oily suspension in which is dissolved or dispersed in oil.
- examples of the aqueous solution, aqueous solution preparation or aqueous suspension include distilled water for injection, and an aqueous or aqueous solution containing a buffer, pH adjuster, stabilizer, isotonic agent and / or emulsifier as appropriate. Suspensions and the like.
- oily compositions examples include compositions containing vegetable oils and fats, animal oils and fats, hydrocarbons, fatty acid esters, phospholipids and the like, and more specifically.
- a composition containing squalene, squalane and the like can be mentioned.
- Examples of the emulsifier include a hydrophilic surfactant and a hydrophobic surfactant.
- composition herein is one or more pharmaceutically acceptable selected from the group consisting of sucrose, sucralose, trehalose, mannitol, glycine, methionine, citric acid, lactic acid, tartaric acid, acetic acid, trifluoroacetic acid and a pH adjuster.
- Carrier may be included.
- the administration method of the composition, kit, etc. in the present application can be appropriately selected according to conditions such as the type of disease, the condition of the subject, and the target site.
- Examples of the administration method include parenteral administration, specifically, intravascular (eg, intravenous), subcutaneous, intradermal, intramuscular, nasal, and transdermal administration.
- the dose of the vaccine adjuvant and vaccine antigen is not particularly limited, and is appropriately selected depending on the administration method, subject, age of subject, dosage form, administration route, and the like.
- the dose is usually 5 to 5000 mg / m 2 (body surface area) for a warm-blooded animal.
- Ie administered in a unit dose ranging from about 0.1 ng / kg to 100 mg / kg, which usually results in a prophylactically effective dose.
- a unit dosage form such as an injection, tablet or capsule usually contains, for example, 1 ng to 250 mg of the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
- it is used in a dose ranging from 1 ng to 50 mg / kg per day.
- the daily dose also has to be varied according to the host to be treated, the particular route of administration and the severity of the disease to be treated.
- the optimal dosage can also be determined by the practitioner treating the individual patient or warm-blooded animal.
- the vaccine adjuvant is administered concurrently with a universal influenza vaccine antigen, for example, according to the universal influenza vaccine antigen administration schedule described below.
- the vaccine adjuvant can be administered in consideration of, for example, the administration schedule of the universal influenza vaccine antigen described below.
- the dose of the universal influenza vaccine antigen (eg, the influenza HA split vaccine of the present invention), the dosage form of the composition, the number of administrations, the time required for one administration, and the like depend on the age of the subject, the target site, and other conditions. It can be appropriately selected depending on the situation.
- the universal influenza vaccine antigen (eg, the influenza HA split vaccine of the present invention) can be administered, for example, only once, or can be used so as to be boosted after a predetermined period of time after the initial vaccination. It is possible.
- the period from the initial inoculation to the additional inoculation is not particularly limited, but is, for example, 20 days to 3 years, preferably 3 months to 2 years, and more preferably 6 months to 1 year. Year.
- the amount of the universal influenza vaccine antigen for the first vaccination and the booster vaccination is not particularly limited, but is, for example, 1 ⁇ g to 200 ⁇ g, preferably 10 ⁇ g to 30 ⁇ g, and more preferably 15 ⁇ g per dose.
- One dose is, for example, 0.5 mL.
- the amount of the substance that enhances the biological activity of TLR7 in the initial inoculation and the booster inoculation is usually 0.0001 mg to 1000 mg, preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 10 mg.
- the administration method is not particularly limited, but for example, nasal, subcutaneous, intradermal, transdermal, intraocular, mucosal or oral administration, preferably intramuscular administration.
- warm-blooded animals include humans and non-human animals.
- Non-human animals include, but are not limited to, mammals such as, for example, non-human primates, sheep, dogs, cats, horses, and cows.
- warm-blooded animals humans, particularly those in need of influenza prevention, are preferred.
- An embodiment of the method for preventing influenza in the present application includes a method comprising administering a composition comprising the above-mentioned universal influenza vaccine antigen and a vaccine adjuvant. Another embodiment includes a method comprising administering a universal influenza vaccine antigen composition and a vaccine adjuvant composition.
- Example 1 Influenza HA split vaccine of the present invention
- H3N2 influenza virus particles X31 strain
- H1N1 influenza virus particles A / Puerto Rico / 8/34 strain
- phosphate buffered saline have a final concentration of 0.1 v Tween 80 was added and suspended so as to be / v%.
- Diethyl ether was added and the mixture was further suspended and allowed to stand until the aqueous layer and the diethyl ether layer were completely separated, and then the diethyl ether layer was removed. After repeating the ether extraction, diethyl ether remaining in the collected aqueous layer was distilled off at normal pressure to obtain an HA split vaccine.
- H3N2 influenza vaccine BALB / c mice (female, 6 to 12 weeks old) were given H3N2-type current HA split vaccine or membrane-fused HA split vaccine ( 10 ⁇ g vaccine + 10 v / v% AddaVax adjuvant (InvivoGen) was dissolved in phosphate buffered saline to make a volume of 200 ⁇ l) intraperitoneally. Twenty-eight days after the first vaccination, the membrane-fused HA vaccine (only 10 ⁇ g vaccine was dissolved in phosphate-buffered saline to make a volume of 200 ⁇ l) was intraperitoneally inoculated. After 14 days from the additional vaccination, blood was collected from the vaccinated mice, and the serum was collected.
- a synthetic peptide (H3; @ Ac-RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENADYKDDDDKC) (SEQ ID NO: 1) corresponding to a part of the stem portion (long @ alpha @ helix) was dissolved in phosphate buffered saline (pH@7.3) at 10 .mu.g / ml, 100 ⁇ l each was added to a 96-well plate. After standing at 4 ° C.
- each well is washed three times with a phosphate buffered saline (containing 0.05 v / v% of Tween 20) and contains 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin Peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) diluted with phosphate buffered saline was added to each well in an amount of 100 ⁇ l.
- a phosphate buffered saline containing 0.05 v / v% of Tween 20
- 0.05 v / v% Tween 20 contains 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin Peroxidase-labeled anti-mouse IgG antibody (Southern Biotech) diluted with phosphate buffered saline was added to each well in an amount of 100 ⁇ l.
- the LAH antibody titer in the serum of BALB / c mice intraperitoneally inoculated with the membrane fusion HA split vaccine was determined by the LAH antibody titer in BALB / c mice inoculated intraperitoneally with the current HA split vaccine. It was significantly higher than the antibody titer.
- H3N2 influenza virus ⁇ ⁇ ⁇ (A / Guizhou / 54/89) with a different antigenicity from the vaccine strain was lethal dose 50 (5% of the virus that causes lethal infection in 50% of mice).
- the virus infection was performed by intranasal administration under anesthesia in (2).
- H1N1 influenza virus particles C57BL / 6 mice (female, 6 to 12 weeks old) were treated with the current H1N1 HA split vaccine or membrane-fused HA split vaccine (10 ⁇ g vaccine + 10 ⁇ g CpG-ODN1760 was suspended in phosphate buffered saline and mixed with an equal volume of Freund's incomplete adjuvant (ROCKLAND) to a volume of 200 ⁇ l) intraperitoneally.
- ROCKLAND Freund's incomplete adjuvant
- a membrane fusion HA split vaccine (similar to the first vaccination, 10 ⁇ g vaccine + 10 ⁇ g CpG-ODN was suspended in phosphate buffered saline, and an equal volume of Freund's incomplete adjuvant (ROCKLAND) To make a volume of 200 ⁇ l). After 14 days from the additional vaccination, blood was collected from the vaccinated mice, and the serum was collected.
- H1 Ac-RIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRSQLKNNADYKDDDDKC
- H1 clone name F2
- the LAH antibody titer in the sera of C57BL / 6 mice inoculated intraperitoneally with the membrane fusion HA split vaccine was determined by the LAH antibody titer in C57BL / 6 mice inoculated intraperitoneally with the current HA split vaccine. It was significantly higher than the antibody titer.
- H1N1 influenza virus ⁇ ⁇ ⁇ (A / Narita / 1/09) with a different antigenicity from the vaccine strain was lethal dose 50 (5% of the amount of virus that causes lethal infection in 50% of mice).
- the virus infection was performed by intranasal administration under anesthesia in (2).
- mice were observed daily for 20 days after virus infection to determine their survival rates. As shown in FIG. 5, in the C57BL / 6 mice inoculated with the membrane fusion HA split vaccine, the decrease in survival rate was significantly suppressed from day 9 after infection with another H1N1 influenza virus having different antigenicity.
- each well was washed three times with phosphate buffered saline, and 150 ⁇ l of phosphate buffered saline containing 1 v / v% bovine serum albumin was added. After allowing to stand at room temperature for 2 hours, each well is washed three times with phosphate buffered saline (containing 0.05 v / v% of Tween 20), and stepwise with a phosphate buffer containing 1 v / v% bovine serum albumin. 50 ⁇ l of the diluted LAH-binding monoclonal antibody was added. After standing at 4 ° C.
- LAAs shown in FIG. 6 the LAH binding monoclonal antibody bound 1.05 to 21 times stronger to the membrane-fused HA split vaccine than the current HA split vaccine. These results indicate that the acidic treatment of the HA split vaccine enhances antibody binding to LAH epitope.
- Example 2 Composition comprising a TLR agonist of the present application and an influenza HA split vaccine
- TLR7 A-1 Preparation of an adjuvant composition containing a compound that enhances the biological activity of TLR7 A-1
- Preparation method 2.96 g dimyristoyl phosphatidylcholine (DMPC), 2.04 g egg yolk phosphatidylglycerol (EPG), and 0.5 g TLR7 agonist ((4E, 8E, 12E, 16E, 20E) -N- ⁇ 2-[ ⁇ 4- [(2-amino-4- ⁇ [(3S) -1-hydroxyhexane-3-yl] amino ⁇ -6-methylpyrimidin-5-yl) methyl] benzyl ⁇ (methyl) amino] ethyl ⁇ -4,8 , 12,17,21,25-Hexamethylhexacosa-4,8,12,16,20,24-hexaenamide) (hereinafter, referred to as compound A) were weighed, and further, cyclohexane 20 g, ethanol 0.4 g Was added
- Filtration was performed using a 0.2 ⁇ m membrane filter and freeze-dried. The dispersion was added using a 9 v / v% aqueous sucrose solution. A liposome solution was prepared using an extruder, passed through a 0.22 ⁇ m filtration filter, and lyophilized. Condensation was performed as needed using distilled water for injection.
- TLR7 agonist methyl (4- ⁇ [[3- (6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl) propyl] (3-piperidin-1- (Ilpropyl) amino] methyldiphenyl) acetate / hydrochloride (hereinafter referred to as compound B) was weighed, and 0.6 mL / v% phosphate buffered saline containing 1 mL of 0.1 V / V% Tween80 (pH 6) was added and suspended.
- compositions containing the influenza HA split vaccine of the present application and a compound that enhances the biological activity of TLR7 X31-type membrane fusion HA split vaccine (10 ⁇ g), phosphate buffered saline, A-1 containing 50 ⁇ g TLR7 agonist (compound A), A-2 containing 200 ⁇ g TLR7 agonist (compound B), 10 ⁇ g A-3 containing the TLR7 agonist (Compound A), an X31-type membrane-fused HA split vaccine solution and an equal volume of AddaVax adjuvant (InvivoGen) or 10 ⁇ g of CpG-ODN1760 are suspended in phosphate buffered saline.
- AddaVax adjuvant InvivoGen
- mice Female, 6 to 12 weeks old were inoculated intradermally or intramuscularly in the thigh (50 ⁇ l per side, total 100 ⁇ l). Three weeks later, the same mouse was boosted with the same antigen and the same conditions, and two weeks after the booster immunization, blood was collected and serum was collected.
- the HA antibody concentration in the serum of the mouse was measured by the ELISA method according to the following method. That is, the recombinant HA protein (X3 strain of H3N2 influenza virus or A / convinced / 716/2007) was dissolved at 10 ⁇ g / ml in phosphate buffered saline (pH 7.3), and 100 ⁇ l was added to a 96-well plate. Was added. After standing at 4 ° C. overnight, each well was washed three times with phosphate buffered saline, and 150 ⁇ l of phosphate buffered saline containing 1 v / v% bovine serum albumin was added.
- each well was washed three times with phosphate buffered saline, and Tween 20 was serially diluted with a phosphate buffer containing 0.05 v / v% and 1 v / v% bovine serum albumin.
- Mouse serum and a standard monoclonal antibody of known concentration (H3; clone name V15-5) were added to each well in an amount of 100 ⁇ l.
- each well is washed three times with phosphate buffered saline (containing 0.05 v / v% of Tween 20), and contains 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin Peroxidase-labeled anti-mouse IgG1 antibody or anti-IgG2c antibody (Southern Biotech) diluted with phosphate buffered saline was added to each well in an amount of 100 ⁇ l.
- phosphate buffered saline containing 0.05 v / v% of Tween 20
- 1 v / v% bovine serum albumin Peroxidase-labeled anti-mouse IgG1 antibody or anti-IgG2c antibody Southern Biotech
- the X31 type HA IgG2c antibody titer of the membrane fusion type HA split vaccination group to which all adjuvants were added was significantly higher than that of the group fusion type HA split vaccination group to which no adjuvant was added.
- the A-1 administration group induced a higher X31 type HA IgG2c antibody titer than CpG-ODN1760 / Freund's incomplete adjuvant (CpG / IFA).
- the A-1 administration group induced the highest X31 type HA ⁇ IgG2c antibody titer.
- it showed completely the same reactivity with the Urg-type HA That is, these results show that the inoculation of the composition of the present application induces a cross-reactive IgG2 antibody having a high protective effect against infection.
- the type X31-derived HA ⁇ IgG1 antibody titer of all the adjuvant-added membrane-fused HA split vaccination groups was significantly increased as compared to the adjuvant-free membrane-fused HA split vaccination group.
- CpG-ODN1760 / Freund's ⁇ incomplete ⁇ adjuvant CpG / IFA
- CpG / IFA CpG-ODN1760 / Freund's ⁇ incomplete ⁇ adjuvant
- the A-1 administered group showed a higher X31 type HA IgG2c / IgG1 antibody ratio than CpG-ODN1760 / Freund's incomplete adjuvant (CpG / IFA). Furthermore, the same ratio of HA-IgG2c / IgG1 antibody to Urg-type HA was obtained. That is, these results show that the inoculation of the composition of the present application induces a cross-reactive antibody with a high protective effect against infection.
- the type X31-derived HAcIgG2c antibody titer of the membrane-fused HA split vaccination group to which all adjuvants were added was significantly lower than that of the group without adjuvant-added membrane-fused HA split vaccination.
- the A-1 and A-3 administration groups induced a higher X31 type HA2IgG2c antibody titer than the AddaVax adjuvant administration group.
- it showed completely the same reactivity with the Urg-type HA. That is, these results show that the inoculation of the composition of the present application induces a cross-reactive IgG2 antibody having a high protective effect against infection.
- the type X31-derived HA ⁇ IgG1 antibody titer of the membrane-fused HA split vaccination group to which all adjuvants were added was significantly lower than that of the group without adjuvant-added membrane-fused HA split vaccination.
- the A-3 and AddaVax adjuvant administration groups induced the highest X31 type HA IgG1 antibody titer.
- it showed completely the same reactivity with the Urg-type HA. That is, these results indicate the induction of cross-reactive antibodies by inoculation of the composition of the present application.
- the A-1 and A-3 administration groups exhibited a higher X31 type HA IgG2c / IgG1 antibody ratio than the AddaVax adjuvant administration group. Furthermore, the same ratio of HA-IgG2c / IgG1 antibody to Urg-type HA was obtained. That is, these results show that the inoculation of the composition of the present application induces a cross-reactive antibody with a high protective effect against infection.
- mice inoculated with the membrane fusion HA split vaccine to which all adjuvants were added the decrease in survival rate was significantly suppressed from day 7 after infection with another H3N2 influenza virus having different antigenicity.
- A-1 and A-3 showed 100% survival rate and showed the strongest protective action against infection.
- mice inoculated with the membrane fusion HA split vaccine to which all adjuvants were added weight loss due to infection with another H3N2 influenza virus having different antigenicity was suppressed.
- A-3 showed a stronger weight loss suppressing effect than the AddaVax adjuvant administration group.
- composition containing the influenza HA split vaccine of the present application and a compound that enhances the biological activity of TLR7 H1N1 influenza virus PR8-derived membrane-fused HA split vaccine (10 ⁇ g) contains phosphate buffered saline, 50 ⁇ g of TLR7 agonist (Compound A) containing A-1, and 10 ⁇ g of TLR7 agonist (Compound A) A-3 or PR8 strain-derived membrane-fused HA split vaccine solution was mixed with an equal volume of AddaVax adjuvant (InvivoGen), respectively.
- mice Male, 6 to 12 weeks old were inoculated intradermally or intramuscularly in the thigh (50 ⁇ l per side, total 100 ⁇ l). Three weeks later, the same mouse was boosted with the same antigen and the same conditions, and two weeks after the booster immunization, blood was collected and serum was collected.
- the HA antibody concentration in the serum of the mouse was measured by the ELISA method according to the following method. That is, the recombinant HA protein (H1N1 influenza virus PR8 strain or A / Narita / 1/09) was dissolved at 10 ⁇ g / ml in phosphate buffered saline (pH 7.3), and 100 ⁇ l was placed in a 96-well plate. Was added. After standing at 4 ° C. overnight, each well was washed three times with phosphate buffered saline, and 150 ⁇ l of phosphate buffered saline containing 1 v / v% bovine serum albumin was added.
- each well was washed three times with phosphate buffered saline, and Tween 20 was serially diluted with a phosphate buffer containing 0.05 v / v% and 1 v / v% bovine serum albumin.
- Mouse serum and a standard monoclonal antibody of known concentration were added to each well in an amount of 100 ⁇ l.
- each well is washed three times with phosphate buffered saline (containing 0.05 v / v% of Tween 20), and contains 0.05 v / v% Tween 20 and 1 v / v% bovine serum albumin Peroxidase-labeled anti-mouse IgG1 antibody or anti-IgG2c antibody (Southern Biotech) diluted with phosphate buffered saline was added to each well in an amount of 100 ⁇ l.
- phosphate buffered saline containing 0.05 v / v% of Tween 20
- 1 v / v% bovine serum albumin Peroxidase-labeled anti-mouse IgG1 antibody or anti-IgG2c antibody Southern Biotech
- the PR8 strain-derived HAGIgG2c antibody titer of the membrane fusion HA split vaccination group to which all adjuvants were added was significantly lower than that of the group without adjuvant added membrane fusion HA split vaccination.
- the A-1 administration group induced a higher PR8 strain-derived HA2IgG2c antibody titer than the AddaVax adjuvant administration group.
- the A-3 administration group induced the highest HA IgG2c antibody titer derived from the PR8 strain. Furthermore, it showed completely the same reactivity with HA derived from A / Narita / 1/09. That is, these results show that the inoculation of the composition of the present application induces a cross-reactive IgG2 antibody having a high protective effect against infection.
- the HA IgG1 antibody titer derived from the PR8 strain of all the adjuvant-added membrane-fused HA split vaccination groups was significantly increased as compared with the adjuvant-free membrane-fused HA split vaccination group.
- the A-3 administration group induced the highest HA IgG1 antibody titer derived from the PR8 strain. Furthermore, it showed completely the same reactivity with HA derived from A / Narita / 1/09. That is, these results indicate the induction of cross-reactive antibodies by inoculation of the composition of the present application.
- the groups administered with A-1 and A-3 exhibited a higher ratio of the HA IgG2c / IgG1 antibody derived from the PR8 strain than the group administered with AddaVax adjuvant. Furthermore, the HA-IgG2c / IgG1 antibody ratio was exactly the same for HA derived from A / Narita / 1/09. That is, these results show that the inoculation of the composition of the present application induces a cross-reactive antibody with a high protective effect against infection.
- mice vaccinated with the membrane-fused HA split vaccine supplemented with A-1 or A-3 adjuvant significantly different from day 7 after infection with another H1N1 influenza virus with different antigenicity. It was able to suppress the decrease in survival rate and showed a strong protective effect on infection.
- mice inoculated with the membrane fusion HA split vaccine to which all adjuvants were added weight loss due to infection with another H1N1 influenza virus having different antigenicity was suppressed.
- the A-1 and A-3 administration groups showed a stronger weight loss suppressing effect than the AddaVax adjuvant administration group.
- the vaccine (post-fix) subjected to formalin treatment after the acid treatment was compared with the vaccine (Pre-fix) treated before the acid treatment.
- LAH binding monoclonal antibody bound more strongly.
- the LAH antibody titer in the serum of BALB / c mice inoculated intraperitoneally with the membrane fusion HA split vaccine was the same as the LAH antibody titer in BALB / c mice inoculated intraperitoneally with the current HA split vaccine. It was higher than the antibody titer. Further, the membrane fusion HA split vaccine (post-fix) had an increased LAH antibody titer compared to the membrane fusion split vaccine (Pre-fix).
- the present invention can be used in the field of influenza vaccine development or production.
- SEQ ID NOs: 1 and 2 synthetic peptide
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Abstract
Description
(1)ユニバーサルインフルエンザワクチン抗原;および
(2)ワクチンアジュバント。
[項3]該インフルエンザHAスプリットワクチン抗原のHA幹領域が外部に露出した形状である、項2に記載の組成物。
[項11]該ワクチンアジュバントが、TLR7、TLR8、およびTLR9からなる群から選択される一つもしくはそれ以上のTLRの生理活性を亢進する物質である、項1~10のいずれか一項に記載の組成物。
[項12]該ワクチンアジュバントが、少なくともTLR7の生理活性を亢進する物質である、項1~11のいずれか一項に記載の組成物。
[項13]該ワクチンアジュバントが、少なくともTLR8の生理活性を亢進する物質である、項1~12のいずれか一項に記載の組成物。
[項14]該ワクチンアジュバントが、少なくともTLR9の生理活性を亢進する物質である、項1~13のいずれか一項に記載の組成物。
[項16]該TLR7の生理活性を亢進する低分子量化合物または該コンジュゲート化合物に含まれる該TLR7の生理活性を亢進する低分子量化合物が、分子量200~600を有し、かつ、アデニン骨格、ピリミジン骨格、イミダゾキノリン骨格、イミダゾピリジン骨格、またはキナゾリン骨格を有する、項15に記載の組成物。
[項17]該TLR7の生理活性を亢進する低分子量化合物または該コンジュゲート化合物に含まれる該TLR7の生理活性を亢進する低分子量化合物が、ピリミジン骨格、アデニン骨格またはイミダゾキノリン骨格を有する、項16に記載の組成物。
[項18]該脂質が、スクワレンまたはスクワランから誘導される脂質である、項15~17のいずれか一項に記載の組成物。
R1は水素原子、またはヒドロキシ基で置換されていてもよい炭素数1~3のアルキル基を表し、
R2は、水素原子、または炭素数1~6のアルキル基を表し、
R3は、炭素数1~3のアルキル基を表し、
R4は、水素原子、ハロゲン原子、ヒドロキシ基、炭素数1~3のアルキル基または炭素数1~3のアルコキシ基を表し、
Y1は、単結合またはメチレンを表し、
Y2は、単結合または-C(O)-を表し、
Lは、炭素数2または3の直鎖アルキレンを表し、
R5およびR6は、独立して水素原子または炭素数1~3のアルキル基を表すか、あるいは、R5は、R6と一緒になって置換もしくは無置換の5~8員の含窒素飽和ヘテロ環を形成してもよく、前記5~8員の含窒素飽和ヘテロ環が置換されている場合、ヒドロキシ基およびハロゲン原子から選択される同一もしくは異なる1~4個の置換基で置換されていてもよく、
mは0または1を表し、
で示される化合物、またはそれらの製薬学的に許容される塩である、項15~18、および20のいずれか一項に記載の組成物。
[項22]該コンジュゲート化合物が、
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンジル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン;
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンゾイル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル](メチル)アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩である、項15~18、20および21のいずれか一項に記載の組成物。
[項23]該TLR7の生理活性を亢進する化合物が、
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩である、項15~18、および20~22のいずれか一項に記載の組成物。
[項26]予防が必要な温血動物に、予防上の有効量のユニバーサルインフルエンザワクチン抗原と、予防上の有効量のワクチンアジュバント(例えばTLR7の生理活性を亢進する物質)を投与することを含む、インフルエンザを予防するための方法。
[項29]ワクチンアジュバント(例えばTLR7の生理活性を亢進する物質)と組み合わせたインフルエンザの予防における使用のための、ユニバーサルインフルエンザワクチン抗原。
[項31]ワクチンアジュバント(例えばTLR7の生理活性を亢進する物質)を含むインフルエンザの予防のための組成物の製造における、ユニバーサルインフルエンザワクチン抗原の使用。
[項32-1]ユニバーサルインフルエンザワクチン抗原がワクチンアジュバント(例えばTLR7の生理活性を亢進する物質)の投与前にもしくは投与後に、または同時に投与される、項25および26に記載の方法、項27に記載のキット、項28に記載のワクチンアジュバント、または項29に記載の該抗原。
a)ホルマリン処理をしていないインフルエンザHAスプリットワクチンに酸性処理を施すことにより、HA幹領域のLAHに結合する抗体を産生するインフルエンザHAスプリットワクチン抗原を製造する工程;および
b)工程a)で得られたワクチン抗原と、ワクチンアジュバントとを混合する工程。
a)インフルエンザHAスプリットワクチンに酸性処理を施す工程、
b)その後ホルマリン処理を施すことにより、HA幹領域のLAHに結合する抗体を産生するインフルエンザHAスプリットワクチン抗原を製造する工程;および
c)工程b)で得られたワクチン抗原と、ワクチンアジュバントとを混合する工程。
a)ホルマリン処理をしていないインフルエンザHAスプリットワクチンに酸性処理を施すことにより、HA幹領域のLAHに結合する抗体を産生するインフルエンザHAスプリットワクチン抗原を製造する工程;および
b)工程a)で得られたワクチンと、ワクチンアジュバントとを組み合わせてキットとする工程。
a)インフルエンザHAスプリットワクチンに酸性処理を施す工程、
b)その後ホルマリン処理を施すことにより、HA幹領域のLAHに結合する抗体を産生するインフルエンザHAスプリットワクチン抗原を製造する工程;および
c)工程b)で得られたワクチンと、ワクチンアジュバントとを組み合わせてキットとする工程。
6-アミノ-9-({6-[2-(ジメチルアミノ)エトキシ]ピリジン-3-イル}メチル)-2-エトキシ-7,9-ジヒドロ-8H-プリン-8-オン;
6-アミノ-2-(ブチルアミノ)-9-({6-[2-(ジメチルアミノ)エトキシ]ピリジン-3-イル}メチル)-7,9-ジヒドロ-8H-プリン-8-オン;
N-(4-{[6-アミノ-2-(ブチルアミノ)-8-オキソ-7,8-ジヒドロ-9H-プリン-9-イル]メチル}ベンゾイル)グリシン;
メチル (3-{[[3-(6-アミノ-2-ブトキシ-8-オキソ-7,8-ジヒドロ-9H-プリン-9-イル)プロピル](3-モルホリン-4-イルプロピル)アミノ]メチル}フェニル)アセテート(英語名:Methyl (3-{[[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl](3-morpholin-4-ylpropyl)amino]methyl}phenyl)acetate);または
メチル (4-{[[3-(6-アミノ-2-ブトキシ-8-オキソ-7,8-ジヒドロ-9H-プリン-9-イル)プロピル](3-ピペリジン-1-イルプロピル)アミノ]メチル}フェニル)アセテート(英語名:methyl (4-{[[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl](3-piperidin-1-ylpropyl)amino]methyl}phenyl)acetate)。
R1は水素原子、またはヒドロキシ基で置換されていてもよい炭素数1~3のアルキル基を表し、
R2は、水素原子、または炭素数1~6のアルキル基を表し、
R3は、炭素数1~3のアルキル基を表し、
R4は、水素原子、ハロゲン原子、ヒドロキシ基、炭素数1~3のアルキル基または炭素数1~3のアルコキシ基を表し、
Y1は、単結合またはメチレンを表し、
Y2は、単結合または-C(O)-を表し、
Lは、炭素数2または3の直鎖アルキレンを表し、
R5およびR6は、独立して水素原子または炭素数1~3のアルキル基を表すか、あるいは、R5は、R6と一緒になって置換もしくは無置換の5~8員の含窒素飽和ヘテロ環を形成してもよく、前記5~8員の含窒素飽和ヘテロ環が置換されている場合、ヒドロキシ基およびハロゲン原子から選択される同一もしくは異なる1~4個の置換基で置換されていてもよく、
mは0または1を表し、
当該含窒素飽和ヘテロ環が置換される場合の置換基として、好ましくは、メチル基、エチル基、プロピル基、ヒドロキシメチル基、ヒドロキシエチル基、カルボニル基、ヒドロキシ基又はハロゲン原子、更に好ましくはヒドロキシ基又はハロゲン原子が挙げられ、同一もしくは異なる1~4個の置換基で置換されていてもよい。
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド(特許文献12、実施例1の化合物、下記式(2));
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンジル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン(特許文献12、実施例2の化合物);
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンゾイル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン(特許文献12、実施例3の化合物);
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド(特許文献12、実施例4の化合物、下記式(3));
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル](メチル)アミノ}エチル)-3-メトキシベンズアミド(特許文献12、実施例5の化合物);
またはそれらの製薬学的に許容される塩が挙げられる。
市販のインフルエンザHAワクチン(販売名)は、生物学的製剤基準(平成16年3月30日 厚生労働省告示 第155号,最終改正:平成30年11月30日 厚生労働省告示 第409号)に記載の通り、エーテル等でウイルスを分解後、脂溶剤を除去した後にホルムアルデヒド又はこれと同等な作用を有する物質により処理する工程が既に施されている。市販のインフルエンザHAワクチン(販売名)はインフルエンザHAスプリットワクチンであるものの、既にホルムアルデヒド等の処理が施されているため、本発明のインフルエンザHAスプリットワクチンの製造には用いないことが好ましい。
使用されるホルマリンのグレードとしては、医療用として使用可能なグレードであることが好ましい。
本発明のインフルエンザHAスプリットワクチンの好ましい例として、H3N2型インフルエンザウイルスおよびH1N1型インフルエンザウイルスに由来するものが挙げられる。
好ましいHA亜型としてはH1型、H3型およびB型が挙げられる。
複数種の亜型を含むワクチンとして接種される場合には、H1型、H3型およびB型からなる群から選択される1~3種の亜型が含まれていることが好ましい。
対しても感染防御効果を有しうる。また例えばH1N1型インフルエンザウイルス粒子(A/Puerto Rico/8/34(H1N1))から現行HAスプリットワクチンを調整し、酸性処理を施した場合、A/Puerto Rico/8/34(H1N1)のみならず、例えば、A/Narita/1/09(H1N1)、A/Beijing/262/95(H1N1)、A/Brazil/11/78(H1N1)、A/Chile/1/83(H1N1)、A/New Jersey/8/76(H1N1)、A/Taiwan/1/86(H1N1)、A/Yamagata/32/89(H1N1)、A/New Caledonia/20/99(H1N1)、A/Solomon Islands/3/2006(H1N1)、A/Brisbane/59/2007(H1N1)またはA/Mexico/4108/2009(H1N1)等に対しても感染防御効果を有しうる。
式(1):
R1は水素原子、またはヒドロキシ基で置換されていてもよい炭素数1~3のアルキル基を表し、
R2は、水素原子、または炭素数1~6のアルキル基を表し、
R3は、炭素数1~3のアルキル基を表し、
R4は、水素原子、ハロゲン原子、ヒドロキシ基、炭素数1~3のアルキル基または炭素数1~3のアルコキシ基を表し、
Y1は、単結合またはメチレンを表し、
Y2は、単結合または-C(O)-を表し、
Lは、炭素数2または3の直鎖アルキレンを表し、
R5およびR6は、独立して水素原子または炭素数1~3のアルキル基を表すか、あるいは、R5は、R6と一緒になって置換もしくは無置換の5~8員の含窒素飽和ヘテロ環を形成してもよく、前記5~8員の含窒素飽和ヘテロ環が置換されている場合、ヒドロキシ基およびハロゲン原子から選択される同一もしくは異なる1~4個の置換基で置換されていてもよく、
mは0または1を表し、
で示される化合物、またはそれらの製薬学的に許容される塩。
ユニバーサルインフルエンザワクチン抗原とワクチンアジュバントの組み合わせの別の態様として、HA幹領域が外部に露出した形状であることによりHA幹領域のLAHの抗原性が高められており、かつ、HA幹領域のLAHに結合する抗体を産生することができるインフルエンザHAスプリットワクチン抗原と、以下から選択されるTLR7の生理活性を亢進する化合物との組み合わせが挙げられる;
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンジル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン;
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンゾイル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル](メチル)アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩。
ユニバーサルインフルエンザワクチン抗原とワクチンアジュバントの組み合わせの別の態様として、HA幹領域が外部に露出した形状であることによりHA幹領域のLAHの抗原性が高められており、かつ、HA幹領域のLAHに結合する抗体を産生することができるインフルエンザHAスプリットワクチン抗原と、以下から選択されるTLR7の生理活性を亢進する化合物との組み合わせが挙げられる;
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩。
ユニバーサルインフルエンザワクチン抗原とワクチンアジュバントの組み合わせの別の態様として、インフルエンザHAスプリットワクチンに酸性処理を施すことにより製造される、HA幹領域のLAHに結合する抗体を産生することができるインフルエンザHAスプリットワクチン抗原と、以下から選択されるTLR7の生理活性を亢進する化合物との組み合わせが挙げられる;
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩。
リン酸緩衝生理食塩水に懸濁したH3N2型インフルエンザウイルス粒子(X31株)もしくはH1N1型インフルエンザウイルス粒子(A/Puerto Rico/8/34株)に、最終濃度が0.1v/v%になるようTween80を添加し懸濁した。ジエチルエーテルを加えさらに懸濁し、水層とジエチルエーテル層が完全に分離するまで静置した後、ジエチルエーテル層を取り除いた。このエーテル抽出を繰り返した後、回収した水層に残存するジエチルエーテルを常圧で留去し、HAスプリットワクチンとした。
HAスプリットワクチンをリン酸緩衝生理食塩水で懸濁後、酸性処理として0.15Mクエン酸緩衝液(pH 3.5)を添加しpHを5.0にした。室温で30分静置した後、1M Tris緩衝液 (pH8.0)を加えて、pHを7.3に戻した。その後、遠心分離を行い、膜融合型HAスプリットワクチンとした。作製した膜融合型HAスプリットワクチンに、最終濃度0.05v/v%になるようホルマリンを加えて数日静置した。
3-1.H3N2型インフルエンザワクチンの接種
BALB/cマウス(メス、6~12週令)に、H3N2型の現行HAスプリットワクチンまたは膜融合型HAスプリットワクチン(10 μgワクチン + 10 v/v % AddaVaxアジュバント(InvivoGen)をリン酸緩衝生理食塩水に溶解し、液量200 μlにする)を腹腔内接種した。初回ワクチン接種28日後に、膜融合型HAワクチン(10 μgワクチンのみをリン酸緩衝生理食塩水に溶解し、液量200 μlにする)を腹腔内接種した。追加ワクチン接種から14日後以降に、ワクチンを接種したマウスより採血を行い、血清を回収した。
下記に示す手法により、ELISA法(Enzyme-Linked Immuno Sorbent Assay)にて、H3N2型の現行HAスプリットワクチンまたは膜融合型HAスプリットワクチンを腹腔内接種したBALB/cマウスの血清中のLAH抗体濃度を測定した。
H3N2型ウイルス感染防御実験では、ワクチン非接種マウス、または、H3N2型の現行HAスプリットワクチン若しくは膜融合型HAスプリットワクチン接種後のマウスより回収した血清を、BALB/cマウス(メス、6~12週令)に200 μlずつ腹腔内投与した。
5-1.H1N1型インフルエンザウイルス粒子
C57BL/6マウス(メス、6~12週令)に、H1N1型の現行HAスプリットワクチンまたは膜融合型HAスプリットワクチン(10 μgワクチン + 10 μg CpG-ODN1760をリン酸緩衝生理食塩水に懸濁し、等量のFreund’s incomplete adjuvant (ROCKLAND) と混合して液量200 μlにする)を腹腔内接種した。初回ワクチン接種28日後に、膜融合型HAスプリットワクチン(初回ワクチン接種と同様に、10 μgワクチン + 10 μg CpG-ODNをリン酸緩衝生理食塩水に懸濁し、等量のFreund’s incomplete adjuvant (ROCKLAND) と混合して液量200 μlにする)を腹腔内接種した。追加ワクチン接種から14日後以降に、ワクチンを接種したマウスより採血を行い、血清を回収した。
下記に示す手法により、ELISA法にて、H1N1型の現行HAスプリットワクチンまたは膜融合型HAスプリットワクチンを腹腔内接種したC57BL/6マウスの血清中のLAH抗体濃度を測定した。
H1N1型ウイルス感染防御実験では、ワクチン非接種マウス、または、H1N1型の現行HAスプリットワクチン若しくは膜融合型HAスプリットワクチン接種後のマウスより回収した血清を、C57BL/6マウス(メス、6~12週令)に200 μlずつ腹腔内投与した。
A-1
調製法
2.96gのジミリストイルホスファチジルコリン(DMPC)、2.04gの卵黄ホスファチジルグリセロール(EPG)、および0.5gのTLR7アゴニスト((4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド)(以下、化合物Aという)をそれぞれはかり取り、さらに、シクロヘキサン20g、エタノール0.4gを加え、溶解した。0.2μmのメンブランフィルターを用いてろ過を行い、凍結乾燥した。9v/v%スクロース水溶液を用いて添加分散した。エクストルーダーを用いてリポソーム液を調製し、0.22μmのろ過フィルターを通した後、凍結乾燥した。復水は注射用蒸留水を用いて要時行った。
A-2
調製法
4mgのTLR7アゴニスト(メチル (4-{[[3-(6-アミノ-2-ブトキシ-8-オキソ-7,8-ジヒドロ-9H-プリン-9-イル)プロピル](3-ピペリジン-1-イルプロピル)アミノ]メチル}フェニル)アセテート・塩酸塩)(以下、化合物Bという)を秤量し、1mLの0.1v/v% Tween80を含有する0.6v/v% リン酸緩衝生理食塩水(pH6)を添加して懸濁した。
A-3
調製法
TLR7アゴニスト(化合物A)、スクワラン、トリオレイン酸ソルビタン、α-トコフェロール、が1:225:25:25の重量比となるように、化合物Aを油性成分(スクワラン、トリオレイン酸ソルビタン及びα-トコフェロール)に溶解させた。化合物A、アスコルビン酸Na、ポリソルベート80、スクロースが1:10:25:500の重量比となるように、注射用水に水性成分(スクロース、ポリソルベート80及びアスコルビン酸ナトリウム)を溶解後、上記油性組成物を加えて予備混合を行い、超高圧乳化分散機を用いて乳化分散させた。0.2μmの滅菌フィルターを通してろ過した後、ガラス製バイアルに1 mLずつ充填し、凍結乾燥した。復水は注射用蒸留水を用いて要時行った。
X31型由来膜融合型HAスプリットワクチン(10μg)に、リン酸緩衝生理食塩水、50μgのTLR7アゴニスト(化合物A)を含むA-1、200μgのTLR7アゴニスト(化合物B)を含むA-2、10μgのTLR7アゴニスト(化合物A)を含むA-3、X31型由来膜融合型HAスプリットワクチン液と等容量のAddaVaxアジュバント(InvivoGen)、もしくは10 μgのCpG-ODN1760をリン酸緩衝生理食塩水に懸濁し等量のFreund’s incomplete adjuvant (ROCKLAND) と混合したものをそれぞれ調製した。C57BL/6マウス(メス、6~12週令)の大腿部皮内もしくは大腿部の筋肉内(片側50 μlで合計100 μl)に接種した。3週間後に同じマウスに同じ抗原および同じ条件で追加免疫を行い、追加免疫2週間後に採血を行い、血清を回収した。
下記に示す手法により、ELISA法にてマウスの血清中のHA抗体濃度を測定した。
即ち、組換えHA蛋白質(H3N2型インフルエンザウイルスのX31株もしくはA/Uruguay/716/2007)を10 μg/mlにてリン酸緩衝生理食塩水(pH 7.3)に溶解し、96ウェルプレートに100 μlずつ添加した。4℃で一晩静置した後、リン酸緩衝生理食塩水にて各ウェルを3回洗浄し、1v/v%牛血清アルブミンを含むリン酸緩衝生理食塩水を150 μlずつ添加した。室温で2時間静置後、リン酸緩衝生理食塩水にて各ウェルを3回洗浄し、Tween20を0.05v/v%と1v/v%牛血清アルブミンを含むリン酸緩衝液にて段階希釈したマウス血清と、濃度既知の標準モノクローナル抗体(H3; クローン名V15-5)を各ウェルに100 μlずつ添加した。室温で2時間静置後、リン酸緩衝生理食塩水(Tween20を0.05v/v%含む)にて各ウェルを3回洗浄し、0.05v/v%Tween20と1v/v%牛血清アルブミンを含むリン酸緩衝生理食塩水にて希釈したペルオキシダーゼ標識抗マウスIgG1抗体もしくは抗IgG2c抗体(Southern Biotech)を各ウェルに100 μlずつ添加した。室温で2時間静置後、リン酸緩衝生理食塩水(Tween20を0.05v/v%含む)にて各ウェルを3回洗浄し、基質としてクエン酸緩衝液(pH 5.0) 60 mlにo-phenylendiamine tablet (シグマ) 30 mgと24 μlの30%過酸化水素水(30%w/w; シグマ)を加えたものを調整し、それを各ウェルに100 μlずつ添加した。発色後50 μlの1mol/L硫酸(和光純薬工業)で反応を止め、Microplate Reader 450型(Biorad)を用いて490 nmの吸光値を測定した。
追加免疫から3週間後、ワクチン株とは抗原性の異なる別のH3N2型インフルエンザウイルス (A/Guizhou/54/89)を5マウスlethal dose50 (50%のマウスに致死性感染をおこすウイルス量の5倍)にて麻酔下において経鼻投与することでウイルス感染を行った。ウイルス感染から14日間、毎日マウスの体重測定および観察を行い、体重の推移と生存率を調べた。25%の体重減少が認められたマウスが生じた場合は、安楽殺処分とした。
H1N1型インフルエンザウイルスのPR8株由来膜融合型HAスプリットワクチン(10μg)に、リン酸緩衝生理食塩水、50μgのTLR7アゴニスト(化合物A)を含むA-1、10μgのTLR7アゴニスト(化合物A)を含むA-3、もしくはPR8株由来膜融合型HAスプリットワクチン液と等容量のAddaVaxアジュバント(InvivoGen)と混合したものをそれぞれ調製した。C57BL/6マウス(メス、6~12週令)の大腿部皮内もしくは大腿部の筋肉内(片側50 μlで合計100 μl)に接種した。3週間後に同じマウスに同じ抗原および同じ条件で追加免疫を行い、追加免疫2週間後に採血を行い、血清を回収した。
下記に示す手法により、ELISA法にてマウスの血清中のHA抗体濃度を測定した。
即ち、組換えHA蛋白質(H1N1型インフルエンザウイルスのPR8株もしくはA/Narita/1/09)を10 μg/mlにてリン酸緩衝生理食塩水(pH 7.3)に溶解し、96ウェルプレートに100 μlずつ添加した。4℃で一晩静置した後、リン酸緩衝生理食塩水にて各ウェルを3回洗浄し、1v/v%牛血清アルブミンを含むリン酸緩衝生理食塩水を150 μlずつ添加した。室温で2時間静置後、リン酸緩衝生理食塩水にて各ウェルを3回洗浄し、Tween20を0.05v/v%と1v/v%牛血清アルブミンを含むリン酸緩衝液にて段階希釈したマウス血清と、濃度既知の標準モノクローナル抗体(H1;クローン名F2)を各ウェルに100 μlずつ添加した。室温で2時間静置後、リン酸緩衝生理食塩水(Tween20を0.05v/v%含む)にて各ウェルを3回洗浄し、0.05v/v%Tween20と1v/v%牛血清アルブミンを含むリン酸緩衝生理食塩水にて希釈したペルオキシダーゼ標識抗マウスIgG1抗体もしくは抗IgG2c抗体(Southern Biotech)を各ウェルに100 μlずつ添加した。室温で2時間静置後、リン酸緩衝生理食塩水(Tween20を0.05v/v%含む)にて各ウェルを3回洗浄し、基質としてクエン酸緩衝液(pH 5.0) 60 mlにo-phenylendiamine tablet (シグマ) 30 mgと24 μlの30%過酸化水素水(30%w/w; シグマ)を加えたものを調整し、それを各ウェルに100 μlずつ添加した。発色後50 μlの1mol/L硫酸(和光純薬工業)で反応を止め、Microplate Reader 450型(Biorad)を用いて490 nmの吸光値を測定した。
追加免疫から4週間後、ワクチン株とは抗原性の異なる別のH1N1型インフルエンザウイルス (A/Narita/1/09)を5マウスlethal dose50 (50%のマウスに致死性感染をおこすウイルス量の5倍)にて麻酔下において経鼻投与することでウイルス感染を行った。ウイルス感染から14日間、毎日マウスの体重測定および観察を行い、体重の推移と生存率を調べた。25%の体重減少が認められたマウスが生じた場合は、安楽殺処分とした。
リン酸緩衝生理食塩水に懸濁したH3N2型インフルエンザウイルス粒子(X31株)に、最終濃度が0.1v/v%になるようTween80を添加し懸濁した。ジエチルエーテルを加えさらに懸濁し、水層とジエチルエーテル層が完全に分離するまで静置した後、ジエチルエーテル層を取り除いた。このエーテル抽出を繰り返した後、回収した水層に残存するジエチルエーテルを常圧で留去した。さらに、最終濃度0.05v/v%になるようホルマリンを加えて数日静置し、ホルマリン前処理HAスプリットワクチンとした。
ホルマリン前処理HAスプリットワクチンをリン酸緩衝生理食塩水で懸濁後、酸性処理として0.15Mクエン酸緩衝液(pH 3.5)を添加しpHを5.0にした。室温で30分静置した後、1M Tris緩衝液 (pH8.0)を加えて、pHを7.3に戻した。その後、遠心分離を行った。
LAH epitopeに対する抗体結合性は、実施例1における7.と同様の操作を実施し、結合性の変化を算出した。ここで、モノクローナル抗体は、図6における#2、#4及び#5と同じ抗体、およびコントロールとしてHAヘッド領域に結合するモノクローナル抗体#6を用いた。
4-1.H3N2型インフルエンザワクチンの接種
BALB/cマウス(メス、6~12週令)に、H3N2型の現行HAスプリットワクチンまたは膜融合型HAスプリットワクチン(10 μgワクチン + 10 v/v % AddaVaxアジュバント(InvivoGen)をリン酸緩衝生理食塩水に溶解し、液量200 μlにする)を腹腔内接種した。接種から12日後以降に、ワクチンを接種したマウスより採血を行い、血清を回収した。
4-2.ELISA法による測定
LAH抗体価は、実施例1における3-2.と同様の操作を実施し、測定した。
Claims (30)
- 以下の(1)および(2)を含む組成物;
(1)ユニバーサルインフルエンザワクチン抗原;および
(2)ワクチンアジュバント。 - 該ユニバーサルインフルエンザワクチン抗原が、HA幹領域のLAHに結合する抗体を産生することができるインフルエンザHAスプリットワクチン抗原である、請求項1に記載の組成物。
- 該インフルエンザHAスプリットワクチン抗原のHA幹領域が外部に露出した形状である、請求項2に記載の組成物。
- 該ユニバーサルインフルエンザワクチン抗原が、HA幹領域が外部に露出した形状であることによりHA幹領域のLAHの抗原性が高められており、かつ、HA幹領域のLAHに結合する抗体を産生することができる、インフルエンザHAスプリットワクチン抗原である、請求項1に記載の組成物。
- 該ユニバーサルインフルエンザワクチン抗原が、インフルエンザHAスプリットワクチンに酸性処理を施すことにより製造される、請求項1~4のいずれか1項に記載の組成物。
- 該ユニバーサルインフルエンザワクチン抗原が、インフルエンザHAスプリットワクチンに酸性処理を施す工程と、その後ホルマリン処理を施す工程とを含む製造プロセスにより製造される、請求項1~5のいずれか1項に記載の組成物。
- 該ワクチンアジュバントが、TLRの生理活性を亢進する物質である、請求項1~6のいずれか一項に記載の組成物。
- 該ワクチンアジュバントが、TLR7、TLR8、およびTLR9からなる群から選択される一つもしくはそれ以上のTLRの生理活性を亢進する物質である、請求項1~7のいずれか一項に記載の組成物。
- 該ワクチンアジュバントが、少なくともTLR7の生理活性を亢進する物質である、請求項1~8のいずれか一項に記載の組成物。
- 該ワクチンアジュバントが、少なくともTLR8の生理活性を亢進する物質である、請求項1~9のいずれか一項に記載の組成物。
- 該ワクチンアジュバントが、少なくともTLR9の生理活性を亢進する物質である、請求項1~10のいずれか一項に記載の組成物。
- 該TLR7の生理活性を亢進する物質が、TLR7の生理活性を亢進する低分子量化合物、またはTLR7の生理活性を亢進する低分子量化合物と脂質とがスペーサーを介して化学的に結合しているコンジュゲート化合物である、請求項8または9に記載の組成物。
- 該TLR7の生理活性を亢進する低分子量化合物または該コンジュゲート化合物に含まれる該TLR7の生理活性を亢進する低分子量化合物が、分子量200~600を有し、かつ、アデニン骨格、ピリミジン骨格、イミダゾキノリン骨格、イミダゾピリジン骨格、またはキナゾリン骨格を有する、請求項12に記載の組成物。
- 該TLR7の生理活性を亢進する低分子量化合物または該コンジュゲート化合物に含まれる該TLR7の生理活性を亢進する低分子量化合物が、ピリミジン骨格、アデニン骨格またはイミダゾキノリン骨格を有する、請求項13に記載の組成物。
- 該脂質が、スクワレンまたはスクワランから誘導される脂質である、請求項12~14のいずれか一項に記載の組成物。
- 該TLR7の生理活性を亢進する化合物が、アデニン骨格を有する低分子量化合物とスクワレンまたはスクワランから誘導される脂質とがスペーサーを介して化学的に結合しているコンジュゲート化合物またはその製薬学的に許容される塩である、請求項12~15のいずれか一項に記載の組成物。
- 該TLR7の生理活性を亢進する化合物が、ピリミジン骨格を有する低分子量化合物とスクワレンまたはスクワランから誘導される脂質とがスペーサーを介して化学的に結合しているコンジュゲート化合物またはその製薬学的に許容される塩である、請求項12~15のいずれか一項に記載の組成物。
- 該TLR7の生理活性を亢進する化合物が、下記式(1):
R1は水素原子、またはヒドロキシ基で置換されていてもよい炭素数1~3のアルキル基を表し、
R2は、水素原子、または炭素数1~6のアルキル基を表し、
R3は、炭素数1~3のアルキル基を表し、
R4は、水素原子、ハロゲン原子、ヒドロキシ基、炭素数1~3のアルキル基または炭素数1~3のアルコキシ基を表し、
Y1は、単結合またはメチレンを表し、
Y2は、単結合または-C(O)-を表し、
Lは、炭素数2または3の直鎖アルキレンを表し、
R5およびR6は、独立して水素原子または炭素数1~3のアルキル基を表すか、あるいは、R5は、R6と一緒になって置換もしくは無置換の5~8員の含窒素飽和ヘテロ環を形成してもよく、前記5~8員の含窒素飽和ヘテロ環が置換されている場合、ヒドロキシ基およびハロゲン原子から選択される同一もしくは異なる1~4個の置換基で置換されていてもよく、
mは0または1を表し、
で示される化合物、またはそれらの製薬学的に許容される塩である、請求項12~15、および17のいずれか一項に記載の組成物。 - 該コンジュゲート化合物が、
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンジル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン;
(4E,8E,12E,16E,20E)-1-(4-{4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-3-メトキシベンゾイル}ピペラジン-1-イル)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエン-1-オン;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル](メチル)アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩である、請求項12~15、17および18のいずれか一項に記載の組成物。 - 該TLR7の生理活性を亢進する化合物が、
(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-アミノ-4-{[(3S)-1-ヒドロキシヘキサン-3-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]ベンジル}(メチル)アミノ]エチル}-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエンアミド;
4-[(2-アミノ-4-{[(2S)-1-ヒドロキシペンタン-2-イル]アミノ}-6-メチルピリミジン-5-イル)メチル]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-ヘキサメチルヘキサコサ-4,8,12,16,20,24-ヘキサエノイル]アミノ}エチル)-3-メトキシベンズアミド;
またはそれらの製薬学的に許容される塩である、請求項12~15、および17~19のいずれか一項に記載の組成物。 - 請求項1~20のいずれか一項に記載の組成物を含む、インフルエンザワクチン。
- 予防が必要な温血動物に、予防上の有効量の請求項1~20のいずれかに記載の組成物を投与することを含む、インフルエンザを予防するための方法。
- 予防が必要な温血動物に、予防上の有効量のユニバーサルインフルエンザワクチン抗原と、予防上の有効量のワクチンアジュバントを投与することを含む、インフルエンザを予防するための方法。
- ユニバーサルインフルエンザワクチン抗原、およびワクチンアジュバントを含む、インフルエンザを予防するためのキット。
- ユニバーサルインフルエンザワクチン抗原と組み合わせたインフルエンザの予防における使用ための、ワクチンアジュバント。
- ワクチンアジュバントと組み合わせたインフルエンザの予防における使用のための、ユニバーサルインフルエンザワクチン抗原。
- ユニバーサルインフルエンザワクチン抗原を含むインフルエンザの予防のための組成物の製造における、ワクチンアジュバントの使用。
- ワクチンアジュバントを含むインフルエンザの予防のための組成物の製造における、ユニバーサルインフルエンザワクチン抗原の使用。
- ユニバーサルインフルエンザワクチン抗原とワクチンアジュバントとが、同時にまたは別々に投与される、請求項22および23に記載の方法、請求項24に記載のキット、請求項25に記載のワクチンアジュバント、または請求項26に記載の該抗原。
- ユニバーサルインフルエンザワクチン抗原がワクチンアジュバントの投与前にもしくは投与後に、または同時に投与される、請求項22および23に記載の方法、請求項24に記載のキット、請求項25に記載のワクチンアジュバント、または請求項26に記載の該抗原。
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Cited By (5)
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WO2020138217A1 (ja) * | 2018-12-26 | 2020-07-02 | 大日本住友製薬株式会社 | ワクチンアジュバントを含む製剤 |
WO2020179797A1 (ja) * | 2019-03-04 | 2020-09-10 | 公益財団法人ヒューマンサイエンス振興財団 | インフルエンザhaスプリットワクチンの製造方法 |
WO2021192393A1 (en) * | 2020-03-27 | 2021-09-30 | Path | Malaria transmission-blocking vaccines |
WO2022003999A1 (en) * | 2020-06-29 | 2022-01-06 | Path | Pre-erythrocytic malaria vaccines |
US11732031B2 (en) | 2017-09-04 | 2023-08-22 | Japan As Represented By Director General Of National Institute Of Infectious Diseases | Method for producing influenza HA split vaccine |
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Cited By (5)
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US11732031B2 (en) | 2017-09-04 | 2023-08-22 | Japan As Represented By Director General Of National Institute Of Infectious Diseases | Method for producing influenza HA split vaccine |
WO2020138217A1 (ja) * | 2018-12-26 | 2020-07-02 | 大日本住友製薬株式会社 | ワクチンアジュバントを含む製剤 |
WO2020179797A1 (ja) * | 2019-03-04 | 2020-09-10 | 公益財団法人ヒューマンサイエンス振興財団 | インフルエンザhaスプリットワクチンの製造方法 |
WO2021192393A1 (en) * | 2020-03-27 | 2021-09-30 | Path | Malaria transmission-blocking vaccines |
WO2022003999A1 (en) * | 2020-06-29 | 2022-01-06 | Path | Pre-erythrocytic malaria vaccines |
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JP7395461B2 (ja) | 2023-12-11 |
TW202011986A (zh) | 2020-04-01 |
CN112839673A (zh) | 2021-05-25 |
SG11202100706UA (en) | 2021-03-30 |
MX2021000951A (es) | 2021-06-23 |
IL280332A (en) | 2021-03-25 |
BR112021001188A2 (pt) | 2021-04-27 |
BR112021001188A8 (pt) | 2022-12-06 |
US20210353737A1 (en) | 2021-11-18 |
CA3107409A1 (en) | 2020-01-30 |
EP3827842A1 (en) | 2021-06-02 |
AU2019311965A1 (en) | 2021-02-25 |
JPWO2020022272A1 (ja) | 2021-08-05 |
KR20210034614A (ko) | 2021-03-30 |
EP3827842A4 (en) | 2022-04-20 |
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