JP6274668B2 - Adjuvant and vaccine containing the same - Google Patents

Description

  The present invention relates to an adjuvant and a vaccine containing the same.

  Immunostimulation is the stimulation and activation of dendritic cells, which means that antibody production (humoral immunity) and immune functions by T cells are enhanced in vivo. Components that exhibit such functions are called adjuvants, immunostimulators, immune enhancers, and the like. In the present specification, the components having the above functions are collectively referred to as “adjuvant”, and the function is referred to as “adjuvant ability”. Specific adjuvants include, for example, precipitating adjuvants such as calcium phosphate, aluminum phosphate, aluminum oxide, etc .; Freund's complete adjuvant containing paraffin oil, heated Mycobacterium tuberculosis, and surfactant; Complete Freund's adjuvant to Mycobacterium tuberculosis Examples include Freund's incomplete adjuvant excluding dead bacteria.

  Conventionally, various components useful as such an adjuvant have been developed, and in particular, intensive research on an adjuvant that can be used together with an antigen as a vaccine is progressing.

  For example, a significant number of ligands for RIG-I-like receptors associated with antiviral innate immune responses have been discovered, and European and US drug licensing agencies have used these ligands as novel adjuvants for nonclinical toxicity studies It is recommended to evaluate with. Some of these ligands are also in clinical trials.

  It is also known that CpG-DNA, poly I: C, flagellin and the like which act as agonists of TOLL-like receptors related to innate immune responses, like RIG-I, have a function as an adjuvant (non-patent) Reference 1).

  Currently, adjuvants included in vaccines specifically used in clinical practice include aluminum salts contained in vaccines shown in Non-Patent Document 2 and Non-Patent Document 3, influenza vaccines sold by Novartis Pharma And MF59 (oil-in-water type adjuvant containing squalene) contained in Celura (registered trademark).

Japan Linfen 69, 9 (2011) 1541-1546 Precipitation purified pertussis diphtheria tetanus mixed vaccine pharmaceutical interview form, Takeda Pharmaceutical Co., Ltd., August 2009 (revised 8th edition) Beamgen (registered trademark) pharmaceutical interview form, Astellas Pharma Inc., April 2012 (revised 15th edition)

  As described above, commercially available vaccines contain an aluminum salt as an adjuvant. However, since this easily causes inflammation, there is a concern that it may adversely affect the human body. In particular, as described in Non-Patent Documents 1 and 2, vaccines containing an aluminum salt as an adjuvant have anaphylactic shock-like symptoms, multiple sclerosis, acute disseminated encephalomyelitis as serious side effects. There is a risk of causing Guillain-Barre syndrome. These may be attributed to the fact that the aluminum salt produces IgE, not IgG that acts in the neutralization reaction of the pathogen.

  The use of aluminum salts is contraindicated for patients undergoing dialysis therapy, and long-term administration of aluminum salts is known to cause aluminum encephalopathy, aluminum osteopathy, aluminum nephropathy, anemia, etc. ing. Furthermore, it cannot be said that the aluminum salt is sufficiently strong as an ability to enhance the immunity.

  In this way, inoculation with an adjuvant as a vaccine component may cause a life-threatening side effect. The current situation is that little is known about the immunological findings for providing low-risk adjuvants.

  In other words, it is safe to develop an adjuvant that has a sufficiently enhanced immune function and has a very low possibility of side effects even when applied to a living body, in particular, a subunit vaccine. It is an important issue in developing and producing peptide vaccines, mucosal vaccines and the like.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research. As a result, phospholipids are important components for activating the IRF3-TBK1 signal cascade, which is a signal cascade that is very important for immunostimulation. I found it. Furthermore, it has been found that the phospholipids are not involved in excessive production of inflammatory cytokines that are thought to cause side effects when used in the immune system, especially as an adjuvant. Based on this finding, when the phospholipid was actually administered to mice together with the antigen, it was confirmed that antibodies against the antigen were sufficiently produced in the mouse serum.

  The present invention has been completed on the basis of these findings, and widely encompasses the embodiments described below.

  Item 1. An adjuvant containing a phospholipid.

  Item 2. The adjuvant according to Item 1, comprising a phospholipid (excluding 3-deacylated-4'-monophosphoryl lipid A).

  Item 3. The adjuvant according to Item 1 or 2, wherein the phospholipid is anionic.

  Item 4. The adjuvant according to any one of Items 1 to 3, wherein the phospholipid is glycerophospholipid, sphingophospholipid, glycerophosphonolipid, sphingophosphonolipid, amino derivatives thereof, or salts thereof.

  Item 5 The phospholipid is phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, bisphosphatidic acid, pyrophosphatidic acid, plasmalogen, ethanolamine plasmalogen, sphingomyelin, ceramide phosphocholine, ceramide Phosphoethanolamine, ceramide phosphoserine, ceramide phosphoglycerol, ceramide phosphoglycerophosphoric acid, ethylamine diacylglycerophosphonic acid, ethylamine monoacylglycerophosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid, ethylamine monoacylglyceroaminoethylphosphonic acid, ethylamine diacylglycero Cholinephosphonic acid Selected from the group consisting of ethylamine monoacylglycerocholine phosphonic acid, ethylamine monoacylglyceroserine phosphonic acid, ethylamine diacylglyceroserine phosphonic acid, ceramide aminoethylphosphonic acid, acylsphingosylaminoethylphosphonic acid, and derivatives and salts thereof Item 5. The adjuvant according to any one of Items 1 to 4 above.

  Item 6. The adjuvant according to any one of Items 1 to 5, wherein the phospholipid is phosphatidylinositol, a phosphatidylinositol ester derivative, or a salt thereof.

Item 7 The phosphatidylinositol or phosphatidylinositol ester derivative is PtdIns, PtdIns (3) P, PtdIns (4) P, PtdIns (5) P, PtdIns (3,4) P ₂ , PtdIns (3,5) P ₂ , and PtdIns (3, 4, 5) adjuvant according to Item 6 is at least one selected from the group consisting of _{P 3.}

  Item 8 The adjuvant according to Item 6 or 7, wherein the phosphatidylinositol or phosphatidylinositol ester derivative is a compound represented by the following formula (1):

(In the formula, R ¹ is an alkyl group which may have an amino group or a hydroxyl group,
The alkyl group may further have one or more alkanoyloxy groups having 3 to 24 carbon atoms or amino acid residues or peptide residues,
R ² , R ³ , and R ⁴ are the same or different and are a residue of the acid obtained by a dehydration condensation reaction between a hydrogen atom or an acid and a hydroxyl group. ).

  Item 9 The adjuvant according to any one of Items 6 to 8, wherein the phosphatidylinositol ester derivative is phosphatidylinositol phosphate.

  Item 10 The adjuvant according to Item 9, wherein the phosphatidylinositol phosphate is a compound represented by the following formula (2);

  Item 11. A vaccine comprising the adjuvant according to any one of Items 1 to 10 and an antigen.

  Item 12 The antigen consists of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell-specific proteins, cancer cell lysates, prions, nucleic acids, renin, angiotensin, and angiotensin receptors Item 12. The vaccine according to Item 11, which is at least one selected from the group.

  Item 13. The vaccine according to Item 12, wherein the antigen is a virus.

  Item 14. An immunostimulation method comprising a step of administering a phospholipid to a living body.

  Item 15. The immunostimulation method according to Item 14, comprising a step of administering a phospholipid (except for 3-deacylated-4'-monophosphoryl lipid A) to a living body.

  Item 16 The immunostimulation method according to Item 14 or 15, wherein the phospholipid is anionic.

  Item 17 The immunostimulation method according to any one of Items 14 to 16, wherein the phospholipid is glycerophospholipid, sphingophospholipid, glycerophosphonolipid, sphingophosphonolipid, amino derivatives thereof, or salts thereof. .

  Item 18 The phospholipid is phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, bisphosphatidic acid, pyrophosphatidic acid, plasmalogen, ethanolamine plasmalogen, sphingomyelin, ceramide phosphocholine, ceramide Phosphoethanolamine, ceramide phosphoserine, ceramide phosphoglycerol, ceramide phosphoglycerophosphoric acid, ethylamine diacylglycerophosphonic acid, ethylamine monoacylglycerophosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid, ethylamine monoacylglyceroaminoethylphosphonic acid, ethylamine diacylglycero Cholinephosphone , Ethylamine monoacyl glycerocholine phosphonic acid, ethylamine diacyl glyceroserine phosphonic acid, ethylamine monoacyl glyceroserine phosphonic acid, ceramide aminoethyl phosphonic acid, and acyl sphingosyl aminoethyl phosphonic acid, or a salt thereof Item 18. The immunostimulation method according to any one of Items 14 to 17 above.

  Item 19. The immunostimulation method according to any one of claims 14 to 18, wherein the phospholipid is phosphatidylinositol, a phosphatidylinositol ester derivative, or a salt thereof.

Item 20 The phosphatidylinositol or the phosphatidylinositol ester derivative is PtdIns, PtdIns (3) P, PtdIns (4) P, PtdIns (5) P, PtdIns (3, 4) P ₂ , PtdIns (3,5) P ₂ , and PtdIns (3, 4, 5) immunostimulatory method according to item 19 is at least one selected from the group consisting of _{P 3.}

  Item 21 The immunostimulation method according to Item 19 or 20, wherein the phosphatidylinositol or phosphatidylinositol ester derivative is a compound represented by the following formula (1):

(In the formula, R ¹ is an alkyl group optionally having an amino group or a hydroxyl group, and the alkyl group further has an alkanoyloxy group having 3 to 24 carbon atoms, an amino acid residue, or a peptide residue. May have one or more groups,
R ² , R ³ , and R ⁴ are the same or different and are a residue of the acid obtained by a dehydration condensation reaction between a hydrogen atom or an acid and a hydroxyl group. ).

  Item 22. The immunostimulation method according to any one of Items 19 to 21, wherein the phosphatidylinositol ester derivative is phosphatidylinositol phosphate.

  Item 23 The immunostimulation method according to Item 22, wherein the phosphatidylinositol phosphate is a compound represented by the following formula (2);

  Item 24. The immunostimulation method according to any one of Items 14 to 23, wherein an antigen is administered together with a phospholipid.

  Item 25 The antigen is composed of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell-specific proteins, cancer cell melts, prions, nucleic acids, renin, angiotensin, and angiotensin receptors. Item 25. The immunostimulation method according to Item 24, which is at least one selected from the group.

  Item 26 The immunostimulation method according to Item 25, wherein the antigen is a virus.

  Item 27 The immunostimulation method according to any one of Items 14 to 26, wherein the living body is a human.

Item 28 A method for screening an adjuvant candidate substance comprising the following step 1 and step 2;
(1) A step 1 for bringing an intracellular component into contact with IRF3 (Interferon regulatory factor 3), and (2) a step 2 for selecting the intracellular component that phosphorylates the IRF3.

Item 29 A method for screening an adjuvant candidate substance comprising the following step 1 and step 2; (I) contacting an intracellular component with IRF3 (Interferon regulatory factor 3) and TBK1 (TANK binding kinase 1); II) Step 2 of selecting the intracellular component that binds to the TBK1 and phosphorylates the IRF3.

  Item 30 The screening method according to Item 28 or 29, wherein the intracellular component is a component contained in the water-insoluble fraction.

    The effects of the present invention are described below. However, the present invention is not limited to the invention that exhibits all of the effects described below, and it is sufficient that at least one of the effects is exhibited.

  The adjuvant according to the present invention has an effect of being easily taken up by dendritic cells, and is excellent in the effect of activating dendritic cells. That is, it is considered that the adjuvant according to the present invention has an action of inducing antibodies and increasing the response of Th1 cells through such activation of dendritic cells. Therefore, the adjuvant which concerns on this invention is useful as a component which comprises a vaccine with an antigen.

  Since the adjuvant according to the present invention is not involved in the production of inflammatory cytokines and does not enhance it, it is very unlikely to cause side effects even as a component constituting a vaccine together with an antigen.

  Since the adjuvant of the present invention originally contains a compound present in the living body, unlike an adjuvant containing a foreign compound, the possibility of causing other side effects such as fever and vomiting other than inflammation is very low.

  From the above, the adjuvant of the present invention or the vaccine comprising the same is safe for the human body and is very excellent in immunostimulatory ability.

  The immunostimulation method by administering the adjuvant of the present invention alone or together with an antigen to the human body is safe for the human body and is useful as a method for improving the immunostimulatory ability of the living body.

FIG. 1 shows the experimental results showing that PtdIns (5) P enhances phosphorylation of IRF3 by TBK1 in vitro. (A), (B), and (D) are in vitro kinase assays with recombinant IRF3 and TBK1 using various reagents. And it is the experimental result which identified phosphorylation of IRF3 by TBK1 using pS394IRF3 antibody. (A) shows an in vitro kinase assay using various reagents, isolated fractions of proteins, or reconstituted proteins. (B) shows an in vitro kinase assay using lipid fractions extracted from HEK293 cells transformed and activated with the plasmids shown in the figure. (C) Lipid and TBK1 protein-lipid overlay assay (left), lipid and IRF3 protein-lipid overlay assay (right). (D) shows an in vitro kinase assay of C16-PtdIns synthesized with PE / PC-based liposomes. FIG. 2 shows the experimental results showing that PIKfive activates the IFNβ promoter or ISRE promoter. (A) A schematic diagram relating to the synthesis of PtdIn (5) P and candidate genes involved in the synthesis. (B) a plasmid that expresses a negative control, PIKfyve, or kinase negative PIKfyve, a plasmid that expresses IFNβ luciferase (top diagram in B), a plasmid that expresses ISRE luciferase (middle diagram in B), or NFκB luciferase The reporter activity in HEK293 cells transformed with the plasmid to be expressed (bottom panel in B) is shown. (C) Shows reporter activity in HEK293 cells transformed with a plasmid encoding a gene thought to be involved in the synthesis of PtdIn (5) P and a plasmid expressing ISRE luciferase. The experimental result which shows that the production of cytokine was suppressed by knocking down PIKfyve. (A)-(F) show that the PIKfive level in MEF is knocked down by electroporation of siRNA. The production amounts of IFNβ, IP-10, and IL-6 were measured using ELISA. (A) Intracellular PIKfyve is concentrated by anti-PIKfyve antibody, and PIKfyve is detected by Western blotting using anti-PIKfyve. (B) Production amounts of IFNβ, IP-10, and IL-6 measured by ELISA of MEF stimulated by poly I: C stimulation and infection by NDV. (C) Poly I: Shows the expression level of IFNβ mRNA measured by QPCR after stimulation with C. (D)-(F) The analysis of the signal molecule group sexualized by the transduction by poly I: C under the knockdown of PIKfive in MEF is shown. (D) Non-denaturing polyacrylamide gel electrophoresis shows that IRF3 forms a dimer. (E) shows phosphorylation of IRF3 and JNK by Western blotting. (F) Localization of RelA after isolation of nuclear fraction (top), total RelA as a control. (G)-(I) The result of knockdown experiment of PIKfive in GM-DC by electroporation of siRNA is shown. (G) GM-DC stimulated by transduction with poly I: C. (H) GM-DC infected with SeV (Cm) or EMCV. (I) GM-DC stimulated by transduction with ISD. The experimental result of C8-PtdIns (5) P producing a cytokine and acting as an adjuvant is shown. (A) IP-10 and RANTES production amounts of GM-DC stimulated with C8 lipid shown in the figure measured by ELISA are shown. (B) and (C) GM-DCs derived from wild type, IRF3 ^{− / −} / IRF7 ^{− / −} , or IPS-1 ^{− / −} mice with 10, 25, and 50 μM C8-PtdIns (5) P. The production amount of IP-10, RNATES, and IFNβ measured by ELISA after stimulation is shown. (D) Immunized intraperitoneally with alum and ovalbumin (OVA) as a positive subject, immunized with OVA with PBS and C8-PtdIns (5) P, or C8-PtdIns (4,5) P ₂ The titer of IgG specific for OVA in serum measured after 5 weeks is given (left). On the right is the titer measured over time. (E) OVA-specific OT-II transgenic CD4-positive T cells were isolated, C8-PtdIns in the presence of OVA (5) P, C8- PtdIns (4,5) P 2, or poly I: C 2 shows the production amount of interferon γ (IFNγ) measured after co-culturing GM-DC treated with 1. The production amount of IL1β measured by ELISA of GM-DC stimulated with C8 lipid shown in the figure is shown. The figure explaining the abbreviation used in this specification, a claim, and drawing. The figure explaining the abbreviation used in this specification, a claim, and drawing.

  The present invention is described in detail below. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the technique that clearly indicates the source.

See, for example, Sambrook and Russell, “Molecular Cloning A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel, F .; M.M. et al. “Current Protocols in Molecular Biology”, John Wiley & Sons, New York,. NY; Molecular Biology of the Cell 5E: Reference Edition Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff; Basic and Clinical Pharmacology E / E
Science) by Bertram Katzung, Susan Masters and Anthony Trevor (Dec 13, 2011).

  In the field of immunity such as vaccines and adjuvants, William E. et al. References such as Paul “Fundamental Immunology”, Lippincott Williams &Wilkins; 2012 may be referred to as appropriate.

  Further, abbreviations and the like described in the present specification, drawings, and the like are illustrated in FIGS. 6-1 and 6-2.

<Adjuvant>
The adjuvant according to the present invention is also called an immunopotentiator or an immunostimulator. All such designations are normally used to mean agents used for the same application as adjuvant.

  The adjuvant according to the present invention comprises phospholipids. Among such phospholipids, adjuvants containing phospholipids other than 3-deacylated-4'-monophosphoryl lipid A are preferable.

  The phospholipid may be a phospholipid that exists naturally, particularly in vivo, or may be an industrially produced phospholipid, and is not particularly limited. Further, the industrially produced phospholipid is not particularly limited to a specific technique such as a biochemical technique or a chemical technique. That is, it may be a phospholipid obtained by synthesis in a cell or a phospholipid synthesized by an in vitro chemical reaction.

  Specific examples of such phospholipids include glycerophospholipid, sphingophospholipid, glycerophosphonolipid, sphingophosphonolipid, and derivatives thereof. Glycerophospholipids and glycerophosphonolipids are phospholipids that contain a glycerol-derived constituent alcohol moiety in the phospholipid molecule, and sphingophospholipids and sphingophosphonolipids are derived from long-chain amino alcohols such as sphingosine and ceramide. A phospholipid containing an alcohol moiety.

  Glycerophospholipid and sphingophospholipid are phospholipids containing a C—O—P bond (phosphate ester bond) in which an oxygen atom is bonded between a carbon atom and a phosphate atom. A phosphonolipid is a phospholipid containing a phosphono group containing a C—P bond in which a phosphorus atom is bonded directly to a carbon atom.

  Specific glycerophospholipids include, for example, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidic acid, bisphosphatidic acid, pyrophosphatidic acid, plasmalogen, ethanolamine plasmalogen And derivatives thereof.

  These glycerophospholipids may be lyso forms. Here, the lyso form means that among the three hydroxyl groups of glycerol, only one of the two hydroxyl groups remaining other than the above-mentioned C—O—P bond formed by ester linkage with phosphoric acid is fatty acid and ester. It is a bonded compound.

  Further, these glycerophospholipids may be amino derivatives in which at least one of the two hydroxyl groups described above is ester-linked with an amino acid or a peptide. This is called an amino derivative of glycerophospholipid in the present invention.

  The amino acids are not only 20 amino acids based on the genetic information of t-RNA, but also selenocysteine, selenomethionine, N-formylmethionine, pyrrolysine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine. , Β-alanine, N-methylglycine, ornithine, statin, citrulline, creatine, γ-aminobutyric acid and the like, and amino acids in a broad sense. Among these amino acids, amino acids containing side chains having a charge such as lysine, arginine, histidine, glutamic acid, aspartic acid and the like are preferable.

  The peptide is a peptide in which about 2 to 10 amino acids described above are peptide-bonded. Among them, a peptide containing an amino acid residue containing a side chain having the above charge is preferable.

  Specific sphingophospholipids include, for example, sphingomyelin, ceramide phosphocholine, ceramide phosphoethanolamine, ceramide phosphoserine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate, and derivatives thereof.

  These sphingophospholipids may be lyso forms. Here, the lyso form is a compound in which the other hydroxyl group used for the above-mentioned C—O—P bond formed by the ester bond with phosphoric acid among the two hydroxyl groups of sphingosine remains as it is.

  Further, these sphingophospholipids may be amino derivatives in which an amino acid or a peptide is ester-bonded to the remaining hydroxyl group. This is referred to as an amino derivative of sphingophospholipid in the present invention.

  The amino acids and peptides are as described in detail for the amino derivatives of glycerophospholipid.

  Specific glycerophosphonolipids include, for example, ethylamine diacylglycerophosphonic acid, ethylamine monoacylglycerophosphonic acid, ethylamine diacylglyceroaminoethylphosphonic acid, ethylamine monoacylglyceroaminoethylphosphonic acid, ethylamine diacylglycerocholinephosphonic acid, ethylamine monoacylphosphonic acid. Examples include acyl glycerocholine phosphonic acid, ethylamine diacyl glyceroserine phosphonic acid, ethylamine monoacyl glyceroserine phosphonic acid, and derivatives thereof.

  These glycerophosphonolipids may be lyso forms. Here, the lyso form is one of the three hydroxyl groups of glycerol and the other two hydroxyl groups remaining in addition to the above-mentioned CP bond formed by directly binding to phosphoric acid, and fatty acid and ester bonds. It is a compound.

  Further, these glycerophosphonolipids may be amino derivatives in which an amino acid or a peptide is ester-bonded to at least one of the two hydroxyl groups described above. This is referred to as an amino derivative of glycerophosphonolipid in the present invention.

  The amino acids and peptides are as described in detail for the amino derivatives of glycerophospholipid.

  Specific examples of sphingophosphonolipids include ceramide aminoethylphosphonic acid, acylsphingosylaminoethylphosphonic acid, and derivatives thereof.

  Further, these sphingophosphonolipids may be lyso forms. Here, the lyso form is a compound in which the other of the hydroxyl groups used for the above-mentioned CP bond formed by directly binding to phosphoric acid among the two hydroxyl groups of sphingosine remains as it is.

  Further, these sphingophosphonolipids may be amino derivatives in which an amino acid or a peptide is ester-bonded to the remaining hydroxyl group. This is called an amino derivative of sphingophosphonolipid in the present invention.

  The amino acids and peptides are as described in detail for the amino derivatives of glycerophospholipid.

  Examples of the phospholipid include compounds represented by the structural formulas shown in Table 1 below. The phospholipids represented by these structural formulas can be purchased from Avanti polar lipid as a salt form (the numbers in the left column indicate product numbers).

  Among these phospholipids, phosphatidylinositol or phosphatidylinositol ester derivatives are preferable.

  Examples of the phosphatidylinositol ester derivatives include phosphatidylinositol (3) monophosphate, phosphatidylinositol (4) monophosphate, phosphatidylinositol (5) monophosphate, phosphatidylinositol (3,4) bisphosphate, phosphatidylinositol (3,5 ) Bisphosphoric acid, phosphatidylinositol (3,4,5) triphosphoric acid, and derivatives thereof.

  Moreover, the compound shown by following formula (1) is also mentioned as a phosphatidylinositol or a phosphatidylinositol ester derivative.

In the above formula, R ¹ is an alkyl group which may have an amino group or a hydroxyl group.

  The alkyl group may further have one or more alkanoyloxy groups having 3 to 24 carbon atoms or amino acid residues or peptide residues.

  Preferred is an alkyl group further having two or more alkanoyloxy groups or amino acid residues or peptide residues. The upper limit of the number of alkanoyloxy groups or amino acid residues or peptide residues that the alkyl group has is not particularly limited as long as it can be retained according to the number of carbon atoms of the alkyl group.

  The alkanoyloxy group preferably has about 4 or more carbon atoms, more preferably about 6 or more, and still more preferably about 8 or more. Further, the upper limit of the carbon number of the alkanoyloxy group is preferably about 20, more preferably about 16, further preferably about 12, and most preferably about 8.

  The alkanoyloxy group may contain one or more double bonds in the carbon-carbon bond. The upper limit number of double bonds is determined according to the number of carbon atoms of the alkanoyloxy group, and preferably about 4 carbon-carbon double bonds may be included. Thus, when including a carbon-carbon double bond, it may be a cis isomer or a trans isomer, and is not limited to any one.

  Although carbon number of the said alkyl group is not specifically limited, Usually, 2-8 may be sufficient, More preferably, it is 3 or more. The upper limit of the number of carbon atoms is preferably 7, more preferably 6, and even more preferably 5.

  When the alkyl group has an alkanoyloxy group, the oxygen atom that is single-bonded to the terminal carbonyl carbon atom in the alkanoyloxy group may be single-bonded to any carbon atom in the alkyl group. When the alkyl group has two or more alkanoyloxy groups, the two or more alkanoyloxy groups described above may be bonded to the same carbon atom in the alkyl group, and may be bonded to different carbon atoms of the alkyl group. The alkanoyloxy group mentioned above may be couple | bonded. The two or more alkanoyloxy groups described above may be the same or different.

  The amino acid residues are not only 20 kinds of amino acids based on the genetic information of t-RNA, but also selenocysteine, selenomethionine, N-formylmethionine, pyrrolysine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O- A substance in which a hydrogen atom is removed from at least one carboxy group of a broadly defined amino acid such as a compound having an amino group and a carboxy group such as phosphoserine, β-alanine, N-methylglycine, ornithine, statin, citrulline, creatine, and γ-aminobutyric acid It is. Among these amino acids, an amino acid residue obtained by removing a hydrogen atom from an amino acid containing a charged side chain such as lysine, arginine, histidine, glutamic acid, and aspartic acid is preferable.

  The peptide residue is a peptide residue obtained by removing a hydrogen atom from at least one carboxy group of a peptide obtained by peptide bonding of the above 2 to 10 amino acids. Such peptide residues preferably include the above-mentioned preferred amino acid residues.

  In the alkyl group, the oxygen atom that is single-bonded to the carbonyl group in the amino acid residue or peptide residue described above may be single-bonded to any carbon atom in the alkyl group. When the alkyl group has two or more amino acid residues or peptide residues, two or more amino acid residues or peptide residues described above may be bonded to the same carbon atom in the alkyl group, The amino acid residue or peptide residue described above may be bonded to different carbon atoms of the alkyl group. Two or more amino acid residues or peptide residues described above may be the same or different.

In the formula, R ² , R ³ and R ⁴ are the same or different and are the residues of the acid obtained by a dehydration condensation reaction between a hydrogen atom or an acid and a hydroxyl group.

  The acid may be an organic acid or an inorganic acid, and is not particularly limited, but is preferably an inorganic acid. Examples of such inorganic acids include phosphoric acid, phosphorous acid, hypophosphorous acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, and boronic acid, and are not particularly limited, but phosphoric acid is preferred.

  Most preferred as the residue is a phosphono group.

  Specific examples of the compound represented by the above formula (1) include compounds represented by the structural formulas shown in Tables 2 to 4 below. The compounds represented by these structural formulas are in the form of salts, which can be purchased from echelon bioscience for Tables 2 to 4, and from Avanti polar lipid for Tables 5 and 6 (the numbers in the left column are Indicates the catalog number.)

Incidentally, based on _H 2 _O 3 P- structures in Table 2-6 are as follows equation.

  As such a phosphatidylinositol ester derivative, phosphatidylinositol phosphate is preferable, and the phosphatidylinositol derivative of the most preferable embodiment is a compound represented by the following chemical formula (2).

  The above-described phospholipid may be in the form of a salt.

  As one embodiment of such a salt, specifically, a salt composed of an anion obtained by removing a proton from any one or more OH groups contained in a phospholipid and a counter cation thereof can be mentioned. More preferably, a salt comprising an anion obtained by removing a proton from an OH group of an alkylphosphono group which may have a phosphono group or one or more alkanoyloxy groups contained in a phospholipid, and a counter cation thereof may be mentioned. It is done.

  The valence of the anion is not particularly limited, but is usually about monovalent to 7-valent.

  The kind of counter cation is not specifically limited, For example, sodium ion, potassium ion, calcium ion etc. are mentioned.

  As a salt of another embodiment, a salt composed of a cation in which a proton is bonded to any one or more amino groups contained in a phospholipid and a counter anion thereof can be mentioned.

  The type of the counter anion is not particularly limited, and examples thereof include fluoride ions, chloride ions, bromide ions, and the like.

  The above-mentioned phospholipid can be produced using a known method. For example, when the phospholipid is a phospholipid having a phosphonooxy group containing a phosphate ester bond as described above, it should be produced by appropriately referring to Experimental Chemistry Course 5th edition (16) “Carboxylic acid / amino acid / peptide”. Can do. It is also possible to purchase and obtain commercially available products.

  The phospholipid contained in the adjuvant according to the present invention may be anionic, cationic or nonionic, but is preferably an anionic phospholipid.

  The adjuvant which concerns on this invention contains the above-mentioned phospholipid, The content is about 0.01-99.99 weight% normally with respect to the said adjuvant. Further, the phospholipid itself may be the adjuvant according to the present invention.

  In the adjuvant according to the present invention, in addition to the above-described phospholipid, a known component usually contained in the adjuvant may be included only within a range not impairing the above-described effect of the adjuvant according to the present invention.

  Examples of such components include preservatives, buffers, preservatives, inactivators, isotonic agents, pH adjusters, and the like.

  As long as the adjuvant according to the present invention does not impair the effect of the adjuvant according to the present invention described above, a known component that exerts an immunostimulatory action usually contained in the adjuvant is included in addition to the above-described phospholipid. It may be.

  Examples of such components include aluminum hydroxide, squalene, α-tocopherol, mineral oil, paraffin oil, single-stranded RNA or analog thereof, double-stranded RNA or analog thereof, flagellin, trehalose derivative, MPL, and the like.

  The adjuvant according to the present invention is effectively used for, for example, laboratory animals generally employed when producing antibodies for commercial use. That is, when an antigen specifically recognizing an antibody is administered to a laboratory animal, the adjuvant according to the present invention is mixed therewith and administered at the same time or separately from the antigen.

  Specific examples of such experimental animals include mice, rats, rabbits, sheep, chickens, donkeys, horses, ostriches, camels, llamas, and alpaca.

  The amount of the adjuvant according to the present invention to be used for these experimental animals may be appropriately set depending on the desired degree of immunostimulation, and is not particularly limited, but in terms of the amount of the phospholipid contained in the adjuvant. Usually, it may be about 0.5 mg to 0.5 g / time per 1 kg of animal individual.

  Moreover, according to the desired degree of immunostimulation, you may use it multiple times with respect to the same animal individual, and what is necessary is just to make the upper limit normally about 2 to 4 times. In addition, when using the adjuvant based on this invention in multiple times with respect to the same solid, the space | interval may be provided suitably, and what is necessary is usually just about 14 to 30 days.

  The use method of the adjuvant according to the present invention when used in the above-mentioned animals is not particularly limited, and examples thereof include oral administration, intravenous administration, transdermal administration, transmucosal administration, sublingual administration, transperitoneal administration. What is necessary is just to use by administration, transmuscular administration, etc.

  In addition, when the adjuvant according to the present invention is administered to a living body together with an antigen, dendritic cells in the living body are activated and an acquired immune effect is exhibited. Therefore, the adjuvant according to the present invention is expected to exhibit a function as a vaccine when clinically used together with an antigen.

<Vaccine>
The vaccine according to the present invention comprises the above-mentioned adjuvant and antigen.

  The antigen is one that is detected by the humoral immunity or cellular immunity system, and an antibody that specifically binds to the antigen is produced, or is preyed on by white blood cells, macrophages, T cells, and the like.

  Specific antigens include bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell-specific proteins, cancer cell lysates, prions, nucleic acids, renin, angiotensin, angiotensin receptors, etc. There is no particular limitation.

  The bacterium is not particularly limited as long as it is a bacterium that causes disease to humans, pets, livestock, and the like. Specifically, streptococci (such as streptococci, pneumococci, etc.), Staphylococcus aureus (MSSA, MRSA, etc.), Staphylococcus epidermidis, enterococci, and bacteria belonging to the genus Listeria (listeria, hereinafter) may be expressed in the same manner. ), Neisseria meningitidis, Neisseria gonorrhoeae, Pathogenic E. coli, Neisseria pneumoniae, Proteus, Pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, M. tuberculosis, Cholera Fungus, plague, diphtheria, shigella, anthrax, treponema, tetanus, leprosy, legionella, leptospira, borrelia, francisella, coccella, rickettsia, chlamydia, rhinomycosis, pylori Etc.

  The virus is not particularly limited as long as it is a virus that causes disease to humans, pets, livestock, and the like. Specifically, influenza virus, papilloma virus, hepatitis virus (A type, B type, C type, D type, E type, F type, G type, TT type, etc.), smallpox virus, measles virus, rubella virus, Poliovirus, varicella-zoster virus, norovirus, norwalk virus, sapovirus, sapporovirus, mumps virus, adenovirus, enterovirus, rotavirus, AIDS virus, rabies virus, T lymphophilic virus, yellow fever virus, cytomegalo Virus, SARS virus, polyoma virus, JC virus, BK virus, herpes virus, lymphocrypt virus, roseovirus, Japanese encephalitis virus, coxsackie virus, dengue virus, West Nile virus, coronavirus, parvovirus Epstein-Barr virus, Marburg virus, Hantavirus, Lassa virus, Chikungunya virus, Hanturn virus, Jumping disease virus, Lymphocytic choriomeningitis virus, Borna virus, Rift Valley fever virus, Togot virus, Dori virus, Foot-and-mouth disease virus, Newcastle virus, Bovine papular stomatitis virus, Rinderpest virus, Swine vesicular disease virus, Calicivirus, Torovirus, African horse sickness virus, Arteri virus, Yokan virus, Capripox virus, Sheep-associated malignant catarrhal fever Examples include viruses, viral hemorrhagic septic viruses, and vesicular stomatitis viruses.

  The fungus is not particularly limited as long as it is a fungus that causes disease to humans, pets, livestock, and the like. Specific examples include Aspergillus, Candida, Cryptococcus, Ringworm mycosis, Histoplasma, Pneumocystis and the like.

  The parasitic protozoa is not particularly limited as long as it is a parasitic protozoa causing disease to humans, pets, livestock and the like. Specifically, amoeba dysentery, malaria, toxoplasma, leishmania, cryptosporidium, trypanosoma and the like can be mentioned.

  The parasitic worms are not particularly limited as long as they are parasitic worms that cause diseases to humans, pets, livestock, and the like. Specific examples include Echinococcus, Schistosoma japonicum, Filaria, roundworm, and broad-headed fibroids.

  The cancer cell is not particularly limited as long as it is a cancerous lesion of a human, pet animal, livestock or the like. Examples of such cancer include leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, brain tumor, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer, appendic cancer, colon cancer, Liver cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck cancer, laryngeal cancer, oral cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer , Sinus cancer, maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, sphenoid sinus cancer, thyroid cancer, kidney cancer, lung cancer, small cell lung cancer, osteosarcoma, prostate cancer, testicular tumor, renal cell cancer, Examples include bladder cancer, rhabdomyosarcoma, skin cancer, anal cancer, brain tumor, osteosarcoma, chondrosarcoma, and synovial sarcoma.

  The cancer cell-specific protein is not particularly limited as long as it is a protein that is specifically expressed in the above-described cancer cells (also referred to as a tumor-associated antigen). Among such proteins, a protein that is specifically expressed on the surface of a cancer cell is preferable. For example, a receptor is also included. Such a protein may be a full-length protein or a partial fragment thereof.

  An example of such a cancer cell-specific protein is PSA if the cancer is prostate cancer.

  The cancer cell lysate is obtained by melting the above cancer cells. The method of thawing may be simply crushing the cancer cells, for example, a method of crushing using a buffer containing a surfactant, an ultrasonic crushing method, a method of crushing using glass beads, or a French press. The method etc. which are crushed are mentioned, You may combine these suitably.

  The above-mentioned prion is an infectious factor composed of a non-wild type protein, which causes diseases by accumulating in humans, pet animals, livestock and the like.

  Such diseases are not particularly limited, and examples include spongiform encephalopathy, kuru, Creutzfeldt-Jakob disease, lethal familial insomnia, Gerstman-Streisler-Scheinker syndrome, Alzheimer's disease, etc. Specific examples of prions include β amyloid and the like.

  The above-mentioned renin works in the renin-angiotensin system related to an increase in blood pressure.

  The angiotensin may be type I or type II, and works in the above-described renin-angiotensin system.

  The above-mentioned angiotensin receptor is a receptor for type I angiotensin and / or type II angiotensin, and may be the full length or a partial fragment thereof.

  The bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell specific proteins, cancer cell lysates, prions, nucleic acids, renin, angiotensin, angiotensin receptors, etc. Two or more types may be combined and used as an antigen contained in the vaccine of the present invention.

  The above-mentioned bacteria, viruses, fungi, parasitic protozoa, parasitic helminths and the like may be used as antigens, or inactivated may be used as antigens.

  As a specific method for inactivation, for example, a method of performing a treatment such as heating, ultraviolet irradiation, addition of an organic solvent, addition of a surfactant or the like can be mentioned.

  Further, when the above-mentioned bacteria, viruses, fungi, parasitic protozoa, and parasitic helminths are used as antigens, it is not necessary to use these as antigens, for example, those obtained by removing nucleic acids such as DNA, RNA, etc. In the case of a virus, a virus coat (which is also called a capsid) may be used as an antigen.

  Furthermore, constituent components such as proteins, carbohydrates, lipids and the like possessed by bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, etc. may be used as antigens, and some of these may be used as antigens. Such components are preferably present on the surface of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths and the like.

  For example, in the case of an influenza virus, various proteins such as HA and NA present on the surface may be used as antigens, and a part of these proteins or a sugar chain contained therein or a part thereof may be used as an antigen.

  The nucleic acid may be ribonucleotide or deoxyribonucleotide. Moreover, such a nucleic acid is not limited to a single-stranded form, and may be a double-stranded form or more. And said nucleic acid is not limited to what contains adenine, guanine, cytosine, thymine, and uracil as a base, The nucleic acid containing these derivatives or the modified base may be sufficient.

  The vaccine according to the present invention is usually suitably used for humans in order to prevent diseases such as infectious diseases and cancers, but in addition to the above-mentioned pet animals (for example, dogs, cats, ferrets, Birds) and the above-mentioned livestock (cow, pig, chicken, goat, ostrich, sheep, horse, etc.).

  Although the content rate of the above-mentioned adjuvant in the vaccine which concerns on this invention is not specifically limited, Usually, it is about 0.01 weight%-99.99 weight% with respect to 100 weight% of vaccines.

  If the ratio of the above-mentioned antigen and the above-mentioned adjuvant is used, the adjuvant is usually about 10 to 1000 parts by weight with respect to 1 part by weight of the antigen.

  The administration method of the vaccine according to the present invention is not particularly limited, and examples thereof include oral administration, intravenous administration, transdermal administration, transmucosal administration, sublingual administration, and transmuscular administration.

  The dose of the vaccine varies depending on the desired degree of immunostimulation, age, sex, etc. of the administration target, and may be set as appropriate. Although not particularly limited, for example, if converted into the amount of phospholipid In general, it may be about 0.5 mg to 0.5 g / time per 1 kg of humans, pets or livestock.

  Further, the vaccine may be administered multiple times to the same human, companion animal, or livestock according to the degree of effect exerted by the desired vaccine, the age of the administration subject, sex, etc., and usually 2 to 4 times. The upper limit may be set as the upper limit. In addition, when administering the vaccine based on this invention in multiple times with respect to the same human, a pet animal, or livestock, the space | interval may be provided suitably and should just be normally about 14 to 30 days.

  Such a vaccine not only exhibits a sufficient preventive effect on diseases (particularly infectious diseases), but also exhibits extremely low side effects caused by administration of the vaccine.

<Immunostimulation method>
The immunostimulation method according to the present invention includes a step of administering the above-described phospholipid to a living body.

  The living body is not particularly limited as long as it is a living body that needs to enhance the immune system. Specifically, as described in detail in <Adjuvant> above, experimental animals used in the production of antibodies for commercial use, and disease (particularly infectious diseases) detailed in <Vaccine> described above are prevented. For example, humans, pets, livestock, etc., to be administered vaccines.

  The dose of phospholipid is not particularly limited, and is appropriately set based on, for example, the amount of adjuvant used as described in detail in <Adjuvant> above. Or you may set based on the dosage of a vaccine explained in full detail in the above <vaccine>.

  Similarly, the number of multiple administrations and the administration interval at the time of multiple administrations may be appropriately set based on the details described in the above <Adjuvant> or <Vaccine>.

  The administration method may be appropriately selected from the administration methods detailed in the above <Adjuvant> or <Vaccine>.

  In the immunostimulation method according to the present invention, it is preferable to administer an antigen together with the above-mentioned adjuvant according to the present invention. The specific antigen may be the same as that described in detail in <Vaccine> above, and the subject to be administered, the administration method, the number of multiple doses and the interval thereof, etc. in <Vaccine> and <Adjuvant> What is necessary is just like description.

<Screening method>
The screening method according to the present invention is a method for screening an adjuvant candidate, which includes the following steps 1 and 2.
Process 1
(1) A step of bringing an intracellular component into contact with IRF3 (Interferon regulatory factor 3)
Process 2
(2) A step of selecting the intracellular component that phosphorylates the IRF3.

In addition, the screening method according to the present invention includes a method for screening an adjuvant candidate in an embodiment including the following step 1 ′ and step 2 ′.
Process 1 ''
(I) Step 1 ′ for contacting an intracellular component with IRF3 (Interferon regulatory factor 3) and TBK1 (TANK binding kinase 1)
Process 2 ''
(II) Step 2 ′ of selecting the intracellular component that binds to the TBK1 and phosphorylates the IRF3.

  The intracellular components used in the above steps 1 and 1 'can be obtained by disrupting cells by a known method.

  Examples of such a method include a method of crushing using a buffer containing a surfactant, an ultrasonic crushing method, a method of crushing using glass beads, a method of crushing using a French press, etc. These may be combined.

  The above-mentioned intracellular components obtained by crushing cells by such means include those obtained by crushing, for example, in a methanol fraction (water-soluble fraction) fractionated with a methanol / chloroform mixed solvent. May be a component contained in the chloroform fraction (water-insoluble fraction), or a component contained in both of them, and is not particularly limited. It is a component contained in the sex fraction. The specific cell origin is not particularly limited and may be appropriately selected.

  The cells may be activated in advance so that IRF3 is easily phosphorylated before disruption. As a specific activation method, for example, a method of introducing a gene expressing IPS-1, TRIF, MyD88, TIRAP or the like into the cell can be mentioned. Preferably, it is a method of introducing a gene that expresses IPS-1.

  As a method for selecting an intracellular component that phosphorylates IRF3 in the above steps 2 and 2 ', the intracellular component may be selected by confirming phosphorylation of IRF3 by a known method. Specific confirmation methods include Western blotting, flow cytometry, ELISA, and the like using an antibody that recognizes phosphorylated IRF3.

The method of binding to TBK1 and phosphorylating IRF3 in the above steps 2 and 2 ′ is as follows.
In addition to the above method for confirming phosphorylation of IRF3, an immunoprecipitation method, a QCM (quartz crystal microbalance) method, an SPR (surface plasmon resonance) method, or the like may be combined.

  The present invention will be described in more detail based on the following experimental examples. However, it goes without saying that the present invention is not limited to the following experimental examples.

  First, materials and experimental methods used in experimental examples to be described later will be described.

(1) About cells and reagents HEK293 cells and MEFs (mouse fetal fibroblasts) use a DMEM medium containing 10% inactivated FCS (Life Technology) at 37 ° C. in a 5% carbon dioxide environment. Was cultured.

Bone marrow-derived dendritic cells (GM-DCs) induced by GM-CSF were obtained from 10% FCS, 100 μM 2-ME, and 10 ng / ml mouse-derived GM-CSF. It was prepared by culturing for 6 to 8 days in an RPMI1640 medium containing (BD Bioscience).

  The ISD sequence was as follows and was prepared according to a conventional method. (Sense: 5'-TACAGATCTACTAGTGATCTATGAACTGATCTGTACATGATCTACCA; SEQ ID NO: 1).

  LPS and poly I: C and poly dA: dT were purchased from Invivogen and used to stimulate cells with Lipofectamine 2000 (Life Technology) and 2: 1 (2 μg: 1 μl) respectively. And stimulated with OPTI-MEM (Life Technology).

  YM-201636, a PIKfyve inhibitor, was purchased from Santa Cruz.

  Hereinafter, the source of the antibody will be described. Anti-PIKfyve antibody (Sigma), anti-pIRF3 antibody (Cell Signaling), anti-pJNK (Cell Signaling), anti-TBK1 (Abcam), anti-pTBK1 (BD Bioscience). For various ELISA methods, kits were purchased from R & D Systems, and experiments were conducted according to the manual.

(2) About Plasmid and Reporter Assay A FLAG-tagged PIKfyve expression plasmid was prepared by amplifying PIKfyve by PCR and then incorporating it into the pFLAG-CMV6 vector. And a kinase expression mutant (K1831M PIKfyve) expression plasmid was prepared using KOD (Toyobo).

  The FLAG-tagged PIP5Kα, β, and γ, expression plasmids were obtained by amplifying gene fragments encoding these from the cDNA of HEK293 cells and incorporating them into the pFlag-CMV6 vector.

  PI3KIII, PTEN, TMEM55α, TMEM55β, and PI4K2α expression plasmids used were those incorporated into a pCMV-SPORT6 vector (Open Biosystems).

  In the reporter assay, HEK293 cells were seeded in a 24-well plate, HEK293 cells seeded with various expression plasmids and reporter luciferase plasmids using Lipofectamine 2000 (Life Technology) were transformed and cultured for 16 hours. Cells were lysed using passive lysis buffer (Promega).

  Thereafter, luciferase activity was measured using a dual luciferase reporter assay kit (Promega). The reporter plasmids IFNα, ISRE, and NFκB were known. The TK promoter that operates Renilla luciferase was used as an internal control.

(3) In vitro phosphorylation assay GRF-tagged IRF3 was produced using E. coli, and after purification, the GST tag was cleaved according to the protocol using precision protease (BD Biosciences). GST-tagged TBK1 was produced using HEK293t cells and purified using 10 mM glutathione.

In vitro kinase assay was performed by incubating 20 ng TBK1 and 100 nmol IRF3 for 30 minutes at 30 ° C. in reaction buffer (5 mM HEPES (pH 7.2) containing 10 μM ATP, 100 mM NaCl, 2 mM MgCl ₂ ). did.

  Liposomes were prepared by sonication by mixing PC: PE: PI at 30: 8: 2, respectively. This was also used at 40 μM for in vitro kinase assay.

  GST-tagged STING was produced using HEK293t cells and purified using 10 mM glutathione sepharose beads. Purified STING was reconstituted by forming liposomes by removing the surfactant with azolectin lipids in a dialysis environment.

(4) Protein Lipid Overlay Assay 100 μM purified IRF3 or GST-TBK1 protein with membrane lipid strip (Echelon Biosciences; P6001) and TBS-T buffer (140 mM NaCl, 2.5 mM KCl, and 0.05% Tween20) In 25 mM Tris (pH 8.0)). Thereafter, the membrane was washed 3 times with TBS-T buffer, incubated with anti-IRF3 antibody or anti-TBK1 antibody, and further treated with a secondary antibody.

(5) RNAi and quantitative PCR (QPCR) Double- stranded stealth RNA was obtained from Life Science Technology. The target sequence of PIKfive is as follows.

  (Sense strand) 5'-UUCAGAUUGAUUCAUUCUCUCUCGG-3 '[SEQ ID NO: 2].

  siRNA was electroporated using Amaxa Nucleofector (Lonza) or NEON (Life Technology). Two days after electroporation, MEF and GM-DC were stimulated with various reagents or viruses. Total RNA was isolated using Trizol reagent (Life Technology), and reverse transcription reaction was performed using River TraAce (Toyobo) according to the protocol. The primers used for quantitative PCR are as follows.

  mIFN; 5'-ATGGTGTCCGAGCAGAGAT-3 '[sense: SEQ ID NO: 3]: 5'-CCACCACTCATTCTGAGGCA-3' [reverse: SEQ ID NO: 4].

  mIP-10; 5'-CCATCAGCACCATGAACCCAAGT-3 ': [sense: SEQ ID NO: 5]: 5'-CACTCCAGTATAGAGGCCCTTTTAGACC-3' [reverse: SEQ ID NO: 6].

  mGAPDH: 5'-TGACGTGCCGCCTGGAGAAA-3 '[sense: SEQ ID NO: 7]: 5'-AGTGTAGCCCAAGATGCCCTCTCAG-3' [reverse: SEQ ID NO: 8].

  mIL-6; 5'-GTAGCTATGGTACTCCAGAAGAC-3 ': [sense: SEQ ID NO: 9]: 5'-ACGATGATGCACTTGCAGAA-3' [reverse: SEQ ID NO: 10].

  mPIKfy; 5'-AAGTCTTACCCCTCACATGAGCTAGTGA-3 '[sense: SEQ ID NO: 11]: 5'-ATCAGCTAGCATTCTACCCAAGGGT-3' [reverse: SEQ ID NO: 12].

(6) Immunization to mice IPS-1 ^{− / −} mice, IRF3 ^{− / −} / IRF7 ^{− / −} mice, and OT-II mice were prepared by known methods. All animal experiments were conducted with the approval of the Animal Experiment Committee of the Institute for Microbial Diseases, Osaka University.

  For immunization of mice, C57BL / 6J mice (CLEA Japan, Inc.) were injected intraperitoneally with 100 μg of OVA (Seikagaku Corporation) and 1 mg of alum (Sigma) every week for a total of 4 immunizations. Or immunized by intramuscular injection with 100 μg OVA / 100 μg C8-PtdIns.

C8-PtdIns (5) P and C8-PtdIns (4,5) _{P 2} was solubilized in PBS, and mixed with OVA for immunization. For co-culture experiments, we derived from 1 × 10 ⁵ OVA-specific OT-II transgenic mice treated with C8-PtdIns (5) P, C8-PtdIns (4,5) P ₂ , or poly I: C. CD4 ⁺ T cells and 1 × 10 ⁵ GM-DCs were cultured for 7 days in the presence of 10 μg of OVA protein, and the amount of IFNγ produced was measured.

Experimental example 1
Identification of PtdIns (5) P Activation of IRF3 accompanying phosphorylation of transcription factor IRF3 by TBK1 is known as one of the signal cascades essential for the activation of dendritic cells. In order to identify cellular components that regulate such a TBK1-IRF3 signal cascade, an in vitro screening system was constructed. Specifically, recombinant TBK1 and IRF3 were prepared and mixed with various reagents in the presence of ATP for experiments.

The activation of IRF3 was evaluated by detecting by Western blotting using an antibody against S396 phosphorylated IRF3 after an in vitro kinase reaction. Purified STING, which is a signal protein that functions when TBK1 and IRF3 are known immune activators DMXAA, dsDNA, high concentrations of KCl, H ₂ O ₂ , and dendritic cells are activated using known DNA viruses as antigens None of these were able to enhance IRF3 phosphorylation when incubated with (FIG. 1A).

  Next, it was tested whether intracellular components are involved in phosphorylation of IRF3. For this, HEK293t cells were separated into a water-soluble fraction and a water-insoluble fraction using chloroform / methanol. The water-insoluble fraction (chloroform side) containing lipid or cholesterol was dried and resuspended in a reaction buffer.

  As a result, it was found that the insoluble fraction containing lipid increases phosphorylation of IRF3 (FIG. 1A).

  Furthermore, when an experiment was performed using an insoluble fraction isolated from HEK293t cells transformed with IPS-1, TRIF, MyD88, or TIRAP to activate the cells, the IRF3 phosphorylation was compared with the control. It became clear that oxidation was strongly induced (FIG. 1B).

  This indicates that components contained in the water-insoluble fraction increased in activated cells are involved in the activation of IRF3 via TBK1. Focusing on lipids as such components, further experiments were conducted to examine them.

  To further identify lipids involved in IRF3 activation, lipids that bind to TBK1 or IRF3 were examined using a protein-lipid overlay assay. As a result, it was found that TBK1 binds to PtdIns (5) P and IRF3 binds to several anionic lipids such as PtdIns (3) P, PtdIns (4) P, and PtdIns (5) P ( FIG. 1C). The binding of these proteins to cationic lipids or any other lipid was undetectable.

  These findings suggest that anionic lipids have the ability to promote IRF3 phosphorylation in vitro. In order to investigate the involvement of anionic lipids in the activation of IRF3, various synthetic phosphatidylinositols, which are anionic lipids, are mixed with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to form liposomes, These were used for in vitro kinase assays.

  As a result, among these, liposomes containing PtdIns (5) P most effectively promoted phosphorylation of IRF3 (FIG. 1D). From the above, it was suggested that PtdIns (5) P may exert an adjuvant ability by regulating the TBK1-IRF3 signal cascade in in vitro experiments.

  The various phospholipids used in Example 1 contain fatty acids having 16 carbon atoms, and specific sources are the following catalog numbers of Avanti Polar Lipid.

PC: 840035C
PE: 840022C
PI: 850149P,
PI (3) P: 850 150 P,
PI (4) P: 840045X,
PI (5) P: 850152P,
PI (3,4) P ₂ : 850153P,
PI (3, 5) P ₂ : 850154P, PI (4, 5) P ₂ : 850155P,
_{PI (3,4,5) P 3: 850156P} .

  Such a method shown in Example 1 is useful as a screening method for an adjuvant candidate.

Experimental example 2
Identification of PIKfyve Next, we attempted to identify a kinase or phosphatase that regulates the TBK1-IRF3 signal cascade through the production of PtdIns (5) P during viral infection in cells.

  Whether or not the candidate gene induces IRF3 activation is determined by a reporter plasmid (IFR stimulation response element (ISRE)) designed to directly monitor the activity of the transcription factor IRF together with the expression plasmid of the candidate gene. ISRE reporter plasmid) was transformed into HEK293 cells and quantified by a reporter assay using intracellular luciferase expression as an index (FIG. 2A).

  Overexpression of PIKfyve in HEK293 cells increased ISRE and IFNβ promoter activity, but overexpression of PIKfyve kinase negative mutant did not activate these promoters. Moreover, overexpression of PIKfyve did not affect the promoter activity of NF-κB, which is a transcription factor essential for cellular response during virus infection (FIG. 2B).

PIKfyve is Kainesu for synthesizing PtdIns (5) P or PtdIns (3,5) _{P 2} by phosphorylation of the 5-position of the inositol ring, overexpression PtdIns (5) Production of P is induced by, specifically It was thought that the promoter activity of ISRE was increased (FIG. 2C).

  It is known that NF-κB and ISRE are cooperatively activated to induce the production of IFNβ, an essential cytokine for antiviral response. From these results, PtdIns (5) P by PIKfyve is known. It was suggested that the production of IFNβ and the like are controlled by specifically regulating the TBK1-IRF3 signal cascade. These facts suggest that PtdIns (5) P may act as an adjuvant specifically targeting TBK1-IRF3.

Experimental example 3
The role of PIKfyve in the detection pathway of nucleic acids derived from cytoplasmic pathogens In order to examine whether the production of PtdIns (5) P via PIKfyve actually regulates the antiviral innate immune response, PIKfyve was used to siRNA An experiment using MEF knocked down was performed (FIG. 3A).

  Knockdown of PIKfyve suppressed the production of IFNβ and IP-10, known as IRF3-dependent after stimulation with poly I: C and infection with the RNA virus NDV, but IL-6 independent of IRF3 Was not affected (FIG. 3B). The mRNA level of IFNβ after stimulation with poly I: C was also reduced by PIKfive knockdown (FIG. 3C).

  Furthermore, phosphorylation of IRF3 following stimulation with poly I: C and dimer formation caused by phosphorylation were also suppressed by PIKfyve knockdown (FIG. 3D).

  In contrast to this, the effects of other signal cascades activated by viral infection were confirmed by phosphorylation of JNK and nuclear translocation of RelA, which is an NF-κB family, but were not prevented by the knockdown of PIKfyve. (FIGS. 3E and 3F). In addition, knockdown of PIKfive in GM-DC (dendritic cells) also suppressed IFNβ and IP-10 production after stimulation with poly I: C and infection with EMCV and SeV (Cm) (FIGS. 3G and 3H). ). And IL-6 production after stimulation with poly I: C was not suppressed in these cells (FIG. 3G).

  To summarize these results, it is shown that PtdIns (5) P is specifically required for the activation of the TBK1-IRF3 signal cascade caused during RNA virus infection and suppresses PIKfive, a metabolic factor of PtdIns (5) P. It is specified by. Furthermore, the addition of PtdIns (5) P suggests the possibility of activating dendritic cells and exerting adjuvant ability through cytokine production.

Experimental Example 4
Cytokine production and adjuvant effect by synthetic C8-PtdIns (5) P PIKfyve is obtained by phosphorylation of PtdIns (5) P from PtdIns (3) P to PtdIns (3,5) by phosphorylation at the 5 ′ position of the inositol ring. resulting in P _2.

  To obtain direct evidence of the involvement of PtdIns (5) P in the TBK1-IRF3 signal cascade, exogenous administration of synthesized PtdIns (5) P induces TBK1-IRF3 signal-dependent cytokine production Investigate whether or not you can.

  Among phosphatidylinositol phosphorylated at the 5 ′ position of the inositol ring, treatment of GM-DC with C8-PtdIns (5) P can induce the production of IP-10 and RANTES, which are IRF3-dependent cytokines. It became clear (FIG. 4A).

  For the production of cytokines in the viral response, the TBK1-IRF3 signal cascade is activated via IPS-1, which is an adapter factor present upstream of TBK1. Therefore, in order to clarify the action point of cytokine production by C8-PtdIns (5) P, production of cytokine in GM-DC derived from IPS-1-deficient mice or IRF3 / IRF7-deficient mice was examined.

As a result, GM-DC derived from wild-type GM-DC and IPS-1 ^{− / −} did not affect the production of IP-10 and RANTES after C8-PtdIns (5) P stimulation. On the other hand, GM-DC derived from IRF3 ^{− / −} / IRF7 ^{− / −} did not show the production of IP-10 or RANTES (FIG. 4B).

In addition, IFNβ production by C8-PtdIns (5) P was also suppressed in GM-DC derived from IRF3 ^{− / −} / IRF7 ^{− / −} , but not changed in GM-DC derived from IPS-1 ^{− / −.} (FIG. 4C).

From these results, it was shown that PtdIns (5) P generated by PIKfyve activates TBK1-IRF3, which is a signal molecule downstream of IPS-1, as a target.
Since C8-PtdIns (5) P induces the production of cytokines of GM-DC, it is strongly expected that C8-PtdIns (5) P will act as an adjuvant in the living body as well as MPLA and poly I: C. It was.

Therefore in C57 / BL6 mice, ovalbumin (OVA) and PBS, C8-PtdIns (5) P, or C8-PtdIns (4,5) or immunized intramuscularly with a _{P 2,} or the positive control Alam Was immunized by intraperitoneal injection with OVA in combination with OVA-specific total IgG in serum after immunization.

OVA and C8-PtdIns (5) mice immunized with a P, produced OVA and PBS or C8-PtdIns (4,5) OVA-specific IgG of high titers than mice immunized with a _{P 2} (FIG. 4D).

Next, in order to investigate what mechanism of action the adjuvant ability is exerted, it was examined whether C8-PtdIns (5) P induces T cell activation. Transgenic CD4 ⁺ T cells expressing OVA-specific T cell receptors were treated with C8-PtdIns (5) P, C8-PtdIns (4,5) P ₂ , or poly I: C in the presence of OVA protein. Co-cultured with GM-DC, and the amount of IFNγ produced was measured as an index of T cell activation (FIG. 4E). Treatment with C8-PtdIns (5) P induced IFNγ production, but treatment with C8-PtdIns (4,5) P ₂ did not.

  Therefore, these findings revealed that C8-PtdIns (5) P exerts adjuvant ability by promoting Th2 cell response.

Various phospholipids used in Example 4 are those containing a fatty acid having 8 carbon atoms, the specific origin, except for PI (3,4,5) _{P 3,} those of Echelon Biosciences Inc. follows catalog number It is.

PI: P-0008a,
PI (3) P: P-3008a,
PI (4) P: P-4008,
PI (5) P: P-5008a,
PI (3,4) P ₂ : catalog number P-3408a,
PI (3,5) _{P 2:} catalog number P-3508,
PI (4, 5) P ₂ : Catalog number P-4508a,
PI (3,4,5) _{P 3} are those Avanti Polar Lipid's catalog number is 850176P.

  To summarize the experimental results in Examples 1 to 4 above, PtdIns (5) P activates an immune activation signal in the same manner as poly I: C, but TBK1-IRF3 is involved in poly I: C. Since it specifically activates only the signal cascade, it indicates that PtdIns (5) P exerts sufficient adjuvant ability while reducing the side effects induced by poly I: C and the like. .

Furthermore, C8-PtdIns (5) but increased levels of IgG ₁ after immunization with P was _observed, for another subclass of IgG _2, IgG 3, IgA, or IgE, etc. were observed. When IgE is produced in a significant amount, there is a high possibility of inducing inflammation, and the immune effect is exerted by IgG ₁ or the like, so C8-PtdIns (5) has side effects such as inflammation. It was revealed that it has an adjuvant ability that does not cause it.

  In addition, the reduction of side effects was supported by the fact that C8-PtdIns (5) P acts on wild-type GM-DC and does not cause the production of IL-1β known as an inflammatory cytokine (FIG. 5). ).

  From the above, it is suggested that C8-PtdIns (5) P can be a safe and useful adjuvant with no side effects due to a specific mechanism of action while having sufficient adjuvant ability.

Claims (5)

Compound represented by the following formula (2)
An adjuvant . A vaccine comprising the adjuvant of claim 1 and an antigen. The antigen is from the group consisting of bacteria, viruses, fungi, parasitic protozoa, parasitic helminths, cancer cells, cancer cell specific proteins, cancer cell lysates, prions, nucleic acids, renin, angiotensin, and angiotensin receptors The vaccine according to claim 2 , which is at least one selected. A method for screening an adjuvant candidate substance comprising the following steps 1 and 2;
(1) A step 1 for bringing an intracellular component into contact with IRF3 (Interferon regulatory factor 3), and (2) a step 2 for selecting the intracellular component that phosphorylates the IRF3. A method of screening an adjuvant candidate substance comprising the following steps 1 ′ and 2 ′;
(I) a step 1 ′ of bringing an intracellular component into contact with IRF3 (Interferon regulatory factor 3) and TBK1 (TANK binding kinase 1); and (II) binding the TBK1 and phosphorylating the IRF3. Step 2 ′ for selecting ingredients.