Particulate powder type mucosal vaccine

Abstract

When this vaccine is administered to the nasal cavity, etc., a vaccine component stays in the nasal cavity or the pharynx, providing a means for heightening the effects of vaccine administration such as induction of immunity. It was confirmed that an antibody-production-inducing effect is greatly enhanced by a synergistic effect achieved by adding a synthetic peptide, a mixed lipid, β-1,3-glucan or inulin, and an amino acid to an antigen protein.

Description

Technical Field

[0001] The present invention relates to a particulate powder-type mucosal vaccine, and more particularly to a particulate powder-type mucosal vaccine including an antigen protein, a synthetic peptide consisting of the amino acid sequence of KnLm, a mixed lipid, β-1,3-glucan or inulin, and an amino acid, and a method for producing the same.

Background Art

[0002] Currently, subcutaneous and intramuscular injection-type vaccines widely used in the world are excellent in inducing anti-pathogen IgG antibodies in the blood and show an effect in preventing the aggravation of infection. However, since there is almost no inductive effect on anti-pathogen specific secretory IgA (s-IgA) antibodies, which play a role in defending against infections in the whole body mucosa where pathogens invade, the infection prevention effect is not expected (see, for example, Patent Documents 1 and 2). In the case of influenza virus, coronavirus, etc., which are considered to have strong infectivity, it is necessary to enhance the infection prevention effect. Regarding COVID-19, which is an urgent issue, the main sites of entry for droplet infection and contact infection are the nasal cavity and oral cavity. ACE2, which is the so-called receptor for the new coronavirus, is abundantly present in the salivary glands, and there are many minor salivary glands in the nasal mucosa and oral mucosa. Therefore, administering an effective vaccine to the nasal cavity and pharynx is also one of the infection prevention measures.

[0003] The inventors have hitherto developed an artificial synthetic lung surfactant that imparts a function of transporting an antigen to antigen-presenting cells based on the composition of lung surfactant that covers the mucosal surface of the lung and trachea and acts as a surfactant. For example, as an artificial synthetic lung surfactant, a synthetic peptide consisting of the amino acid sequence of KnLm is used, and a carboxyvinyl polymer (CVP) as a lipid and a thickener is added, and a trans-mucosal administration-type influenza vaccine that is safe and effective has been reported (see, for example, Patent Documents 3 and 4).

[0004] Such transmucosal vaccines have been confirmed to exhibit antigen-specific IgG induction in serum, antigen-specific secretory IgA induction in the respiratory tract mucosa (nasal lavage fluid) (see, for example, Patent Document 4 and Non-Patent Document 1), and cellular immunity induction (see, for example, Non-Patent Document 2).

[0005] However, for example, the nasal mucosa has cilia and is covered with a mucus layer full of mucus. Therefore, when conventional liquid transmucosal vaccines are administered to the nasal mucosa, even if they adhere to the mucus, they are quickly expelled into the throat by a strong expulsive action along with tiny dust particles and bacteria from the air that enter the nasal cavity. As a result, practical application has been largely impossible. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Open Brochure WO2005 / 097182 [Patent Document 2] International Open Brochure WO2007 / 018152 [Patent Document 3] International Open Brochure WO2009 / 123119 [Patent Document 4] International Open Brochure WO2011 / 108521 [Non-patent literature]

[0007] [Non-Patent Document 1] Vaccine 2019;37: 612-22. [Non-Patent Document 2] PLoS One 2018; 13:e0191133. [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a means for enhancing the effects of vaccine administration, such as immune induction, by ensuring that vaccine components remain in the nasal cavity or pharynx when the vaccine is administered to the nasal cavity or the like. [Means for solving the problem]

[0009] The inventors decided to re-examine the dosage forms used for (trans)mucosal vaccines. As a result, they conceived the idea of ​​creating a microparticle powder type mucosal vaccine as an alternative to the conventional liquid mucosal vaccine. First, they attempted to create a powder vaccine by freeze-drying a conventional liquid vaccine containing an antigen protein, a synthetic peptide consisting of the amino acid sequence of KnLm, mixed lipids, and CVP. However, they were unable to form microparticles, and instead formed clumps or string-like compositions, which could not be administered into the nasal cavity or dispersed. Therefore, they removed CVP, a polymer that is highly viscous and is thought to be the cause of string formation during freeze-drying, from the conventional liquid vaccine, and attempted to administer a dried powder formulation, which was freeze-dried to produce a composition (AD Vehicle) containing the antigen protein, a synthetic peptide consisting of the amino acid sequence of KnLm, and mixed lipids, into the nasal cavity of mice. However, this composition alone was inefficient in dispersing with the airflow into the nasal cavity and did not reach the mucous membrane.

[0010] Therefore, we added glycine, a neutral amino acid with a small molecular weight and known to be commonly used as an excipient in dry powder formulations for its dispersing and scattering properties, to the above-mentioned antigen protein and AD vehicle complex as a dispersant. When this composition was freeze-dried and powdered, it was administered to the nasal cavity of mice, and it was able to reach the nasal mucosa and spread appropriately throughout the nasal cavity. When the concentrations of s-IgA in the nasal lavage solution and IgG in the serum of the administered mice were measured, they were higher than expected, suggesting that the addition of glycine may enhance the antibody production induction effect. Therefore, we decided to conduct further investigations.

[0011] First, various polysaccharides, which have been conventionally used as excipients and bulking agents in dried powder formulations, were added to the above-mentioned antigen protein and AD vehicle complex along with glycine, and then the composition was freeze-dried and powdered, which was then administered into the nasal cavity of mice.

[0012] When cellulose, a glucose polymer classified as β-1,4-glucan, and sodium alginate, a uronic acid polymer, were selected as polysaccharides, no effect in inducing antibody production was observed in either the mucous membrane or the serum. However, when a glucose polymer containing β-1,3-glucan such as curdlan, or inulin, was added to the above-mentioned antigen protein and AD Vehicle complex together with glycine, and the resulting powdered composition was administered to the nasal cavity of mice, it was found that the levels of s-IgA in the nasal lavage fluid and IgG in the serum increased. Therefore, by continuing to measure the levels of s-IgA and IgG with various combinations of AD Vehicle, various β-1,3-glucans or inulin, and various amino acids, it was confirmed that the antibody production induction effect was significantly enhanced by a synergistic effect when synthetic peptides, mixed lipids, β-1,3-glucans or inulin, and amino acids were added to the antigen protein, thus completing the present invention.

[0013] In other words, the present invention is defined by the following: [1] A microparticle powder type mucosal vaccine comprising an antigen protein and an adjuvant containing the following (A) to (D). (A) Synthetic peptides consisting of the amino acid sequence of (A) KnLm (where n is an integer between 4 and 8, and m is an integer between 11 and 20); (B) Mixed lipids; (C)β-1,3-glucan or inulin; (D) Amino acids; [2] The microparticle powder type mucosal vaccine according to [1] above, characterized in that the synthetic peptide consisting of the amino acid sequence of KnLm (where n is an integer between 4 and 8, and m is an integer between 11 and 20) consists of the amino acid sequence shown in SEQ ID NO: 1 or 2. [3] The fine particle powder type mucosal vaccine according to [1] or [2] above, characterized in that the mixed lipids contain one or more phospholipids. [4] The microparticle powder type mucosal vaccine described in [3] above, characterized in that one or more phospholipids are selected from dipalmitoylphosphatidylcholine, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. [5] A fine particle powder type mucosal vaccine according to any one of [1] to [4] above, characterized in that the mixed lipid contains one or more selected from lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. [6] A microparticle powder type mucosal vaccine according to any one of [1] to [5] above, characterized in that the antigen protein is a protein derived from a pathogen, an inactivated antigen protein, a recombinant antigen protein, or a detoxified toxin protein. [7] A microparticle powder type mucosal vaccine according to any of [1] to [6] above, characterized in that β-1,3-glucan is one or more selected from curdlan, laminaran, zymosan, and yeast β-glucan. [8] A microparticle powder type mucosal vaccine according to any one of [1] to [6] above, characterized in that β-1,3-glucan is curdlan. [9] A microparticle powder type mucosal vaccine according to any one of the above [1] to [8], characterized in that the amino acid is one or more amino acids selected from glycine, alanine, serine, threonine, leucine, isoleucine, phenylalanine, and methionine.

[10] A microparticle powder type mucosal vaccine according to any one of [1] to [9] above, characterized in that it is used to enhance the production of antigen-specific s-IgA in the mucosa or to enhance the production of antigen-specific IgG in the blood. The particulate powder dosage form mucosal vaccine according to any one of [1] to

[10] above, which is an inhaled or sprayable uniform particulate powder that exhibits an infection prevention effect against airway infections via the nasopharynx.

[0014] In addition, the present invention is specified by the following matters.

[12] (1) Antigen protein; (2) A synthetic peptide consisting of the amino acid sequence of KnLm (where n represents an integer of 4 - 8 and m represents an integer of 11 - 20); (3) Mixed lipid; (4) β-1,3-glucan or inulin; (5) Amino acid; A method for producing a freeze-dried particulate powder dosage form mucosal vaccine containing the following, characterized by comprising the following steps (a) to (e). The production method as described above, characterized by comprising the following steps (a) to (e). (a) A step of preparing an AD-vehicle suspension by suspending the synthetic peptide and the mixed lipid in water; (b) A step of adding an antigen protein to the AD-vehicle suspension and performing heating and stirring one or more times to form an antigen protein-AD vehicle complex; (c) A step of preparing a glucan-added antigen protein-AD vehicle complex by adding β-1,3-glucan or inulin to the antigen protein-AD vehicle complex and then stirring; (d) A step of preparing a vaccine solution by adding an amino acid to the glucan-added antigen protein-AD vehicle complex and then stirring; (e) A step of freeze-drying the vaccine solution to form a particulate powder dosage form;

[13] The method for producing a particulate powder dosage form mucosal vaccine according to

[12] above, characterized in that the mixed lipid is a three-lipid mixed lipid of dipalmitoyl phosphatidylcholine, phosphatidyl glycerol, and palmitic acid.

[14] The method for producing a particulate powder dosage form mucosal vaccine according to

[12] or

[13] above, characterized in that in step (b) of forming the antigen protein-AD vehicle complex, the temperature setting range for heating is 25 - 75°C.

[15] A method for producing a microparticle powder type mucosal vaccine according to any of

[12] to

[14] above, comprising an amount of antigen protein that, on its own, does not produce an effective immune induction and protective effect against infection, and being used to produce an effective immune induction and protective effect by combining it with a synthetic peptide, a mixed lipid, β-1,3-glucan or inulin, and an amino acid.

[0015] When the vaccine of the present invention is administered, for example, to the nasal cavity, the production of antigen-specific s-IgA antibodies in mucous membranes throughout the body, including the nasal cavity mucosa, and antigen-specific IgG antibodies in the serum are significantly increased, resulting in excellent infection protection and antibody production induction effects against infectious diseases. [Brief explanation of the drawing]

[0016] [Figure 1] This figure shows the results of a study on adding curdlan to a microparticle powder type mucosal vaccine. [Figure 2] This graph shows the results of particle size measurements of a fine-particle powder-type mucosal vaccine using a scanning electron microscope. [Figure 3] These are images of the nasal cavity, pharynx, trachea, bronchi, and lungs of test mice 20 minutes after administration of a fine particle powder formulation composition via nasal orifice. [Modes for carrying out the invention]

[0017] The vaccine of the present invention is not particularly limited as long as it is a fine particle powder type mucosal vaccine comprising an antigen protein; and an adjuvant comprising (A) a synthetic peptide consisting of the amino acid sequence of KnLm (where n is 4-8 and m is 11-20), (B) a mixed lipid, (C) β-1,3-glucan, and (D) an amino acid. However, in the present invention, the adjuvant refers to a combination of components that, when administered together with the antigen protein (peptide), induce the production of antibodies against the antigen and increase the production of various antibodies, and / or synergistically increase the production of antibodies, particularly when the antigen protein alone contains an amount that does not effectively produce antigen-specific s-IgA in the mucosa and does not produce antigen-specific IgG in the blood.

[0018] The synthetic peptide consisting of the above KnLm amino acid sequence is not particularly limited as long as it is an amino acid sequence consisting of n K (lysine: Lys(K)) residues at the N-terminus and m L (leucine: Leu(L)) residues at the C-terminus, where n is 4-8 and m is 11-20 amino acid sequences. Specifically, the following peptides can be given as examples. Here, amino acid residues are indicated by single-letter symbols. Sequence ID 1:(K6L16):KKKKKKLLLLLLLLLLLLLLLL Sequence ID 2:(K6L11):KKKKKKLLLLLLLLLLL

[0019] The above-mentioned synthetic peptide can be prepared according to known chemical synthesis methods, and it is preferable to use one with a purity of 95% or higher.

[0020] The above-mentioned synthetic peptide is preferably dissolved in an organic solvent such as methanol, ethanol, or trifluoroacetic acid that does not affect the synthetic peptide structure, and added to the vaccine as a K6L16 peptide-containing solution. For example, it can be used as a K6L16 peptide-containing solution dissolved in methanol to a concentration of 3 mg to 7 mg / mL.

[0021] The lipids included in the above mixed lipids include lipids containing two or more types of lipids. The two or more types of lipids include combinations of one or more phospholipids and one or more non-phospholipid lipids, combinations of two or more phospholipids, and combinations of two or more non-phospholipid lipids. A combination of one or more phospholipids and one or more non-phospholipid lipids is preferred, and a combination of two phospholipids and one non-phospholipid lipid is more preferred.

[0022] The phospholipids mentioned above are preferably those contained in natural lung surfactants derived from mammals. Specifically, examples include phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin. These phospholipids can be used individually or as a mixture of two or more. Among the two or more phospholipids, a combination of dipalmitoylphosphatidylcholine and phosphatidylglycerol is particularly preferred.

[0023] When the phospholipids contained in the above mixed lipid are dipalmitoylphosphatidylcholine and phosphatidylglycerol, examples of the mixing ratios for dipalmitoylphosphatidylcholine:phosphatidylglycerol include 10:1 to 1:10, 5:1 to 1:5, 4:1 to 1:1, and 3.5:1 to 2.5:1.

[0024] Lipids other than the phospholipids mentioned above include lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, but palmitic acid is preferred.

[0025] When the above mixed lipids contain phospholipids and non-phospholipid lipids, the ratio of phospholipid content to non-phospholipid content (mass) can be 100:1 to 30, preferably 100:5 to 15, more preferably 100:8 to 12, even more preferably 100:9 to 11, and particularly preferably 100:9.5 to 10.5.

[0026] In the present invention, a composition comprising the above-mentioned mixed lipids and a synthetic peptide consisting of the amino acid sequence of KnLm, or a composition containing the above-mentioned mixed lipids and a synthetic peptide consisting of the amino acid sequence of KnLm, may be referred to as "AD Vehicle". The mass ratio of mixed lipids to synthetic peptides in AD Vehicle, i.e., mixed lipids:synthetic peptide, can be 100:0.1 to 50, preferably 100:0.5 to 10, more preferably 100:1 to 5, and even more preferably 100:1.5 to 3.

[0027] The above-mentioned antigen proteins are not particularly limited as long as they are proteins that can be used as vaccine antigens. Examples include those that can induce immunity (antibodies) against the antigen protein in the body, thereby preventing or curing diseases caused by pathogens from which the antigen protein originates. Examples include proteins derived from common pathogens, as well as inactivated antigen proteins, purified antigen proteins, partially purified antigen proteins, recombinant antigen proteins, detoxified toxin proteins, allergens, etc. Furthermore, the term "antigen proteins" can include not only whole (mature) proteins, but also preproteins, preproproteins, and functional or immunodominant antigen peptides thereof.

[0028] Examples of the above-mentioned pathogens include viruses, bacteria, and parasites.

[0029] Examples of the viruses mentioned above include varicella virus, measles virus, poliovirus, rotavirus, influenza virus, herpesvirus, severe acute respiratory infection syndrome (SARS) virus, coronavirus (COVID-19), Ebola virus, West Nile virus, hantavirus, dengue virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and HIV virus.

[0030] Examples of bacteria include Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Neisseria pneumoniae, and Vibrio cholerae. Examples of fungi include Trichophyton, Candida, and Aspergillus. Examples of parasites include malaria pathogens and sleeping sickness pathogens.

[0031] When the above-mentioned pathogen is the influenza virus, specific examples of antigenic proteins include antigenic glycoproteins present on the surface of the virus, such as hemagglutinin (HA) antigenic protein, neuraminidase antigenic protein, and M protein, as well as nucleoproteins inside the virus.

[0032] In the vaccine of the present invention, the content (mass) of the antigen protein can be expressed, taking the case where hemagglutinin is used as the antigen protein as an example, as the HA content measured quantitatively by the Single radial immunodiffusion (SRD), Fahey, J. et al., J.Immunol., 94, 84-90 (1965) method. Alternatively, after the HA content has been measured once by the SRD method, as long as the same HA stock solution is used, the expression can be expressed as the total amount of protein, which is easier to measure than SRD quantification, representing the total amount of hemagglutinin antigen protein and other antigen proteins. In the latter case, the mass of HA antigen protein in an HA antigen protein solution using hemagglutinin (HA) of the influenza virus can also mean the total mass of protein including HA antigen protein and antigenic proteins other than HA antigen protein, and in that case, in the present invention, it is expressed as the mass (HA antigen protein amount) or concentration of HA antigen protein. Other antigenic proteins besides the HA antigen protein include M protein, neuramylase, and nucleoprotein.

[0033] The HA antigen protein in the vaccine of the present invention is not particularly limited as long as it achieves the effects of the present invention. However, in terms of being an amount that does not enhance the immune response to the extent of inducing antibody production, the amount of HA antigen protein (A) can be exemplified as 0.01 μg to 3 μg / 20g mouse body weight, preferably 0.05 μg to 2 μg / 20g mouse body weight, and more preferably 1.2 μg to 1.8 μg / 20g mouse body weight. Since young mature mice are often used in typical immune response tests, mice weighing approximately 20g at 7-8 weeks of age are used, and therefore, the amount is expressed here as 20g mouse body weight.

[0034] Furthermore, the mass of the HA antigen molecule itself in the above-mentioned HA antigen protein can range from 5 to 90% by mass, preferably 10 to 80% by mass, preferably 20 to 70% by mass, and preferably 30 to 60% by mass.

[0035] The method for producing the above-mentioned antigen protein (antigen peptide) is not particularly limited as long as it is a known method, but a method that can secure a sufficient amount of antigen protein for vaccine production is preferred, and examples include methods produced by genetic engineering or chemical synthesis.

[0036] The mass ratio of the above mixed lipids to the antigen protein is not particularly limited as long as the effects of the present invention can be achieved, but the amount of mixed lipids can be 0.1 to 20 times that of the antigen protein, preferably 1 to 18 times, more preferably 5 to 15 times, even more preferably 8 to 12 times, and even more preferably 9 to 11 times.

[0037] The β-1,3-glucan used in this invention is not particularly limited as long as it is a polysaccharide whose main constituent sugar is glucose, including a glucose polymer linked by β-1,3-glycosidic bonds, and which can perform the role described above in this invention. Examples include curdlan, a water-insoluble polysaccharide having a linear β-1,3-glucan structure; laminaran, a glucose polymer known as a storage polysaccharide found in seaweed and mushrooms, which consists of a glucose main chain with β-1,3 and β-1,6 bonds, with a ratio of β-1,3 to β-1,6 bonds of approximately 3:1, and is soluble in water; zymosan, a suspension of polysaccharides derived from the cell wall of budding yeast such as yeast, which contains mannan and has β-1,3-glucan as its main component polysaccharide as an active ingredient; and yeast β-glucan.

[0038] Inulin, as mentioned above, is a type of dietary fiber that has a structure in which approximately 2 to 60 fructose molecules are linked to one glucose molecule.

[0039] The amount of β-1,3-glucan or inulin added to the vaccine of the present invention is not particularly limited as long as it is an amount that can effectively exert its adjuvant effect. Examples include an amount 5 to 50 times the mass of the antigen protein-AD vehicle complex, preferably 10 to 30 times, or an amount 10 to 1000 times the mass of the antigen protein, preferably 25 to 500 times, more preferably 50 to 200 times, and even more preferably 75 to 150 times.

[0040] The amino acids used in the vaccine of the present invention are not particularly limited as long as they can be used as excipients to facilitate the delivery of the administered fine particle powder type mucosal vaccine of the present invention to mucous membranes such as the nasal cavity via airflow, and can enhance antibody production induction effects such as increasing s-IgA production in mucous membranes and significantly increasing IgG production in serum, without reducing the effect of inducing protective antibodies against infection. However, due to their availability, 20 amino acids that are major building blocks of proteins in living organisms are used. The α-amino acids are preferred, including neutral amino acids with nonpolar side chains such as glycine (Gly), alanine (Ala), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), proline (Pro), tryptophan (Trp), valine (Val), and methionine (Met); neutral amino acids with polar neutral side chains such as asparagine (Asn), glutamine (Gln), serine (Ser), threonine (Thr), and tyrosine (Tyr); and acidic amino acids such as aspartic acid (Asp) and glutamic acid (Glu). Acidic amino acids having a neutral side chain; basic amino acids having a basic side chain such as arginine (Arg), lysine (Lys), histidine (His) can be given as examples, but one or more amino acids selected from glycine, alanine, serine, threonine, leucine, isoleucine, phenylalanine, and methionine are preferred, one or more amino acids selected from glycine, alanine, serine, threonine, leucine, isoleucine, and phenylalanine are more preferred, one or more amino acids selected from glycine, alanine, leucine, and phenylalanine are even more preferred, glycine or phenylalanine are even more preferred, one or more amino acids selected from glycine, serine, leucine, and phenylalanine are even more preferred, one or more amino acids selected from glycine, serine, leucine, and phenylalanine are even more preferred, glycine, alanine, leucine, and / or phenylalanine are preferred, glycine or phenylalanine are most preferred, and phenylalanine is particularly preferred. Alternatively, it may be desirable to remove cysteine, and in some cases, it may be desirable to remove amino acids that have an SH group in their side chain, such as methionine or cysteine.

[0041] The amount of amino acids added to the vaccine of the present invention is not particularly limited as long as the effects of the present invention are achieved as described above, but examples include an amount 15 to 35 times the mass of the antigen protein-AD vehicle complex, preferably 25 to 30 times, or an amount 10 to 3000 times the mass of the antigen protein, preferably 100 to 1500 times, more preferably 150 to 750 times, and even more preferably 200 to 400 times.

[0042] The number of doses of the vaccine of the present invention is not particularly limited as long as the vaccine of the present invention is effective, but can be one dose (primary immunization only) or two or more doses, preferably two doses (primary immunization and secondary immunization) or three doses (primary immunization, secondary immunization, and tertiary immunization), preferably the second dose is administered 1 week to 1 month after the first dose, preferably 10 days to 3 weeks after, and more preferably 2 weeks after, and the third dose is also preferably 1 week to 1 month after the second dose, more preferably 10 days to 3 weeks after, and even more preferably 2 weeks after.

[0043] The present invention's microparticle powder-type mucosal vaccine contains an amount of antigen protein that does not produce effective immune induction and infection protection effects. It is used to produce effective immune induction and infection protection effects by combining synthetic peptides, mixed lipids, β-1,3-glucan or inulin, and amino acids, thereby enhancing the production of antigen-specific s-IgA in the mucosa or enhancing the production of antigen-specific IgG in the blood. It can also be said to be a use invention with limited applications.

[0044] In the present invention, examples of cases in which the antibody production induction effect is significantly increased include, for example, a 4-fold or greater increase in s-IgA production in mouse nasal lavage fluid compared to the administration of antigen protein, AD vehicle, and amino acids, preferably a 5-fold or greater increase, more preferably a 10-fold or greater increase, and / or a 1.5-fold or greater increase in IgG production in serum, preferably a 2.5-fold or greater increase, more preferably a 3-fold or greater increase, more preferably a 5-fold or greater increase, and even more preferably a 10-fold or greater increase.

[0045] Alternatively, in the vaccine of the present invention, the amount of antibody induced that produces an effective immune induction can be exemplified by a value of the viral infection inhibitory effect HI that is equal to or greater than the international evaluation standard for influenza vaccines (HI ≥ 40).

[0046] The method for producing the microparticle powder type mucosal vaccine of the present invention is as follows: (1) Antigen protein; (2) Synthetic peptides consisting of the amino acid sequence KnLm (where n is 4-8 and m is 11-20); (3) mixed lipids; (4) Amino acids; (5) β-1,3-glucan or inulin; A method for producing a lyophilized particulate powder type mucosal vaccine containing, (a) A step of preparing an AD-vehicle suspension by suspending the synthetic peptide and the mixed lipids in water; (b) A step of adding an antigen protein to the AD-vehicle suspension and repeating heating and stirring one or more times to form an antigen protein-AD-vehicle complex; (c) A step to prepare a homogeneous glucan-added antigen protein-AD vehicle complex by adding β-1,3-glucan to the antigen protein-AD vehicle complex and stirring it; (d) A step of adding amino acids to the glucan-added antigen protein-AD vehicle complex and stirring to prepare a homogeneous vaccine crude solution; An example of a method for producing a microparticle powder type mucosal vaccine is that which includes the step of (e) preparing a microparticle powder type mucosal vaccine by freeze-drying the crude vaccine solution to form a microparticle powder dosage form.

[0047] Furthermore, after step (b) above, (b') A step to prepare a freeze-dried antigen protein-AD vehicle by freeze-drying the antigen protein-AD vehicle complex prepared in step (b); And, before step (c) above, The process may also include a step of suspending the lyophilized antigen protein-AD vehicle prepared in step (b)(b') in water or physiological saline to prepare an antigen protein-AD vehicle complex (homogeneous suspension);

[0048] The AD-vehicle suspension prepared in step (a) above is not particularly limited as long as it is a liquid prepared by suspending a synthetic peptide consisting of an amino acid sequence of KnLm (where n is 4-8 and m is 11-20) and a mixed lipid in water. The mass ratio of the mixed lipid to the synthetic peptide in the AD-vehicle suspension, i.e., mixed lipid:synthetic peptide, can be 100:0.1 to 50, preferably 100:0.5 to 10, more preferably 100:1 to 5, and even more preferably 100:1.5 to 3. The above mixed lipids and synthetic peptides are conveniently dissolved in an organic solvent such as chloroform or methanol (mixed lipids:organic solvent = 100:1-50, preferably 100:2-25, more preferably 100:5-15, even more preferably 100:8-12), and then suspended in water after removing the organic solvent by vacuum drying or freeze-drying. In this case, an AD-vehicle suspension is obtained containing the synthetic peptide consisting of the amino acid sequence KnLm and the lipid solution.

[0049] The antigen protein-AD vehicle complex prepared in step (b) above can be a complex formed by hydrophobic interactions, etc., by heating and stirring the AD vehicle suspension prepared in step (a) above one or more times. The stirring can be done by adding the antigen protein in a water bath and shaking and mixing for 5 to 20 minutes, preferably 8 to 12 minutes. The heating can generally be done in a water bath at 20 to 60°C, preferably 40 to 50°C, but it is desirable to select the heating temperature considering the thermal stability of the antigen protein. In the case of influenza vaccine antigen, 35 to 43°C is preferred, and 41 to 42°C is more preferred.

[0050] The glucan (or inulin)-added antigen protein-AD vehicle complex prepared in step (c) above can be a homogeneous glucan-added antigen protein-AD vehicle complex obtained by adding β-1,3-glucan to the antigen protein-AD vehicle complex and then stirring. It is preferable to add β-1,3-glucan or inulin after the antigen protein-AD vehicle complex has been formed. If β-1,3-glucan or inulin is added before the antigen protein-AD vehicle complex is formed, the adjuvant effect of the AD vehicle may be lost. To prepare a homogeneous glucan-added antigen protein-AD vehicle complex, it is preferable to use a homogenizer, mixer, shaker, stirrer, etc.

[0051] The vaccine crude solution prepared in step (d) above is not particularly limited as long as it is a homogeneous vaccine crude solution obtained by adding one or more amino acids to the glucan-added antigen protein-AD vehicle complex and then stirring, and it is preferable to add the amino acids after the antigen protein-AD vehicle complex has been formed by the antigen protein and the AD vehicle. If amino acids are added before the formation of the antigen protein-AD vehicle complex, the adjuvant effect of the AD vehicle may be lost. To prepare a homogeneous glucan-added antigen protein-AD vehicle complex, it is preferable to use a homogenizer, mixer, shaker, stirrer, etc. Therefore, it is also possible to prepare the vaccine crude solution by performing step (d) after step (b) above, and then performing step (c) after step (d).

[0052] The microparticle powder type mucosal vaccine prepared in step (e) above is not particularly limited as long as it is a vaccine obtained by freeze-drying the crude vaccine solution to form a microparticle powder. Freeze-drying can be carried out using a known freeze-dryer, and the method is not particularly limited as long as it can freeze-dry the crude vaccine solution to form a microparticle powder. For example, it is preferable to freeze the crude vaccine solution at -50 to -80°C for 5 hours or more in a pre-freezing process, then insert the frozen sample into a freeze-dryer and freeze-dry it for 10 hours or more under the conditions of a vacuum of 4.0 to 6.0 Pa, a trap temperature of -47 to -50°C, and an ambient temperature of room temperature to form a microparticle powder. Freeze-drying can be carried out using an apparatus capable of freeze-drying under the above conditions.

[0053] The antigen protein-AD vehicle freeze-dried product prepared in step (b') above is not particularly limited as long as it is a solid obtained by freeze-drying the antigen protein-AD vehicle complex. The pre-freezing process temperature for freeze-drying can be -80°C to -50°C, and such freeze-dried product is preferably stored at -20°C to -30°C.

[0054] Furthermore, as a homogeneous suspension of the antigen protein-AD vehicle complex in step (b) above, an example of such a suspension can be obtained by suspending the freeze-dried antigen protein-AD vehicle prepared in step (b') in water or physiological saline and adjusting it to a predetermined concentration suitable for vaccine formulation.

[0055] Whether or not the above-mentioned antigen protein-AD vehicle complex is formed can be confirmed, for example, by detecting the hydrophobic interaction signal between the antigen protein and the AD vehicle molecule using a calorimeter. A calorimeter can measure the heat of reaction generated when the antigen protein and the AD vehicle interact at a constant temperature, and by measuring this heat of reaction, the intermolecular interaction can be confirmed. Furthermore, the quantitative measurement of the amount of antigen protein-AD vehicle complex formed can be calculated from the amount of antigen protein recovered in the AD vehicle fraction that precipitates by centrifugation, based on the amount of antigen protein in the supernatant before and after centrifugation. In the present invention, it is preferable to determine that a sample in which the complex has been formed is used after confirming that 70% or more of the antigen protein is recovered in the AD vehicle fraction. Below is an example of a method for calculating the binding rate by centrifugation to calculate the proportion in which the above-mentioned antigen protein-AD vehicle complex is formed (binding rate calculation method).

[0056] As an example of the above method for calculating the binding ratio, HA (Solution A) and [HA + AD Vehicle] (Solution B) are prepared, each is shaken and mixed, each solution is centrifuged, and the supernatant samples after centrifugation are separated and designated as the centrifuged samples (Solution C) and (Solution D). The protein concentration of each sample is measured using a BCA protein assay kit, and the amount of bound HA protein that co-precipitates with the AD Vehicle, which is recovered in the precipitate fraction after centrifugation, can be measured to calculate the binding ratio between HA and the AD Vehicle that form the complex. The formula for calculating the binding ratio is shown below.

[0057]

number

[0058] While known methods can be used for administering the above vaccine, for example, when administering it into the nasal cavity of a test animal such as a mouse, the required amount of crude vaccine obtained in step (d) can be filled into an Eppendorf tube with its tip closed, and step (e) can be performed to obtain an "Eppendorf tube filled with crude vaccine microparticle powder formulation." One example of this method is to open the tip of the Eppendorf tube before administration, press it against the entrance of the test animal's nose, and forcefully push the Eppendorf tube with your fingers to spray the microparticle powder into the nasal cavity using the airflow. For administration to humans, methods using known means or unit-dose spraying devices suitable for intranasal administration of a dry powder formulation for nasal spraying can be mentioned.

[0059] Furthermore, administration via the nasal cavity can also be performed by nasal inhalation, tracheal or bronchial inhalation, and pulmonary inhalation. In these cases, the target destination can be determined by the preferred particle size of the microparticles. When the nasal cavity is the main destination, the particle size of the microparticle powder is preferably 5.0 μm to 80 μm, more preferably 6.5 μm to 50 μm, and even more preferably 6.5 μm to 40 μm. When tracheal or bronchial inhalation is the main destination, the particle size of the microparticle powder is preferably 3.0 μm to 5.0 μm, and when the alveoli are the main destination, the particle size of the microparticle powder is preferably 0.5 μm to 3 μm. Microparticles smaller than 0.5 μm may be easily expelled from the body by exhalation.

[0060] In the present invention, examples of mucous membranes include the mucous membrane lining the nasal cavity; tracheal mucosa; bronchial mucosa; alveolar mucosa; as well as mucous membranes located throughout the body, such as the vaginal mucosa, small intestinal mucosa, and large intestinal mucosa. The presence or absence and amount of antigen-specific secretory IgA (s-IgA) antibody production secreted in the mucous membrane can be measured by measuring the amount of s-IgA antibody in the secretions secreted in the mucous membrane. Methods for measuring the amount of antibody in the secretions include measuring the amount of antibody in nasal lavage fluid in the nasal cavity, vaginal lavage fluid in the vaginal mucosa, small intestine lavage fluid in the small intestine, stool in the large intestine, tracheal lavage fluid in the tracheal mucosa, bronchial lavage fluid in the bronchi, and alveolar lavage fluid in the alveoli.

[0061] Antigen-specific s-IgA (secretary immunoglobulin A) antibodies, which are induced to be produced in the mucous membrane after administration of the vaccine of the present invention and are found in large quantities in secretions secreted in the mucous membrane, have an infection-protective effect by binding to pathogens and toxins on the mucosal surface and inactivating their functions. Furthermore, antigen-specific IgG antibodies found in the blood after administration of the vaccine of the present invention are antibodies produced in the splenic lymphoid tissue and released into the bloodstream after antigen information is transmitted and stored in the splenic lymphoid tissue via the mucosal lymphoid tissue of the vaccination site. These antibodies bind to and inactivate pathogens that have entered the bloodstream, thereby suppressing the progression of infection. This antigen information is stored and produced for a relatively long period in the splenic lymphoid tissue, even as it decays, and in the event of reinfection, antibody production is rapidly initiated by the stored antigen information, enabling the capture of the antigen.

[0062] The vaccine of the present invention can be formulated by known means, but pharmacologically acceptable bases and / or additives may be added as appropriate.

[0063] Examples of pharmacologically acceptable bases and / or additives include lubricants, binders, solvents, isotonic agents, buffers, analgesics, and stabilizers. Additional additives such as preservatives, pH adjusters, cooling agents, antioxidants, humectants, and deodorizers may also be included as needed.

[0064] In one embodiment, the vaccine of the present invention is an inhaled or sprayed vaccine in the form of a uniform fine particle powder that exerts an infection-protective effect against respiratory tract infections transmitted from the nasal cavity through the pharynx.

[0065] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. [Examples]

[0066] [Example 1] [Antibody-inducing experimental animals] Using mice, the induction effects of a microparticle powder-type mucosal vaccine on anti-influenza virus-specific s-IgA in nasal lavage fluid and anti-influenza virus-specific IgG antibodies in serum were evaluated. All subsequent animal experiments were conducted at the Infectious Animal House (P2 level) of the Experimental Animal Center, Tokushima University School of Medicine, in accordance with the guidelines of the Animal Experiment Committee, Tokushima University School of Medicine.

[0067] The mice used were BALB / c mice (7-8 weeks old, female, average weight 20g) purchased from Charles River Japan Co., Ltd.

[0068] [Production of microparticle powder vaccines] To verify the effectiveness of influenza virus hemagglutinin (HA) as an antigenic protein, various microparticle powder vaccines were prepared for administration into the nasal cavity of mice.

[0069] (Preparation of mixed lipid solution) A mixed lipid solution was prepared by suspending dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and palmitic acid (PA) in a chloroform:methanol (2:1 (v / v)) mixture in a ratio of 75:25:10 (w / w / w) so that the phospholipid consisting of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG) had a final concentration of 10 mg / mL.

[0070] (Preparation of K6L16 peptide-containing solution) A synthetic peptide K6L16 (KKKKKKLLLLLLLLLLLLLLLL) (SEQ ID NO: 1) (manufactured by GenScript), with a purity of 95% or higher, was dissolved in methanol to a concentration of 5.0 mg / mL to prepare a K6L16 peptide-containing solution.

[0071] (Preparation of AD-Vehicles) The above mixed lipid solution and the K6L16 peptide-containing solution were mixed in a ratio (mass ratio) of mixed lipid solution:K6L16 peptide-containing solution = 100:2. 4 mg of this mixed lipid-K6L16 mixture was dried to dryness at approximately 40°C using a rotary evaporator, then resuspended in 1 mL of 10% ethanol and shaken in a water bath at approximately 45°C for about 15 minutes to prepare a homogeneous suspension of the lipid solution-K6L16 mixture (AD-vehicle suspension). This AD-vehicle suspension was freeze-dried using a freeze-dryer and stored as freeze-dried AD-vehicle at -30°C.

[0072] (Preparation of antigen proteins) Hemagglutinin (HA) of the influenza virus was used as the antigen protein. Daiichi Sankyo Co., Ltd. prepared the vaccine antigen solution from a vaccine strain provided by the National Institute of Infectious Diseases, and then prepared the HA antigen protein solution from the A / Singapore / GP1908 / 2015(H1N1) strain, which was provided to Tokushima University on a paid basis.

[0073] (Formation of antigen-protein-AD vehicle complex) The freeze-dried AD-vehicle was returned to room temperature and suspended in ultrapure water to prepare another AD-vehicle suspension. The HA antigen protein solution was added to this AD-vehicle suspension so that the ratio of the amount of HA antigen protein (A) to the mass of phospholipids in the mixed lipids (V), which is V / A, was 10 (i.e., the amount of HA antigen protein (A): the mass of phospholipids in the mixed lipids (V) = 1:10). The suspension was prepared by suspending it in a vortex mixer until homogeneous. This suspension was then shaken and mixed in a 42°C water bath for 10 minutes to bind the antigen protein and AD-vehicle and form an antigen protein-AD-vehicle complex (HA+AD-vehicle). This antigen protein-AD-vehicle complex was freeze-dried and stored at -30°C as a freeze-dried antigen protein-AD-vehicle product. When using, it was thawed and suspended in pure water to be used as an antigen protein-AD-vehicle complex suspension. From this point forward, the ratio of the amount of HA antigen protein (A) in the influenza vaccine to the mass of phospholipids in the mixed lipids (V) was fixed at V / A = 10. The amount of HA antigen molecules themselves in the antigen protein was approximately 36% of the total antigen protein amount in the lot used.

[0074] (Lyophilization) Freeze-drying was performed using a tabletop freeze-dryer (Labuconco FZ-4.5 vacuum freeze-dryer, Asahi Life Science Co., Ltd.). After pre-freezing the vaccine crude solution at -80°C for more than 5 hours, the frozen sample was placed in the freeze-dryer and freeze-dried for more than 10 hours under the conditions of a vacuum of 5.0 Pa, a trap temperature of -50 to -47°C, and an ambient temperature of room temperature.

[0075] (Method of administration to mice) [Administration method for microparticle powder type mucosal vaccine] For administration of the microparticle powdered mucosal vaccine, an Eppendorf tube filled with the microparticle powdered mucosal vaccine was placed in the entrance of one nostril of each test mouse anesthetized with ketalar (62.6 mg / kg mouse body weight) and seractal (12.4 mg / kg mouse body weight), and the vaccine was administered by pushing it out with a finger, allowing the dry powder to be carried by the airflow. Each group consisted of 4 to 6 mice. Immunization was performed by intranasally administering the same amount of a sample with the same composition to each group as a secondary and tertiary immunization two weeks after the initial immunization. Nasal lavage fluid and blood samples were collected two weeks after the final immunization.

[0076] [Preparation of mouse nasal lavage fluid and serum samples] Nasal lavage fluid and serum were prepared from mice two weeks after third immunization, according to the method described in J Immunol. 2006; 176: 1122-1130, and antigen-specific s-IgA and IgG levels were measured. Specifically, each vaccinated mouse was anesthetized with pentobarbital, and the trachea was incised. An atom venous catheter was inserted through the incised trachea towards the nasal cavity, and 1 mL of 0.1% BSA (bovine serum albumin) saline solution was injected. The fluid that came out of the nose was collected and used as the mouse nasal lavage fluid sample. Furthermore, blood was collected from the heart of each mouse, and serum samples were prepared by centrifugation at 5000 rpm for 10 minutes.

[0077] [Method for quantifying antigen-specific antibody titers] Antigen (pathogen)-specific antibody titers (amounts) were measured by quantifying the anti-influenza s-IgA and IgG content in nasal lavage fluid samples and serum samples using an ELISA assay, as described in J Immunol. 2006; 176: 1122-1130. For the ELISA assay, 0.1 μg of HA antigen protein was added to each well of a 96-well immunoplate (Nalgene Nunc International, USA), followed by 1 μg / mL of bovine serum albumin (BSA, SIGMA) and 100 μL of PBS solution. The plate was then solidified overnight at 4°C. The plate was then rinsed three times with a washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0). 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl and 200 μL of 1% BSA was added to each well, and a blocking reaction was performed at room temperature for 1 hour. After rinsing each well three times with washing solution, 100 μL of nasal lavage solution or serum diluted appropriately with sample binding buffer (50 mM Tris, 0.15 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) was added, and the mixture was reacted at room temperature for 2 hours. Goat anti-mouse IgA or IgG-horse radish peroxidase (HRP) (BETHYL LABORATORIES INC.) was used as the secondary antibody, and a color reaction was performed using the TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Inc., USA). The reaction was stopped by adding 100 μL of 2 M H2SO4 (Wako Pure Chemical Industries, Ltd.) to each well, and the absorbance at 450 nm was measured using SPECTRAmaxPLUS 384. As a standard for quantification, a calibration curve of absorbance obtained in the same manner for 10 ng each of anti-influenza IgA and IgG, which were affinity-purified according to the description in J Immunol. 2006; 176: 1122-1130, was used.

[0078] [Example Test] (Antigen protein-AD vehicle complex microparticle powder) When we attempted to administer a freeze-dried powder of the antigen protein-AD vehicle complex into the nasal cavity of mice, it did not reach the mice's nasal cavity. We hypothesized that this was because it lacked a component that acts as a dispersant.

[0079] (Addition of amino acids) To the above antigen protein-AD vehicle complex solution, glycine, which has the smallest molecular weight among amino acids, was added in an amount 300 times the concentration of the HA antigen protein ([HA]), and the mixture was stirred in a homogenizer to prepare a vaccine solution. This solution was then freeze-dried to produce a microparticle powder type mucosal vaccine (HA + ADvehicle + Gly) (Comparative Example 1).

[0080] (Addition of sugars) Since inulin is used as an excipient in some known powder-type vaccines, we investigated whether there are any suitable sugars as dispersants. Cellulose (034-22221, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a polysaccharide in which glucose is linked by β-1,4-glucosidic bonds; sodium alginate (194-13321, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), which has a structure in which two types of uronic acids, mannuronic acid and guluronic acid, are linearly polymerized; inulin (9005-80-5, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.); curdlan (030-09903, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.); laminaran (PS131, manufactured by DEXTRA); zymosan (NBP2-26233, manufactured by NOVUS); and yeast β-glucan (OG30829, manufactured by Carbosynth) were each added to an antigen protein-AD vehicle complex solution to which glyceride was added, and after mixing with a homogenizer, freeze-drying was performed. Specifically, microparticle powder-type mucosal vaccines were prepared, each consisting of glycine-added antigen protein-AD vehicle complex (HA + ADvehicle + Gly) + cellulose (Comparative Example 2), HA + ADvehicle + Gly + sodium alginate (Comparative Example 3), HA + ADvehicle + Gly + inulin (Example 1), HA + ADvehicle + Gly + curdlan (Example 2), HA + ADvehicle + Gly + laminaran (Example 3), HA + ADvehicle + Gly + zymosan (Example 4), and HA + ADvehicle + Gly + yeast β-glucan (Example 5). These vaccines were administered to each test mouse, and the amount of s-IgA in the nasal lavage fluid and IgG in the serum were measured. The amount of each polysaccharide added was 100 times the concentration of the HA antigen protein ([HA]). The results are shown in Table 1.

[0081] [Table 1]

[0082] (result) When glycine was added to the antigen protein (HA) (1.5 μg)-AD vehicle complex (HA + ADvehicle), HA + ADvehicle + Gly (Comparative Example 1) resulted in nearly five times higher s-IgA production in nasal lavage fluid and approximately 1.7 times higher IgG production in serum compared to HA + Gly (Comparative Example 11). However, neither the s-IgA production in nasal lavage fluid nor the IgG production in serum was sufficiently high. When a vaccine containing cellulose was administered to HA + ADvehicle + Gly (Comparative Example 2), the amount of s-IgA production in nasal lavage fluid was lower than when physiological saline (a negative control) was administered (Comparative Example 0), confirming that antibody production induction was suppressed. Furthermore, when sodium alginate was added to HA + ADvehicle + Gly (Comparative Example 3), neither the s-IgA production in nasal lavage fluid nor the IgG production in serum was particularly high.

[0083] Furthermore, in the above study, it was confirmed that more than 70% of the added antigen protein (HA) subsequently formed a complex with the AD vehicle antigen protein (HA) by the following method.

[0084] The binding rate was confirmed by detecting the hydrophobic interaction signal between the antigen protein and the AD vehicle molecule using a calorimeter (Malvern Panalytical). For the calculation of the binding rate, 1 mL solutions each of 0.4 mg HA (protein amount) / mL (ultrapure water) (Solution A) and [0.4 mg HA (protein amount) + 4 mg AD vehicle (phospholipid amount)] / mL (ultrapure water) (Solution B) were prepared and mixed by shaking at 42°C for 10 minutes. 0.05 mL of each mixed solution was taken for protein amount measurement, and these were designated as the (Solution A) and (Solution B) samples before centrifugation. Next, each solution was centrifuged in a centrifuge (TOMY, MX-305) at 20400 × g for 15 minutes at 4°C. 0.05 mL of each supernatant standard was taken after centrifugation, and these were designated as the (Solution C) and (Solution D) samples after centrifugation. The protein concentration of each sample was measured using the BCA protein assay kit (Thermo Scientific, 23227).

[0085] The binding rate was calculated by measuring the amount of bound HA protein co-precipitating with the AD beak recovered in the precipitate fraction by centrifugation. The formula for calculating the binding rate is shown below.

[0086]

number

[0087] When a vaccine containing inulin, a fructose polymer, was administered to HA + ADvehicle + Gly (Comparative Example 1) (Example 1), the amount of s-IgA produced in nasal lavage fluid was 4.3 times higher and the amount of IgG produced in serum was 1.7 times higher compared to HA + ADvehicle + Gly, confirming a significant enhancement of the antibody production induction effect.

[0088] 1) When a vaccine containing curdlan was administered to HA + ADvehicle + Gly (Example 2), the amount of s-IgA produced in nasal lavage fluid was approximately 9.9 times higher and the amount of IgG produced in serum was approximately 13 times higher compared to HA + ADvehicle + Gly (Comparative Example 1), confirming a significant enhancement of the antibody production induction effect. 2) When a vaccine containing laminaran was administered to HA + ADvehicle + Gly (Example 3), the amount of s-IgA produced in nasal lavage fluid was approximately 3.6 times higher and the amount of IgG produced in serum was approximately 12.9 times higher compared to HA + ADvehicle + Gly (Comparative Example 1), confirming a significant enhancement of the antibody production induction effect. 3) When a vaccine containing zymosan was administered to HA + ADvehicle + Gly (Example 4), the amount of s-IgA produced in nasal lavage fluid was approximately 3.3 times higher and the amount of IgG produced in serum was approximately 1.9 times higher compared to HA + ADvehicle + Gly (Comparative Example 1), confirming a significant enhancement of the antibody production induction effect. 4) When a vaccine containing yeast β-glucan was administered to HA + ADvehicle + Gly (Example 5), the amount of s-IgA produced in nasal lavage fluid was approximately 3.6 times higher and the amount of IgG produced in serum was approximately 18.9 times higher compared to HA + ADvehicle + Gly (Comparative Example 1), confirming a significant enhancement of the antibody production induction effect.

[0089] Therefore, among polysaccharides, it was confirmed that the antibody production induction effect was significantly enhanced when inulin and β-1,3-glucans (or similar polysaccharides) containing glucose polymers linked by β-1,3-glycosidic bonds, such as curdlan, laminaran, zymosan, and yeast β-glucan, were added. It was also confirmed that cellulose, in which glucose is polymerized by β-1,4-glycosidic bonds, did not contribute to the antibody production induction effect.

[0090] [Example of comparative analysis] Regarding the polysaccharides examined above, we investigated the antibody production induction effect compared to administering hemagglutinin (HA), the antigen protein, with glycine added, but without ADvehicle, to the nasal cavity of mice after lyophilization (Comparative Example 11). The dosage per mouse was 1.5 μg for HA, 450 μg for Gly, and 150 μg for each polysaccharide. The results are shown in Table 2 below.

[0091] [Table 2]

[0092] (result) When glycine was added to the antigen protein hemagglutinin (HA) without adding AD Vehicle, and cellulose (Comparative Example 4) or sodium alginate (Comparative Example 5) was added, the degree of enhancement of the antibody production induction effect was very small. Similarly, when inulin (Comparative Example 6), curdlan (Comparative Example 7), laminaran (Comparative Example 8), zymosan (Comparative Example 9), and yeast β-glucan (Comparative Example 10) were added, the degree of enhancement of the antibody production induction effect was small compared to when AD Vehicle was added to each of the Comparative Examples 6-10 (Examples 1-5).

[0093] Furthermore, adding only amino acids to the antigen protein (Comparative Example 11), or adding inulin (Comparative Example 6) or β-1,3-glucan (Comparative Examples 7-10), did not enhance the antibody production induction effect. Also, as confirmed above, adding glycine to the antigen protein (HA) (1.5 μg)-AD vehicle complex (HA + AD vehicle) (Comparative Example 1) did not significantly enhance the antibody production induction effect. However, when AD vehicle was added further (Examples 1-5), a strong enhancement of the antibody production induction effect was confirmed. Therefore, in the present invention, it was confirmed that AD vehicle, amino acids, and β-1,3-glucan or inulin are essential combination components that produce a synergistic effect as adjuvants that enhance the antibody production induction effect.

[0094] (Considering card runs) As is clear from Figure 1, which summarizes the data focusing on curdlan in the previous studies, the degree of enhancement in antibody production induction was significantly greater in Example 2, which added glycine to the antigen protein (HA) (1.5 μg)-AD vehicle complex (HA + ADvehicle) with 150 μg of curdlan, compared to Comparative Example 1, which added glycine to the antigen protein (HA) (1.5 μg)-AD vehicle complex (HA + ADvehicle). Almost no antibody production was observed with antigen protein (HA) and glycine (Comparative Example 11). Antibody production did not increase significantly even when glycine and curdlan were added to antigen protein (HA) (1.5 μg) (Comparative Example 7). Therefore, it was confirmed that the AD vehicle complex, glycine, and curdlan, through their combined synergistic effect, increase the production of antigen-specific s-IgA antibodies in mucous membranes throughout the body, including the nasal mucosa, and antigen-specific IgG antibodies in serum.

[0095] Next, we investigated the optimal concentration of curdlan to be added. Vaccines were prepared by adding 75, 150, 300, and 450 μg of curdlan to each mouse, and the antibody induction effect was examined. The results are shown in Table 3 below.

[0096] [Table 3]

[0097] (result) As is clear from Table 3, the s-IgA concentration in the nasal lavage fluid showed an increasing trend depending on the curdlan concentration. On the other hand, the serum IgG concentration plateaued at a curdlan addition amount of 150 μg (Example 2). Therefore, it was determined that the optimal amount of curdlan to add is approximately 150 μg, which is 100 times the HA antigen protein concentration ([HA]). Furthermore, when glycine and curdlan were added but AD Vehicle was not added (Comparative Example 12), no significant antibody production enhancement effect was observed.

[0098] (Consideration of amino acids) Next, we investigated amino acids other than glycine. To an antigen protein (1.5 μg)-AD vehicle complex (HA + AD vehicle) to which 150 μg of curdlan had been added, 450 μg each of glycine (Example 2), alanine (Example 9), serine (Example 10), threonine (Example 11), leucine (Example 12), isoleucine (Example 13), phenylalanine (Example 14), methionine (Example 15), and cysteine ​​(Comparative Example 13) were added to prepare vaccines, and their antibody-inducing effects were examined. The results are shown in Table 4 below.

[0099] [Table 4]

[0100] As is clear from Table 4, when each amino acid except cysteine ​​was added, the levels of s-IgA in nasal lavage fluid and IgG in serum increased significantly, similar to glycine. Specifically, amino acids such as phenylalanine, leucine, and alanine induced antibodies equivalent to or better than glycine in HA-specific s-IgA induction, while amino acids such as serine, leucine, phenylalanine, and methionine induced antibodies equivalent to or better than glycine in HA-specific IgG induction in serum. No particularly significant differences were observed in HA-specific s-IgA induction and IgG induction for any of the amino acids except cysteine, and it was determined that any of the amino acids except cysteine ​​can be used as adjuvant components by combining AD Vehicle with sugars such as β-1,3-glucan.

[0101] [HI value] It has been reported that vaccines containing high molecular weight protein antigens may show a decrease in antibody titers, an indicator of infection protection, due to conformational changes associated with freeze-drying. Therefore, we decided to measure the hemagglutinin inhibition (HI) titer, an indicator of infection protection, for the prepared vaccines. To measure the HI titer, we used Denka Seiken's "Class III Immunotherapy Series Influenza Virus Kit / Influenza Virus HI Reagent 'Seiken'" to measure the influenza virus HI antibody titer in serum. For the implementation, we used a sample in which 10 μg of poly(I:C), which has been used in many studies as an adjuvant to stimulate dendritic cells, was added to 1.5 μg of antigen protein as a positive control, and physiological saline as a negative control. We compared HA+ADvehicle and HA+ADvehicle+Cardlan+Gly (Example 2). Since it was difficult to administer the HA+poly(I:C) and physiological saline samples as freeze-dried powders via nasal spray, they were administered intranasally as an administerable liquid. Six mice were used in each group. The results are shown in Table 5.

[0102] [Table 5]

[0103] (result) The vaccine of Example 2, in which curdlan and glycine were added to the antigen protein-AD vehicle complex, was confirmed to have a HI titer twice as high as that of HA+poly(I:C) used as a positive control. Therefore, it was confirmed that the vaccine of the present invention not only satisfies the international evaluation standard of HI≧40 even after freeze-drying, but can also maintain a high antibody titer. One reason for this excellent effect is thought to be that the AD vehicle in the present invention can form a complex with the antigen protein through weak hydrophobic interactions, thus providing a protective effect that avoids the decrease in the HI titer of the HA antigen due to the freeze-drying process.

[0104] [s-IgA antibody induction effect in systemic mucosal tissues] Following administration of the vaccine of the present invention, the production of s-IgA antibodies in various mucosal tissues throughout the body, not just intranasally, was compared and examined. The vaccine of Example 2, in which curdlan and glycine were added to the antigen protein-AD vehicle complex, was administered intranasally a total of three times at two-week intervals. Comparative Example 7, which did not contain AD vehicle, was used as a comparison. Two weeks after the final immunization, nasal fluids, vaginal fluids, small intestinal fluids, and feces were collected, and the s-IgA antibody titer of each specific anti-influenza A / California / 7 / 2009(H1N1)pdm09 contained in the supernatant of each fluid after centrifugation was measured. The above data are shown as the total volume of each fluid per animal, but the total volume of collected samples was 1 mL for nasal fluid, 1 mL for vaginal fluid, 2 mL for lung lavage fluid, 2 mL for small intestinal mucosal fluid, and 500 μL for the large intestine. The results are shown in Table 6.

[0105] [Table 6]

[0106] (result) As is clear from Table 6, the mice in Example 2 showed a higher antibody production per mouse than the mice in Comparative Example 7. Furthermore, when the sample from Example 2 was administered to mice, the amount of antibody produced in vaginal douching fluid was similar to that in nasal douching fluid, and the production of s-IgA in the small intestinal mucosal fluid and feces in the large intestine increased significantly. Therefore, it was confirmed that the microparticle powder type mucosal vaccine of the present invention can produce s-IgA, which has an infection-protective effect, in mucosal tissue throughout the body even through local administration.

[0107] [Checking the size of fine powder particles] 1.5 μg of the fine particle powder type mucosal vaccine from Example 2 was sprayed into a plastic bag, and the scattered powder was transferred onto a measurement stage covered with conductive double-sided tape. The stage on which the scattered powder was transferred was placed in a gold deposition apparatus and gold deposition was performed. After the gold deposition was completed, the measurement stage was removed and inserted into a scanning electron microscope (JCM5700, manufactured by JEOL Ltd.), and the particle size of the fine particle powder was read from the obtained SEM image using the attached measuring device and graphed. The measurement was performed on an image area of ​​3.5 × 10⁻⁶. -6 m 2 The procedure was performed on 244 fine powder particles identified in the sample. The longest axis was used as the particle diameter, and a particle diameter number distribution table was created. The results are shown in Figure 2.

[0108] (result) As is clear from Figure 2, the particle sizes of the fine powder of Example 2 immobilized by the gold deposition method were as follows: the most numerous particles were 10-20 μm, followed by 6.5-10 μm, then 20-30 μm, and then 5-6.5 μm, with approximately 80% of the total falling within the 5-40 μm range.

[0109] [Confirmation of vaccine delivery site] The destination site of a microparticle powder vaccine administered into the nasal cavity of mice was confirmed. The vaccine was administered intranasally by pressing an Eppendorf tube filled with a composition identical to that of Example 2 (1.5 μg OVA antigen + 15 μg AD Vehicle + 150 μg Curdlan + 450 μg Gly) against the nostril of the test mouse and spraying it with the airflow. Twenty minutes after intranasal administration, images of the nasal cavity, pharynx, trachea, bronchi, and lungs of the test mice were taken using an in vivo imaging system (IVIS spectrum, PerkinElmer). The images are shown in Figure 3.

[0110] (result) As is clear from Figure 3, the administered particulate powder was found to be distributed in the nasal cavity and pharynx of the test mice. However, the particulate powder did not reach the trachea, bronchi, or lungs. This result is consistent with the range reached by the above-mentioned particulate powders of 5 μm or larger. [Industrial applicability]

[0111] The vaccine of the present invention is extremely useful in the medical field. Furthermore, since its effectiveness can be confirmed, for example, by mouse experiments using human-derived virus strains that have been adapted to infect mice, in this art, the dosage of the vaccine can be estimated for humans by known methods such as the HED conversion method, which estimates the dose that will produce an equivalent effect in humans from the body surface area of ​​test animals such as mice, or by referring to data on peak blood concentration (Cmax) and area under the time curve (AUC), and then the dosage for humans can be determined by further accumulating experimental data.

Claims

1. A microparticle powder-type mucosal vaccine containing an antigen protein and an adjuvant containing the following (A) to (D). (A) Synthetic peptides consisting of the amino acid sequence KnLm (where n is an integer between 4 and 8, and m is an integer between 11 and 20); (B) Mixed lipid; (C) Cardran or Inulin; (D) Amino acids;

2. The microparticle powder type mucosal vaccine according to claim 1, characterized in that the synthetic peptide consisting of the amino acid sequence KnLm (where n is an integer between 4 and 8, and m is an integer between 11 and 20) consists of the amino acid sequence shown in SEQ ID NO: 1 or 2.

3. A microparticle powder type mucosal vaccine according to claim 1 or 2, characterized in that the mixed lipids contain one or more phospholipids.

4. The fine particle powder type mucosal vaccine according to claim 3, characterized in that one or more phospholipids are selected from dipalmitoylphosphatidylcholine, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid.

5. A fine particle powder type mucosal vaccine according to any one of claims 1 to 4, characterized in that the mixed lipid contains one or more selected from lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid.

6. A microparticle powder type mucosal vaccine according to any one of claims 1 to 5, characterized in that the antigen protein is a protein derived from a pathogen, an inactivated antigen protein, a recombinant antigen protein, or a detoxified toxin protein.

7. A microparticle powder type mucosal vaccine according to any one of claims 1 to 6, characterized in that the amino acid is one or more amino acids selected from glycine, alanine, serine, threonine, leucine, isoleucine, phenylalanine, and methionine.

8. A microparticle powder type mucosal vaccine according to any one of claims 1 to 7, characterized in that it is used to enhance the production of antigen-specific s-IgA in the mucosa or to enhance the production of antigen-specific IgG in the blood.

9. A microparticle powder type mucosal vaccine according to any one of claims 1 to 8, characterized in that it is an inhaled or sprayed type uniform microparticle powder that exerts an infection-protective effect against respiratory tract infections that pass from the nasal cavity through the pharynx.

10. (1) Antigen protein; (2) Synthetic peptides consisting of the amino acid sequence KnLm (where n is an integer between 4 and 8, and m is an integer between 11 and 20); (3) Mixed lipids; (4) Cardran or inulin; (5) Amino acids; A method for producing a lyophilized, fine particle powder type mucosal vaccine, comprising: The manufacturing method is characterized by comprising the following steps (a) to (e). (a) A step of preparing an AD-vehicle suspension by suspending the synthetic peptide and the mixed lipids in water; (b) A step of adding an antigen protein to the AD-vehicle suspension, heating and stirring once or twice or more to form an antigen protein-AD-vehicle complex; (c) A step of preparing a curdlan or inulin-added antigen protein-AD vehicle complex by adding curdlan or inulin to the antigen protein-AD vehicle complex and then stirring; (d) A step of preparing a vaccine solution by adding amino acids to a curdlan or inulin-added antigen protein-AD vehicle complex and then stirring it; (e) A step of freeze-drying the vaccine solution to form a fine particle powder;

11. A method for producing a microparticle powder type mucosal vaccine according to claim 10, characterized in that the mixed lipid is a mixed lipid of three types of lipids: dipalmitoylphosphatidylcholine, phosphatidylglycerol, and palmitic acid.

12. A method for producing a microparticle powder type mucosal vaccine according to claim 10 or 11, characterized in that the heating temperature setting range is 25 to 75°C in step (b) of forming the antigen protein-AD vehicle complex.

13. A method for producing a microparticle powder-type mucosal vaccine according to any one of claims 10 to 12, comprising an amount of antigen protein that, on its own, does not produce an effective immune induction and infection-protective effect, and used to produce an effective immune induction and infection-protective effect by combining it with a synthetic peptide, a mixed lipid, curdlan or inulin, and amino acids.

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