Subcutaneous vaccine

Abstract

Provided is vaccine for subcutaneous administration that uses an antigen protein in a smaller amount than in conventional vaccines, that has the superior effect of inducing antigen-specific blood IgG antibody, and that is free of problems such as formation of a painful lump at the site of administration. Vaccine for subcutaneous administration was produced, the vaccine containing an antigen protein, a synthetic peptide comprising an amino acid sequence of KnLm, a phospholipid-containing liquid, and a carboxy vinyl polymer.

Description

[Technical field]

[0001] The present invention relates to a subcutaneously administered vaccine that effectively induces immune IgG in the blood. [Background technology]

[0002] Immune enhancers (adjuvants) that have been used in subcutaneous vaccines, which are delivered through the skin barrier using a syringe or the like to present antigen information to antigen-presenting cells in the tissue, include aluminum salts (aluminum chloride / aluminum hydroxide / aluminum phosphate / aluminum sulfate, etc.), AS04 (containing phospholipid A, a bacterial substance), and emulsifier adjuvants such as squalene (AS03, MF59, etc.), which are said to have the functions of retaining antigens in the tissue for a long time, stimulating immune cells for a long period of time by gradually releasing antigens, and activating natural immune responses. Of these, aluminum salts are the most widely used adjuvant for subcutaneous vaccines in humans (Non-Patent Document 1). However, recent academic research has shown that their immune induction amplification effect is not necessarily high, and problems have been pointed out such as the possibility of causing symptoms such as pain at the administration site and leaving a lump behind.

[0003] On the other hand, mucosally administered vaccines that enhance the function of presenting antigen information to antigen-presenting cells in mucosal tissues have attracted attention because they can induce secretory IgA antibodies as well as serum IgG antibodies. It has also been proposed to use pulmonary surfactant, which acts as a surfactant and covers the surface of the lungs and airway mucosa, as a mucosal adjuvant in mucosally administered vaccines (see, for example, Patent Documents 1 and 2), and it has been confirmed that they exhibit antigen-specific IgG induction in serum, antigen-specific secretory IgA induction in airway mucosa (nasal lavage fluid) (see, for example, Patent Document 4 and Non-Patent Document 2), and cellular immunity induction (see, for example, Non-Patent Document 3).

[0004] The inventors have proposed an artificial synthetic pulmonary surfactant based on the composition of the above-mentioned pulmonary surfactant, which has been endowed with the function of transporting antigens to antigen-presenting cells. By adding carboxyvinyl polymer (CVP), a thickening agent that has both the function of retaining antigens on the mucosal surface and the function of sustained release, they have previously reported an influenza vaccine administered intranasally (through the airway) as a safe and effective mucosal adjuvant (see, for example, Patent Documents 3 and 4).

[0005] However, for example, the nasal mucosa has cilia and is covered with a mucus layer that is filled with mucus, so a transmucosal vaccine administered to the nasal mucosa adheres to the mucus along with small dust particles and bacteria in the air that enters the nasal cavity, and is then quickly expelled to the throat by a strong excretory action. Therefore, in order to increase the effectiveness of transmucosal vaccines, it is necessary to increase the amount of antigen and to have a means to retain the antigen in the mucosa for a long period of time, but practical application has hardly been achieved yet. [Prior art documents] [Patent documents]

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

[0007] [Non-Patent Document 1] Yakugaku Zasshi 2011; 131(12):1723-31. [Non-Patent Document 2] Vaccine 2019;37:612-22. [Non-Patent Document 3] PLoS One 2018;13:e0191133. Summary of the Invention [Problem to be solved by the invention]

[0008] Antigen viruses mutate in various ways, such as the influenza virus that causes seasonal influenza epidemics around the world every year, and COVID-19, which is an urgent issue. Therefore, vaccine manufacturers are always faced with the problem of having to secure large amounts of appropriate antigens. In this situation, the challenge is to provide a subcutaneous vaccine that can vaccinate many people by using a smaller amount of antigen protein than before, has excellent antigen-specific blood IgG antibody induction effect, and does not leave a painful lump at the administration site. [Means for solving the problem]

[0009] The present inventors have re-examined the administration route of the vaccine for the components used in the mucosal vaccine. For example, they have found that SF-10, which has been used in the mucosal vaccine, is extremely effective when used in the subcutaneous vaccine, and that the subcutaneous vaccine has excellent effects even when the amount of antigen protein used is small compared to the mucosal vaccine. They have also confirmed that the vaccine of the present invention is superior to the subcutaneous vaccine using a conventional aluminum salt as an adjuvant in terms of the antigen-specific IgG antibody induction promotion effect, and that it leaves almost no lumps at the subcutaneous administration site, thereby completing the present invention.

[0010] That is, the present invention is specified by the following items. [1] A subcutaneous vaccine comprising an antigen protein and an adjuvant comprising the following (a) to (c): (a) a synthetic peptide consisting of the amino acid sequence KnLm (wherein n is 4-8 and m is 11-20); (b) phospholipid-containing solution; (c) Carboxyvinyl polymer [2] The subcutaneous vaccine according to [1] above, which induces circulating IgG antibodies but does not induce IgA antibodies. [3] A subcutaneous vaccine according to [1] or [2] above, characterized in that the synthetic peptide consisting of the amino acid sequence of KnLm consists of the amino acid sequence shown in SEQ ID NO: 1 or 2. [4] A subcutaneous vaccine according to any one of [1] to [3] above, characterized in that the synthetic peptide consisting of the amino acid sequence of KnLm is present in a mass ratio of the phospholipid-containing liquid to the synthetic peptide of 100:0.1-50. [5] The subcutaneous vaccine according to any one of [1] to [4] above, characterized in that the phospholipid in the phospholipid-containing liquid is one or more types selected from dipalmitoylphosphatidylcholine, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. [6] A subcutaneous vaccine according to any one of [1] to [5] above, characterized in that the phospholipid-containing liquid further contains one or more types of lipids other than phospholipids selected from lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, and oleic acid. [7] The subcutaneous vaccine according to any one of [1] to [6] above, wherein the antigen protein is a protein derived from a pathogen, an inactivated antigen protein, a recombinant antigen protein, or a detoxified toxin protein. [8] The subcutaneous vaccine according to any one of [1] to [7] above, characterized in that the antigen protein is contained at 0.05 μg to 5 μg / 100 μL. [9] The subcutaneous vaccine according to any one of [1] to [8] above, wherein the mass ratio of the phospholipid-containing liquid to the antigen protein is 0.1 to 20.

[10] The subcutaneous vaccine according to any one of [1] to [9] above, characterized in that the carboxyvinyl polymer is contained in an amount of 0.02% by mass to 2.0% by mass.

[11] A method for producing a subcutaneous vaccine comprising the following steps (a) to (c), in that order: (a) preparing an SSF suspension by suspending a synthetic peptide consisting of the amino acid sequence of KnLm (wherein n is 4-8 and m is 11-20) and a phospholipid-containing liquid in water; (b) adding an antigen protein to the SSF suspension prepared in step (a) and shaking and mixing to prepare an antigen protein-SSF uniform suspension; (c) adding a carboxyvinyl polymer solution to the antigen protein-SSF uniform suspension prepared in step (b) to prepare a subcutaneous administration vaccine containing the antigen protein and an adjuvant;

[12] After step (b), (b') freeze-drying the antigen protein-SSF homogeneous suspension prepared in step (b) to prepare a freeze-dried antigen protein-SSF; and Prior to step (c), (b") is a step of suspending the lyophilized antigen protein-SSF prepared in step (b') in water or physiological saline to prepare a uniform suspension of the antigen protein-SSF; The method for producing a subcutaneous vaccine according to

[11] above, further comprising: Effect of the Invention

[0011] The subcutaneous vaccine of the present invention causes no pain or lump at the vaccine administration site, and by using the adjuvant of the present invention containing CVP, which has been added to conventional mucosal vaccines, rather than vaccines containing aluminum hydroxide salts, which have been conventionally used as an adjuvant, it brings about effects such as significantly more effective induction of IgG antibodies in the blood, increased survival rate after viral infection, and weight recovery. [Brief description of the drawings]

[0012] [Figure 1](a) The results of a subcutaneous vaccine using serum anti-HA IgG antibody titer as an index. The amount of influenza virus HA antigen was varied in concentration from 0.1 to 1.0 μg, and the graph shows the results of comparing the effects of adding no adjuvant, an aluminum hydroxide salt adjuvant, and an SF-10 adjuvant. (b) The graph shows the results of the results of Fig. 1(a) using the antibody titer converted into the amount of anti-HA IgG antibody as an index. [Diagram 2] This graph shows the results when the antigen protein concentration [HA] of the HA solution was fixed at 1.0 μg / 100 μL and the CVP concentration contained in SF-10 was changed within the range of 0.01 to 0.5% by mass, using serum anti-HA IgG antibody titer as an indicator. [Diagram 3] Graphs showing (A) anti-HAIgG antibody titer in serum of mice vaccinated with a subcutaneous vaccine and then nasally infected with the virus, and (B) survival rate over time up to two weeks after nasal infection with the virus. [Figure 4] 1 is a graph showing changes in body weight of mice vaccinated with a subcutaneously administered vaccine and then intranasally infected with the virus. [Figure 5-1] 1 is a graph showing (A) anti-HAIgG antibody titer in serum and (B) survival rate over time up to two weeks after nasal infection with the virus, when the CVP concentration was fixed (0.05% by mass). [Figure 5-2] 1 is a graph showing (A) anti-HA IgG antibody titer in serum and (B) survival rate over time up to two weeks after nasal infection with the virus, when the CVP concentration was fixed (0.01% by mass). [Figure 6] These are the results of evaluating the anti-HA IgA antibody titer induced in the serum of mice after inoculation with a subcutaneous vaccine. As a control group, the results are shown for a mucosally administered (oral, airway) vaccine. Note that in order to keep the experimental conditions constant, the V / A ratio was set to 10 even in the case of mucosal administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The vaccine of the present invention is not particularly limited as long as it is a subcutaneously administered vaccine comprising an antigen protein; and (a) a synthetic peptide consisting of the amino acid sequence of KnLm (wherein n is 4-8 and m is 11-20), (b) a phospholipid-containing liquid, and (c) an adjuvant having an immune enhancing effect comprising a carboxyvinyl polymer (solution) (sometimes referred to as "SF-10"). A characteristic of the vaccine of the present invention is that it has a sufficient infection protection effect, such as an IgG induction effect, while not causing, or is very unlikely to cause, a lump or hardening at the administration site.

[0014] Subcutaneous administration vaccines (sometimes referred to as transdermal administration vaccines) in the present invention include subcutaneous administration vaccines in which a drug is administered (preferably by injection) into the fatty tissue under the skin, as well as intradermal administration vaccines in which a drug is administered (preferably by injection) into the dermal tissue of the skin, and intramuscular injection vaccines.

[0015] The synthetic peptide consisting of the amino acid sequence KnLm is not particularly limited as long as it is an amino acid sequence consisting of n consecutive K (lysine: Lys) residues on the N-terminus and m consecutive L (leucine: Leu) residues on the C-terminus, where n is 4-8 and m is 11-20, but specific examples include the following peptides. The amino acid residues are indicated by single letter codes. SEQ ID NO:1: (K6L16): KKKKKKLLLLLLLLLLLLLL SEQ ID NO:2: (K6L11): KKKKKKLLLLLLLLLLL

[0016] The above synthetic peptides are preferably prepared according to known chemical synthesis methods and have a purity of 95% or more.

[0017] The above synthetic peptide is preferably dissolved in an organic solvent such as methanol, ethanol, trifluoroacetic acid, etc., which does not affect the synthetic peptide structure, and added to the vaccine as a K6L16 peptide-containing liquid. For example, it can be used as a K6L16 peptide-containing liquid dissolved in methanol to a concentration of 3 mg to 7 mg / mL.

[0018] The phospholipid-containing liquid in the present invention is not particularly limited as long as it is a liquid containing phospholipids, and may further contain lipids other than phospholipids. However, the phospholipid is preferably a phospholipid contained in a natural pulmonary surfactant, and specific examples thereof include phosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, etc., and such phospholipids may be used alone or as a mixture of two or more types, and a preferred example is a combination of dipalmitoylphosphatidylcholine and phosphatidylglycerol.

[0019] When the phospholipids contained in the phospholipid-containing liquid are dipalmitoylphosphatidylcholine and phosphatidylglycerol, the mixing ratio of dipalmitoylphosphatidylcholine:phosphatidylglycerol can be exemplified as 10:1 to 1:10, 5:1 to 1:5, 4:1 to 1:1, or 3.5:1 to 2.5:1.

[0020] Examples of lipids other than phospholipids contained in the phospholipid-containing liquid include lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, and oleic acid.

[0021] The ratio (by mass) of phospholipids to lipids other than phospholipids contained in the phospholipid-containing liquid can be, for example, 100:1 to 30 (phospholipids: lipids other than phospholipids), 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.

[0022] In the present invention, a composition containing or consisting of the above-mentioned phospholipid-containing liquid, a synthetic peptide consisting of the amino acid sequence of KnLm, and an appropriate solvent may be referred to as "SSF (synthetic pulmonary surfactant)" or "AD vehicle." The mass ratio of the phospholipid-containing liquid to the synthetic peptide in the SSF, i.e., phospholipid-containing liquid:synthetic peptide, can be 100:0.1-50, preferably 100:0.5-10, more preferably 100:1-5, and even more preferably 100:1.5-3.

[0023] The above-mentioned antigen protein(s) are not particularly limited as long as they are proteins that can be used as antigens for vaccines, and examples thereof include those that are expected to prevent or cure diseases caused by the pathogen from which the antigen protein is derived by inducing immunity against the antigen protein in the body. In addition to proteins derived from common pathogens, examples of such antigen proteins include inactivated antigen proteins, recombinant antigen proteins, and detoxified toxin proteins. Furthermore, antigen proteins may include whole (mature) proteins, as well as preproteins, preproproteins, and functional or immunodominant antigen peptides thereof.

[0024] Examples of the pathogen include viruses, bacteria, and parasites.

[0025] Examples of the above viruses include chickenpox virus, measles virus, poliovirus, rotavirus, influenza virus, herpes virus, severe acute respiratory syndrome (SARS) virus, novel coronavirus (COVID-19), Ebola hemorrhagic fever virus, West Nile virus, hantavirus, dengue virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and HIV virus.

[0026] Examples of the bacteria include Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Pneumoniae, and Vibrio cholerae, and examples of the parasites include malaria pathogens and sleeping sickness pathogens.

[0027] When the pathogen is an influenza virus, specific examples of antigenic proteins include hemagglutinin (HA) antigenic protein, neuraminidase antigenic protein, M protein, and other antigenic glycoproteins present on the surface of the virus, as well as internal nucleoproteins.

[0028] The content (mass) of the antigen protein in the vaccine of the present invention is not particularly limited as long as the effect of the present invention is exhibited, but for example, in the case where the above-mentioned hemagglutinin is used as an antigen protein, the content of the hemagglutinin (HA) antigen protein can be expressed as the HA content obtained by quantitatively measuring the amount of HA by the method of Single radial immunodiffusion (SRD), Fahey, J., et al., J.Immunol., 94, 84-90 (1965), or, once the HA content is measured by the SRD method, as long as the same HA stock solution is used, the notation can be expressed by the protein amount indicating the total amount of the hemagglutinin antigen protein and other antigen proteins, which is easier to measure than SRD quantification. That is, in the latter case, the mass of the HA antigen protein in the HA antigen protein solution using influenza virus hemagglutinin (HA) can also mean the mass of the total protein including the HA antigen protein and antigenic proteins other than the HA antigen protein, and in that case, in the present invention, it is expressed as the mass (HA antigen protein amount) or concentration of the HA antigen protein.

[0029] The HA antigen protein concentration in the vaccine of the present invention can be 0.05μg to 5μg / 100μL, preferably 0.06μg to 2μg / 100μL, more preferably 0.07μg to 1.5μg / 100μL, more preferably 0.08μg to 1.1μg / 100μL, even more preferably 0.08 to 0.5μg / 100μL, and particularly preferably 0.09 to 0.12μg / 100μL. More specifically, in order to increase the survival rate of the vaccine administration subject, 0.08μg to 0.15μg / 100μL is more preferable, 0.08 to 0.12μg / 100μL is even more preferable, and 0.09 to 0.11μg / 100μL is particularly preferable. For significant induction of antigen-specific IgG in the serum of a vaccine subject, 0.6 μg to 2 μg / 100 μL is preferable, 0.7 μg to 1.5 μg / 100 μL is more preferable, and 0.8 μg to 1.2 μg / 100 μL is even more preferable.

[0030] The production method for ensuring a sufficient amount of the above-mentioned antigen protein (or antigen peptide) for vaccine production is not particularly limited as long as it is a known method, and examples of the method include a production method using gene recombination technology and a production method using chemical synthesis.

[0031] The mass ratio of the phospholipid-containing liquid to the antigen protein (phospholipid-containing liquid / antigen protein) is not particularly limited as long as the effects of the present invention can be achieved, but examples of the ratio include 0.1 to 20, preferably 1 to 18, more preferably 5 to 15, even more preferably 8 to 12, and even more preferably 9 to 11.

[0032] The CVP is a hydrophilic polymer obtained by polymerizing acrylic acid, and the molecular weight and the like are not particularly limited, but it is preferable that the CVP conforms to the Pharmaceutical Excipients Standards 2018. Commercially available products include 971P NF (0.5%, 25°C, 4,000 to 11,000 cps), 974P NF (0.5%, 25°C, 29,400 to 39,400 cps), and 71G NF (0.5%, 25°C, 4,000 to 11,000 cps), which are part of the CARBOPOL (registered trademark) series of Lubrizol Advanced Materials, Inc. NF (0.5%, 25℃, 4,000~11,000cps), 980 (0.2%, 20℃, 14,000~32,000cps), 981 (0.2%, 20℃, 1,500~7,500cps), 2984 ( 0.2%, 20℃, 1,500~9,000cps), 5984 (0.2%, 20℃, 6,000~22,000cps), Ultrez10 (0.2%, 20℃, 10,000~36,000cps), 980 NF (0.5%, 25℃, 40,000~60,000cps), 981 NF (0.5%, 25℃, 4,000~10,000cps), Ultrez10 NF (0.5%, 25°C, 45,000 to 65,000cps) can be mentioned. In addition, HIVISUWAKO 103, HIVISUWAKO 104, and HIVISUWAKO 105 manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., and pAA130 (Sigma, St. Louis, MO, Cat No. 181293), pAA450 (Sigma, Cat No. 181285), and pAA1250 (Sigma, Cat No. 306215) manufactured by Sigma can be used, and CARBOPOL (registered trademark) 971P NF POLYMER manufactured by Lubrizol Advanced Materials, Inc. can be preferably mentioned.

[0033] The concentration of CVP in the subcutaneous vaccine of the present invention is not particularly limited as long as the effect of the present invention is exhibited, but may be 0.02 to 2.0 mass%, preferably 0.05 to 2.0 mass%, and a CVP concentration of 0.01 mass% or less may result in a low survival rate. In addition, 0.05 to 0.1 mass% is preferable in that it is unlikely to cause induration at the administration site while sufficiently inducing IgG antibodies in the blood. In order to maintain the antigenicity of the antigen protein, it is preferable to adjust the pH of the CVP solution, and such a pH may be 5.0 to 10.5, preferably 6.8 to 8.0, and more preferably 7.0 to 7.2.

[0034] The number of times the vaccine of the present invention is administered is not particularly limited as long as the vaccine of the present invention is effective, but can be one or more times, preferably one or two times, and preferably two times. It is preferable that the second administration is performed one week to one month after the first administration, preferably 10 days to two weeks after the first administration.

[0035] An example of a method for producing the subcutaneous administration vaccine of the present invention includes a production method comprising the following steps (a) to (c) in order: (a) preparing an SSF suspension by suspending a synthetic peptide consisting of the amino acid sequence of KnLm (wherein n is 4-8 and m is 11-20) and a phospholipid-containing liquid in water; (b) adding an antigen protein to the SSF suspension prepared in step (a) and shaking and mixing to prepare an antigen protein-SSF uniform suspension; (c) adding a carboxyvinyl polymer solution to the antigen protein-SSF uniform suspension prepared in the step (b) to prepare a subcutaneous administration vaccine containing the antigen protein and an adjuvant;

[0036] Furthermore, after step (b), (b') freeze-drying the antigen protein-SSF uniform suspension prepared in the step (b) to prepare a freeze-dried antigen protein-SSF; And, before step (c), (b") is a step of suspending the lyophilized antigen protein-SSF prepared in step (b') in water or physiological saline to prepare a uniform suspension of the antigen protein-SSF; The device may further include:

[0037] The SSF suspension prepared in the above step (a) is not particularly limited as long as it is prepared by suspending a synthetic peptide consisting of the amino acid sequence of KnLm (wherein n is 4-8, m is 11-20) and a phospholipid-containing solution in water. The mass ratio of the phospholipid-containing solution to the synthetic peptide in the SSF suspension, i.e., the phospholipid-containing solution:synthetic peptide, can be 100:0.1-50, preferably 100:0.5-10, more preferably 100:1-5, and even more preferably 100:1.5-3. The phospholipid-containing solution is further dissolved in an organic solvent such as chloroform or methanol (phospholipid-containing solution:organic solvent=100:1-50, preferably 100:2-25, more preferably 100:5-15, and even more preferably 100:8-12) and used as a lipid solution, which is convenient when suspending in water. In this case, the SSF suspension contains or consists of a synthetic peptide consisting of the amino acid sequence of KnLm and a lipid solution.

[0038] An example of the antigen protein-SSF uniform suspension prepared in the above step (b) is a liquid prepared by adding an antigen protein to the SSF suspension prepared in the above step (a) while heating in a water bath at, for example, 30 to 60°C, preferably 40 to 50°C, and then shaking, stirring, and mixing for 5 to 20 minutes, preferably 8 to 12 minutes.

[0039] The subcutaneous vaccine prepared in the above step (c) can be an SF-10-added final vaccination liquid, which is a subcutaneous vaccine containing an SF-10-type adjuvant and an antigen protein, including a synthetic peptide consisting of the amino acid sequence of KnLm, a phospholipid-containing liquid, and CVP, by further adding CVP to the antigen protein-SSF uniform suspension. The concentration of CVP in the vaccine can be 0.02 to 2.0% by mass, preferably 0.05 to 2.0% by mass. In order to uniformly mix the antigen protein-SSF uniform suspension and CVP, it is preferable to use a homogenizer, a mixer, a shaker, a stirrer, or the like. It is also preferable to add pure water and / or physiological saline to CVP and treat it with a homogenizer, and then first prepare a CVP solution by adjusting the pH with a NaOH neutralizing solution, and then add the antigen protein-SSF uniform suspension.

[0040] Furthermore, step (b) may be followed by step (b') or step (b"). The antigen protein-SSF freeze-dried product prepared in step (b') is not particularly limited as long as it is a solid obtained by freeze-drying the antigen protein-SSF uniform suspension. The freezing process temperature for the freeze-drying may be -80°C to -60°C, and the freeze-dried product is preferably stored at -30°C.

[0041] Furthermore, the antigen protein-SSF uniform suspension in step (b") can include an antigen protein-SSF uniform suspension prepared by suspending the antigen protein-SSF lyophilized product prepared in step (b') in water and / or physiological saline to adjust the concentration to a predetermined concentration suitable for formulating a subcutaneous vaccine.

[0042] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0043] [Development of subcutaneous influenza vaccine] In mice, we evaluated the effect of subcutaneous influenza vaccines on the induction of anti-influenza virus-specific IgG antibodies. All subsequent animal experiments were performed in the infectious animal house (P2 level) of the Laboratory Animal Center, Faculty of Medicine, Tokushima University, in accordance with the guidelines of the Animal Experiment Committee, Faculty of Medicine, Tokushima University.

[0044] The mice used were BALB / c mice (6-8 weeks old, female, average body weight 20 g) purchased from Japan SLC Co., Ltd.

[0045] [Example 1] [Preparation of various solutions for subcutaneous administration to mice] To verify the efficacy of influenza virus hemagglutinin (HA) as a vaccine, various solutions were prepared for subcutaneous administration to the back neck of mice.

[0046] (Preparation of lipid solutions) Dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and palmitic acid (PA) were mixed to prepare a phospholipid-containing liquid. For the phospholipid-containing liquid, dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and palmitic acid (PA) were suspended in a chloroform:methanol (2:1 (v / v)) mixture at a ratio of 75:25:10 (w / w / w) so that the phospholipid consisting of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG) was at a final concentration of 10 mg / mL, to prepare a lipid solution.

[0047] (Preparation of synthetic pulmonary surfactant) A synthetic peptide K6L16 (KKKKKKLLLLLLLLLLLLLLLL) (GenScript) with a purity of 95% or more, which is similar to surfactant protein C, was dissolved in methanol to a concentration of 5.0 mg / mL to prepare a K6L16 peptide-containing solution.

[0048] The lipid solution and the K6L16 peptide-containing solution were mixed to a mixing ratio (mass ratio) of phospholipid-containing solution:K6L16 peptide=100:2 (lipid solution:K6L16 peptide=110:2) to prepare a homogeneous suspension of lipid solution-K6L16 mixture (SSF suspension). The SSF suspension was freeze-dried using a freeze dryer and stored at -30°C as freeze-dried SSF.

[0049] [Preparation of HA+SSF subcutaneous solution] Hemagglutinin (HA) of influenza virus was used as an antigen for influenza vaccine. Daiichi Sankyo Co., Ltd. prepared a vaccine antigen liquid from a vaccine strain provided by the National Institute of Infectious Diseases, and an HA antigen protein liquid was prepared from the A / Singapore / GP1908 / 2015 (H1N1) strain provided to Tokushima University for a fee. The freeze-dried SSF was returned to room temperature and suspended in ultrapure water to prepare an SSF suspension again, and the HA antigen protein liquid was added to the SSF suspension so that the ratio of the mass (V) of the phospholipid-containing liquid to the amount (A) of the HA antigen protein was 10 (i.e., the amount (A) of the HA antigen protein: the mass (V) of the phospholipid-containing liquid = 1:10). The suspension was suspended in a vortex mixer until it became uniform, and then the suspension was shaken and mixed in a water bath at 42°C for 10 minutes to bind the antigen protein and SSF, and an antigen protein-SSF uniform suspension (HA+SSF) was prepared. The HA+SSF was freeze-dried and stored as a freeze-dried antigen protein-SSF at -30°C, and was thawed before use. From this point on, the amount of HA antigen protein in the influenza vaccine (A) and the mass of phospholipids in the phospholipid-containing solution (V) were fixed at V / A = 10.

[0050] (Preparation of HA+SF-10-containing solution 1) For vaccine administration, 100 μL of HA+SF-10-containing solution was prepared per dose. The above HA+SSF was dissolved in physiological saline to adjust the HA antigen protein concentration ([HA]) of the HA antigen protein solution to 0.1 μg / 100 μL, 0.5 μg / 100 μL, and 1.0 μg / 100 μL, respectively, and a carboxyvinyl polymer (Carboxyl Vinyl Polymer: CVP) (CARBOPOL (registered trademark) 971P NF POLYMER, manufactured by Lubrizol Advanced Materials, Inc.) having sustained release action was added to a concentration of 0.1% by mass to prepare each of the HA+SF-10-containing solutions (a) to (c) containing the adjuvant (SF-10) added to each HA as a subcutaneous administration solution. Details are shown in Table 1 below.

[0051] [Table 1]

[0052] [Comparative Example 1] (Preparation of HA solution alone) To compare the effect of inducing anti-influenza IgG antibodies in the blood when only the HA antigen was administered subcutaneously, the antigen protein concentration ([HA]) of the HA antigen protein solution in physiological saline was adjusted to 0.1 μg / 100 μL, 0.5 μg / 100 μL, and 1.0 μg / 100 μL, and each HA only solution (d) to (f) was prepared as a solution for subcutaneous administration. Details are shown in Table 2 below.

[0053] [Table 2]

[0054] [Comparative Example 2] (Preparation of HA+Alm-containing solution) Instead of SSF, aluminum hydroxide salt (Alm), which has been used as a subcutaneous adjuvant, was added as an adjuvant to prepare a subcutaneous administration liquid. As the aluminum hydroxide salt, Alhydrogel (registered trademark) adjuvant 2% (Cat No. vac-alu-250, InvitroGen, San Diego, USA) was used. The protein concentration ([HA]) of the HA antigen protein solution in physiological saline was adjusted to 0.5 μg / 100 μL or 1.0 μg / 100 μL, and aluminum hydroxide salt (Alm) was added to a final concentration of 1 mass%, and the solution was suspended until homogeneous using a Vortex mixer, and 100 μL of each of the HA + Alm-containing solutions (g) and (h) was prepared as a subcutaneous administration liquid. The aluminum hydroxide salt was dissolved in MilliQ water before being added, so that the NaCl concentration here was 0.45 mass%. Details are shown in Table 3 below.

[0055] [Table 3]

[0056] [Example 2] [Vaccination of mice 1] The above (a) to (h) subcutaneous administration solutions were administered as vaccines to evaluate the effect of inducing serum anti-HA specific IgG antibodies. BALB / c female mice aged 6 to 8 weeks (Japan SLC Co., Ltd.) were used.

[0057] Each administration group consisted of 5-6 mice, and 100 μL of each of the above subcutaneous administration solutions (a) to (h) was administered subcutaneously to the back of the neck of each group of mice as a first immunization. Two weeks after the first immunization, 100 μL of the mouse subcutaneous administration solution of the same composition was administered subcutaneously to the back of the neck of the mice that had been administered the vaccine as a first immunization as a second vaccine (secondary immunization).

[0058] (Preparation of serum 1) Two weeks after the secondary immunization, mice from each group that had been vaccinated twice were euthanized by intraperitoneal overdose of Tytozol (200 μL), and blood samples were taken from the heart, and the coagulated blood samples were centrifuged at 5000 rpm for 10 minutes to prepare serum. Although the vaccine was administered twice in total, it was determined that a further booster immunization test was unnecessary because sufficient results were obtained from the secondary immunization in the infection protection test to investigate the survival rate of the mice.

[0059] (Evaluation of anti-HA specific IgG antibody titer 1) To quantify the anti-HA-specific IgG antibody titer, serum was prepared from each of the mice described above, and then HA antigen-specific IgG was measured according to the method described in J Immunol. 2006;176:1122-30.

[0060] For the serum prepared from the blood of each group of mice, ELISA was performed using Mouse ELISA quantitation (BETHYL LABORATORIES, USA) according to the attached instruction manual. Specifically, 0.1 μg of HA antigen protein was added as an antigen to each well of a 96-well Nunc immunoplate (Nalgen Nunc International, USA), and then 1 μg / mL of bovine serum albumin (BSA, SIGMA, USA) and 100 μL of PBS solution were added, and the solid-phase reaction was carried out overnight at 4°C. The plate was then rinsed five times with a washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0). Then, 200 μL of 50 mM Tris-HCl buffer (pH 8.0) containing 0.14 M NaCl and 200 μL of 1% BSA was added to each well, and a blocking reaction was carried out at 4°C for 18 to 24 hours. After that, each well was rinsed five times with the above-mentioned washing solution, and 100 μL of serum was added with sample binding buffer (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0), and then serial two-step dilutions were carried out to prepare specimen dilutions with a final dilution ratio of 128,000 to 1,280,000. The specimen dilutions were reacted with the solid-phase antigen at 4°C for 16 to 18 hours. Goat anti-mouse IgA-horse radish peroxidase (HRP-IgA) or goat anti-mouse IgG-horse radish peroxidase (HRP-IgG) (BETHYL LABORATORIES INC.) was used as the secondary antibody, and the color reaction was carried out using TMB Microwell Peroxidase Substrate System (Kirkegaard & Perry Laboratories, Inc., USA). The reaction was stopped by adding 50 μL of 2 M H2SO4 (Wako Pure Chemical Industries, Ltd.) to each well, and the absorbance at 450 nm was measured using SPECTRA max PLUS 384 (MOLECULAR DEVICES). The results of the antibody titer measurement (titer notation) are shown in Figure 1 (a).

[0061] Furthermore, the antibody titer measurements in WO2011 / 08521 were performed according to the method described in J. Immunol. 2006;176:1122-30. A purified standard anti-HAIgG antibody against A / California (H1N1) antigen protein was prepared in advance and used as a calibration curve for ELISA, and the results were shown as the amount of anti-HAIgG antibody (μg / mL) calculated from the above-mentioned absorbance values ​​shown by the samples. In accordance with this method, the antibody titer shown in Figure 1(a) was recalculated in terms of titer (μg / mL), and the results are shown in Figure 1(b).

[0062] (result) The following description will be given with reference to FIG. 1) Group administered HA solution alone (reference example) In the HA alone administration groups administered with HA alone solution in which the antigen protein concentration [HA] of the HA solution in Comparative Example 1 above was (d) 0.1 μg / 100 μL, (e) 0.5 μg / 100 μL, and (f) 1.0 μg / 100 μL, the antibody titers (titers) of anti-HA-specific IgG antibodies were all 36,758, with no statistically significant difference between the respective groups, suggesting that the antigen protein concentration [HA] of the HA solution in the HA alone administration group plateaued at 0.1 μg / 100 μL or higher.

[0063] 2) HA + Alm-containing solution administration group (reference example) In the HA+Alm-containing solution administration group in which the antigen protein concentration [HA] of the HA solution in Comparative Example 2 was (g) 0.5 μg / 100 μL and (h) 1.0 μg / 100 μL, the antibody titers (titers) of anti-HA specific IgG antibodies were 97006 and 73517, but there was no statistically significant difference between the two groups, suggesting that the antigen protein concentration [HA] of the HA solution in the HA+Alm-containing solution administration group shows a plateau at 0.5 μg / 100 μL or more. In addition, when compared with the above-mentioned solution administered with HA alone, no significant difference was observed in the IgG antibody induction effect (P = 0.072), but it was confirmed that it was slightly amplified.

[0064] 3) Group 1 administered with HA+SF-10 solution In the HA+SF-10-containing solution administration group in which the antigen protein concentration [HA] of the HA solution in Example 1 above was (a) 0.1 μg / 100 μL, (b) 0.5 μg / 100 μL, or (c) 1.0 μg / 100 μL, and in which the CVP concentration was 0.1 mass% and the NaCl concentration was 0.9 mass%, the antibody titer (titer) of anti-HA-specific IgG antibody increased as the HA concentration increased, but was 235253 in both cases when [HA] was (b) 0.5 μg / 100 μL and (c) 1.0 μg / 100 μL, suggesting that the titer nearly plateaued at 0.5 μg / 100 μL or above. This plateau value was 6.4 times (P = 0.0012) higher than the IgG antibody titer of 36,758 in the HA alone solution-administered groups (d) to (f), and 3.2 times (P = 0.031) higher than the HA + Alm ([HA] 1.0 μg / 100 μL)-administered group (h) in the HA + Alm-containing solution-administered group, demonstrating a significant difference in the high induction effect of anti-HA-specific IgG antibodies in the HA + SF-10-containing solution-administered group.

[0065] The following description will be made with reference to FIG. In the HA+SF-10-containing solution administration group, in which the antigen protein concentration ([HA]) of the HA solution in Example 1 was (a) 0.1 μg / 100 μL, (b) 0.5 μg / 100 μL, or (c) 1.0 μg / 100 μL, and in which the CVP concentration was 0.1 mass% and the NaCl concentration was 0.9 mass%, the antibody titer (μg / mL) of anti-HA-specific IgG antibody increased as the HA concentration increased. In the case of (a) 0.1 μg / 100 μL, it was 2597 μg / mL, in the case of (b) 0.5 μg / 100 μL, it was 4997 μg / mL, and in the case of (c) 1.0 μg / 100 μL, it was confirmed that it almost plateaued at 0.5 μg / 100 μL or more.

[0066] In this example (Figure 1(b)), the antibody titer against the A / Singapore / GP1908 / 2015 (H1N1) strain was quantified using the ELISA standard curve for the A / California (H1N1) strain shown in WO2011 / 08521, which is a different virus strain but the same H1N1 strain, since there is no purified standard antibody. Since there is almost no difference in the HA molecular weight between H1N1 strains, the antibody titer (μg / mL) value is considered to be within the margin of error.

[0067] The above results were compared with the serum immune IgG induction effect of the mucosal vaccine described in WO2011 / 08521. In Table 3 of WO2011 / 08521, when 0.1 μg of [HA], 1 μg of phospholipid, and 20 μg of CVP (0.5% by mass) were administered to mice as a nasal mucosal vaccine, the antibody titer (μg / mL) of anti-HA specific IgG antibodies induced in the serum was 588.61 μg / mL, whereas in the administration group (a) of FIG. 1(b), when [HA] was 0.1 μg / 100 μL, the titer was 2597 μg / mL. This confirmed that the IgG induction effect of subcutaneous inoculation was about 4.4 times better than that of mucosal (intranasal) inoculation. Both groups had the same conditions of 0.1 μg [HA] and 1 μg phospholipid, but the CVP concentration was 0.5% by mass in Table 3 of WO2011 / 08521 and 0.1% by mass in Figure 1(b). However, as shown in Figure 2, there was no significant difference between the CVP concentrations of 0.1% and 0.5% by mass for subcutaneous administration, so the above conclusion was inferred to be correct.

[0068] [Example 3] [Study on CVP and NaCl concentrations in HA+SF-10-containing solution] Since the thickening and sustained release effects of CVP may depend on the NaCl concentration as well as the CVP concentration, we decided to explore the optimal combination of CVP and NaCl concentrations in the HA+SF-10-containing administration solution. The amount of HA antigen protein contained in 100 μL of the HA+SF-10-containing administration solution was fixed at 1 μg, the amount of phospholipid was fixed at 10 μg, and the V / A ratio was set to 10. The CVP concentrations were 0.01 mass%, 0.1 mass%, or 0.5 mass%. As the concentrations of the NaCl solution in which the CVP and the antigen HA are dissolved, two types were used: physiological saline (0.9% NaCl aqueous solution) and a 0.1% NaCl aqueous solution obtained by diluting this with MilliQ water. The details of the combinations of the formulations of each of the HA+SF-10-containing solutions prepared (i) to (l) are shown in Table 4 below. Note that the pH of the CVP solution of HA+SF-10 was adjusted to pH 7.0 to pH 7.2 by adding NaOH as necessary to maintain the antigenicity of HA.

[0069] [Table 4]

[0070] [Example 4] [Vaccination of mice 2] Each administration group consisted of 5 to 6 mice, and 100 μL of each of the above mouse subcutaneous administration solutions (i) to (l) was administered subcutaneously to the back of the neck of each group of mice as a first immunization. Two weeks after the first immunization, 100 μL of the mouse subcutaneous administration solution of the same composition was administered to the back of the neck of the mice that had been administered the vaccine as a first immunization as a second vaccine (secondary immunization).

[0071] (Serum preparation 2) Serum from each mouse for evaluation of the antibody titer of anti-HA-specific IgG antibody was prepared according to the procedure described in (Serum Preparation 1) in Example 2 above.

[0072] (Evaluation of anti-HA specific IgG antibody titer 2) Quantitation of the antibody titer of anti-HA-specific IgG antibody was also performed according to the procedure described in (Evaluation of antibody titer of anti-HA-specific IgG antibody 1) in Example 2 above. The results are shown in Figure 2.

[0073] (result) As is clear from FIG. 2, it was confirmed that in each of the above HA+SF-10-containing solution administration groups (i) to (l), as the CVP concentration increased to 0.01% by mass, 0.1% by mass, and 0.5% by mass, the anti-HA-specific IgG antibody induction effect tended to become higher. It was also shown that the CVP concentration plateaued at 0.1% by mass or more. Among the above combination conditions (i) to (l), when 0.1% by mass of CVP and 0.1% by mass of NaCl solution were used in (k), the IgG antibody titer was 531,400, showing the highest IgG antibody induction effect. This value was 14 times higher than the antibody titer of 38,055 in the HA only solution administration group (f) and 8.3 times higher than the antibody titer of 64,000 in the HA+Alm solution administration group (h). The above (i) to (l) groups administered subcutaneously with HA+SF-10 showed a significantly higher IgG antibody induction effect than the groups administered with HA alone and with HA+Alm-containing solution.

[0074] [Example 5] [Verification of infection prevention effect] The infection protection effect of subcutaneous influenza vaccine was verified using HA+SF-10-containing solution (a) (0.1 μg / 100 μL [HA], 0.1% CVP by mass, 0.9% NaCl by mass), which showed a high IgG antibody induction effect, and HA+SF-10-containing solution (c) HA+SF-10 (1.0 μg / 100 μL [HA], 0.1% CVP by mass, 0.9% NaCl by mass). The HA+Alm-containing solution (h) (1.0 μg / 100 μL [HA], 1% Alm by mass, 0.NaCl) and HA only solution (f) (1.0 μg / 100 μL [HA], 0.9% NaCl by mass) were used for comparison.

[0075] [Vaccination of mice 3] Each administration group consisted of 5-6 mice, and 100 μL of each of the above (a), (c), (h), and (f) mouse subcutaneous administration solutions was administered subcutaneously to the back of the neck of each group of mice as a first immunization. Two weeks after the first immunization, 100 μL of the mouse subcutaneous administration solution of the same composition was administered subcutaneously to the back of the neck of the mice that had been administered the vaccine as a first immunization as a second vaccine (secondary immunization).

[0076] [Nasal infection of influenza virus] 20×LD on the 14th day after the second vaccination (28th day after the first day). 50 Each mouse was intranasally infected with 20 PFU (plaque forming unit) of influenza virus (A / PR8(N1H1) / 20 μL), which corresponds to 100 μL of influenza virus per nasal passage, and the changes in the antibody titer and survival rate of the mice over the following 14 days were examined. The results regarding the antibody titer of anti-HA-specific IgG antibody are shown in FIG. 3(A), and the results regarding the survival rate are shown in FIG. 3(B). The PR8 strain is derived from the human-derived Spanish flu H1N1 strain, but is a highly virulent laboratory virus strain that has been adapted to infect mice, and is widely and commonly used in animal experiments of human influenza viruses.

[0077] (Evaluation of anti-HA specific IgG antibody titer 3) Serum was prepared from blood collected from the hip vein of each mouse immediately before the virus infection according to the procedure described in Example 2 (Serum Preparation 1), and the anti-HA IgG antibody titer induced in the mouse blood immediately before the infection was quantified according to the procedure described in Example 2 (Evaluation of Anti-HA-specific IgG Antibody Titer 1). The results are shown in Figure 3(A). The survival rate curve of the infected mice over time is shown in Figure 3(B).

[0078] (result) As is clear from Figure 3(A), the anti-influenza HA IgG antibody titer was 6.4 times higher in the HA+SF-10-containing solution (c) administration group (titer: 235,253) than in the HA only solution (f) administration group (36,758), and was 3.2 times higher than in the HA+Alm-containing solution (h) administration group (titer: 73,517), in the same way as the results shown in Figure 1. The HA+SF-10-containing solution (a) administration group (titer: 84,449), in which the amount of HA antigen was reduced to 1 / 10 (0.1 μg), also showed an anti-influenza HA-specific IgG antibody titer that was 1.15 times higher than in the HA+Alm-containing solution administration group (h), indicating that even if the amount of HA antigen administered is reduced to 1 / 10, the vaccine is expected to be as effective as or more effective than conventional subcutaneous influenza vaccines.

[0079] As is clear from Figure 3 (B), which shows the survival rate of mice over time, the group of mice administered HA+Alm-containing solution (h) and the group of mice administered HA only solution (f) both showed a survival rate of 40% on day 14 after virus infection, whereas the group of mice administered HA+SF-10 (a) and the group of mice administered HA+SF-10 (c) both showed a survival rate of 100%, demonstrating superior infection protection and survival rate among the groups administered subcutaneously with HA+SF-10. Therefore, it was determined that subcutaneous administration of 0.1 μg of antigen protein from HA solution was sufficient to maintain survival.

[0080] (Weight change after intranasal infection with influenza virus) The weight changes after influenza infection were examined for each group of mice administered HA alone (f), HA+Alm-containing solution (h), HA+SF-10-containing solution (a) (0.1 μg / 100 μL [HA], 0.1% CVP by mass, 0.9% NaCl by mass), and HA+SF-10-containing solution (c) (1.0 μg / 100 μL [HA], 0.1% CVP by mass, 0.9% NaCl by mass). The results are shown in Figure 4.

[0081] As is clear from Figure 4, differences in weight change during the recovery period from infection on the 8th day after influenza infection were evident between the groups. Among them, the group administered with HA only solution (f) had the worst weight recovery, which was presumably related to the fact that it had the lowest increase in antibody titer. In terms of antibody induction effect, the HA+SF-10-containing solution (c) group (1.0 μg / 100 μL [HA], 0.1% CVP by mass, 0.9% NaCl by mass) showed the highest value compared to the other groups, but in terms of weight gain during the recovery period, there was no significant difference between the HA+SF-10-containing solution (a) group (0.1 μg / 100 μL [HA], 0.1% CVP by mass, 0.9% NaCl by mass), and it was presumed that if the antibody induction effect reached the level shown by group (a), the survival rate would be 100% and weight recovery would also be at a constant rate. In the case of the HA+Alm-containing solution (h) group, the antibody induction effect was similar to that of the (a) group, but the survival rate was a low value of 40%, demonstrating the significantly beneficial effect of the HA+SF-10-containing solution (a) on the survival rate.

[0082] [Example 6] [Study of CVP concentration in vaccine administration solution] In an infection experiment using a subcutaneous vaccine containing SF-10, we investigated the minimum CVP concentration that would achieve 100% mouse survival rate.

[0083] [Vaccination of mice 4] In Example 3, the CVP concentration and NaCl concentration of the HA+SF-10-containing solution were examined. When the CVP concentration exceeded 0.1% by mass, especially at 0.5% by mass, induration may occur after subcutaneous vaccination, and in rare cases, even at 0.1% by mass, some induration-like phenomenon was observed. This could not be denied as the possibility that some CVP, which takes time to be absorbed at the vaccination site, remained. Therefore, a CVP concentration with a 100% survival rate and no possibility of induration was examined. Details are shown in Table 5 below.

[0084] [Table 5]

[0085] Six mice were treated as one administration group, and 100 μL of each of the above mouse subcutaneous administration solutions (m) to (p) was administered subcutaneously to the neck and back of the mice in each group as a first immunization. Two weeks after the first immunization, 100 μL of the mouse subcutaneous administration solution of the same composition was administered subcutaneously to the neck and back of the mice that had been administered the vaccine as a first immunization as a second vaccine (secondary immunization). The results are shown in Figures 5-1 and 5-2.

[0086] (result) As shown in Figure 5-1, the IgG antibody induction values ​​of the HA+SF-10 containing solution administration group (m) (0.1μg HA, 0.05% CVP by mass, 0.1% NaCl by mass) and the HA+SF-10 containing solution administration group (n) (0.1μg HA, 0.05% CVP by mass, 0.9% NaCl by mass) tended to be higher in the vaccine solution containing 0.1% NaCl by mass than in the vaccine solution containing 0.9% NaCl by mass, although there was no significant difference. However, the infection protection effect showed a 100% survival rate in both groups, and no difference was observed.

[0087] FIG. 5-2 shows the results when the CVP concentration was further reduced and fixed at 0.01% CVP by mass. The IgG antibody induction value shown by the HA+SF-10-containing solution administration group (o) (0.1 μg HA, 0.01% CVP by mass, 0.1% NaCl by mass) and the HA+SF-10-containing solution administration group (p) (0.1 μg HA, 0.01% CVP by mass, 0.9% NaCl by mass) was only about twice as high as the antibody induction value shown by HA alone in group (f), indicating that the CVP concentration has a large effect on antibody induction. Here again, the vaccine solution containing 0.1% NaCl by mass showed a slightly higher tendency than the vaccine solution containing 0.9% NaCl by mass, although there was no significant difference. As for the infection protection effect, the survival rate was reduced to 35% in both groups (o) and (p), and it was determined that the conditions were not strong enough to show an infection protection effect.

[0088] From the above results, it was found that the CVP concentration is higher than 0.01% CVP by mass, and the optimum concentration is between 0.1 and 0.05% by mass, and a clear decrease in survival rate is observed with 0.01% CVP by mass. In addition, in the optimal condition of 0.1 to 0.05% CVP by mass solution, both 0.1% NaCl by mass and 0.9% NaCl by mass are acceptable, but as shown in the example of Figure 5-1, the 0.1% NaCl solution tended to show a higher value in the anti-influenza HA IgG antibody induction value compared to 0.9% NaCl by mass, although there was no significant difference. In either case, no induration was observed at the administration site. From the above, it was determined that the risk of induration can be avoided while maintaining a high survival rate by setting the CVP concentration to 0.1 to 0.05% by mass.

[0089] [Reference example] On the other hand, Fig. 6 shows the results of calculating the anti-influenza HA IgA antibody titer in the serum when two immunizations (on days 0 and 14) were performed under the same conditions as in Example 2. For comparison, tests were performed with orally administered HA+SF-10 and airway administered HA+SF-10, which are known to significantly induce anti-influenza HA IgA antibody titers in the serum, with a V / A ratio of 10.

[0090] The dose of antigen protein in the HA solution was 1.0μg for both oral and airway administration, the same as for subcutaneous administration. The vaccine dose was changed depending on the method of administration, with 100μL for subcutaneous administration, 200μL for oral administration, and 15μL for airway administration, for a total of 30μL. As for CVP, the concentrations of 0.01%, 0.1%, and 0.5% were examined for the subcutaneous vaccine, as in Figure 2, contained in HA+SF-10, and combinations of 0.9% NaCl and 0.1% NaCl were examined. The CVP for the oral administration solution was 0.5% and 0.9% NaCl, and the CVP for the airway administration was 0.1% and 0.1% NaCl. The results are shown in Figure 6.

[0091] (result) As is clear from Figure 6, the serum anti-influenza HA IgA antibody titer did not increase in either the subcutaneously administered HA+SF-10 vaccine or the HA antigen alone, and it was determined that these were ineffective in inducing IgA antibody titers. On the other hand, a clear induction effect was confirmed with the oral administration of the HA+SF-10 vaccine and the airway administration of the HA+SF-10 vaccine, but the airway administration was observed to induce approximately 2.7 times higher IgA antibody titers than the oral administration. [Industrial Applicability]

[0092] The vaccine of the present invention is very useful in the medical field. It is common practice in the art to estimate the dose to be administered to humans using various known methods, such as the HED conversion method, which estimates the dose that produces the same effect in humans from the body surface area of ​​a test animal such as a mouse, and to accumulate further experimental data to advance practical application.

Claims

1. A subcutaneously administered vaccine comprising an antigen protein and an adjuvant comprising the following (a) to (c): (a) a synthetic peptide consisting of the amino acid sequence of KnLm (wherein n is 4-8 and m is 11-20); (b) a phospholipid-containing liquid; (c) Carboxyvinyl polymer

2. 2. The subcutaneous vaccine according to claim 1, which induces circulating IgG antibodies but not IgA antibodies.

3. 3. The subcutaneous vaccine according to claim 1 or 2, wherein the synthetic peptide consisting of the amino acid sequence of KnLm consists of the amino acid sequence shown in SEQ ID NO: 1 or 2.

4. 4. The subcutaneous vaccine according to claim 1, wherein the synthetic peptide consisting of the amino acid sequence of KnLm is present in a mass ratio of the phospholipid-containing liquid to the synthetic peptide of 100:0.1-50.

5. The subcutaneous vaccine according to any one of claims 1 to 4, characterized in that the phospholipid in the phospholipid-containing liquid is one or more selected from the group consisting of dipalmitoylphosphatidylcholine, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid.

6. The subcutaneous vaccine according to any one of claims 1 to 5, characterized in that the phospholipid-containing liquid further contains one or more types of lipids other than phospholipids selected from lauric acid, myristic acid, palmitic acid, stearic acid, palmitoleic acid, and oleic acid.

7. 7. The subcutaneous vaccine according to claim 1, wherein the antigen protein is a protein derived from a pathogen, an inactivated antigen protein, a recombinant antigen protein, or a detoxified toxin protein.

8. 8. The subcutaneous vaccine according to claim 1, wherein the antigen protein is contained in an amount of 0.05 μg to 5 μg / 100 μL.

9. 9. The subcutaneous vaccine according to claim 1, wherein the mass ratio of the phospholipid-containing liquid to the antigen protein is 0.1 to 20.

10. 10. The subcutaneous vaccine according to claim 1, wherein the carboxyvinyl polymer is contained in an amount of 0.02% by mass to 2.0% by mass.

11. A method for producing a subcutaneous vaccine comprising the following steps (a) to (c) in sequence: (a) preparing an SSF suspension by suspending a synthetic peptide consisting of the amino acid sequence of KnLm (wherein n is 4-8 and m is 11-20) and a phospholipid-containing liquid in water; (b) adding an antigen protein to the SSF suspension prepared in step (a), and shaking and mixing to prepare an antigen protein-SSF uniform suspension; (c) adding a carboxyvinyl polymer solution to the antigen protein-SSF uniform suspension prepared in step (b) to prepare a subcutaneous administration vaccine containing the antigen protein and an adjuvant;

12. After step (b), (b') freeze-drying the antigen protein-SSF uniform suspension prepared in the step (b) to prepare a freeze-dried antigen protein-SSF; and, prior to step (c), (b") a step of suspending the antigen protein-SSF lyophilized product prepared in step (b') in water or physiological saline to prepare a uniform suspension of the antigen protein-SSF; The method for producing a subcutaneous vaccine according to claim 11, further comprising:

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