WO2018225785A1 - Vaccine composition - Google Patents

Abstract

The purpose of the present invention is to provide an excellent vaccine composition. This vaccine composition contains an antigen for inducing immunity, and a gas component which is a gas at 25°C and 100kPa.

Description

Vaccine composition

The present invention relates to a vaccine composition.

Conventionally, many vaccine compositions have been used for the prevention or treatment of infectious diseases. On the other hand, in recent years, it has become clear that molecules that are gases at normal temperature and pressure, such as nitric oxide, carbon monoxide, carbon dioxide, hydrogen sulfide, and methane, act on the innate immune mechanism.

Christian Bogdan, Nitric oxide and the immuno response, Nature Immunology, vol. 2, no. 10, 2001 Madhav Bhatia, Role of Hydrogen Sulfide in the Pathology of Inflation, Scientifica, Volume 2012

The object of the present invention is to provide an excellent vaccine composition.

Aspects of the present invention are as follows, for example.
[1] A vaccine composition comprising an antigen for immunity induction and a gas component which is a gas at 25 ° C. and 100 kPa.
[2] The vaccine composition according to [1], wherein the gas component is an immune signaling substance.
[3] The vaccine composition according to [2], wherein the immune signaling substance is a factor that acts on keratinocytes, monocytes, lymphocytes, or granulocytes.
[4] The vaccine composition according to any one of [1] to [3], further comprising a metal organic structure.
[5] The vaccine composition according to [4], wherein at least a part of the gas component is contained in pores of the metal organic structure.
[6] The vaccine composition according to any one of [1] to [5], which is configured to be administered onto the skin and / or mucous membrane.
[7] The vaccine composition according to any one of [1] to [5], which is configured to be administered by intradermal injection, subcutaneous injection, or intramuscular injection.

According to the present invention, it is possible to provide an excellent vaccine composition.

FIG. 1A is a CO adsorption profile of metal organic structure AP004 [MIL-100 (Fe)]. FIG. 1B is a NO adsorption profile of metal organic structure AP004 [MIL-100 (Fe)]. FIG. 2 is a NO adsorption profile of metal organic structure AP104 (BioMIL-3). FIG. 3 is a view showing the measurement results of antigen-specific antibody titers in mouse serum. FIG. 4A is a diagram showing the results of measurement of OVA-specific cytokine production. FIG. 4B is a diagram showing the measurement results of OVA-specific cytokine production.

Hereinafter, the vaccine composition according to one embodiment of the present invention will be described.

The vaccine composition according to the present invention contains an antigen for inducing immunity and a gas component that is a gas at 25 ° C. and 100 kPa (SATP).

Antigens include any substance that can induce an immune response. Although the said antigen is not specifically limited, For example, protein or a peptide is mentioned. In transdermal administration where the skin permeability of the antigen is required, it is preferable to use an antigen having a low molecular weight, for example, a peptide consisting of about 8 to 12 amino acids can be used. In addition, cancer antigen peptides, infectious pathogen-derived antigens, and the like can also be used as the antigen.

Alternatively, self antigens (for example, antigens related to autoimmune diseases), endogenous antigens (for example, antigens derived from cancer), foreign antigens (for example, antigens related to allergies, and antigens derived from viruses and bacteria) can be used as antigens. .

Antigens related to autoimmune diseases include, for example, amyloid β and / or precursors thereof and fragment proteins and peptides thereof, which are considered to be the cause of Alzheimer's disease, α-synuclein and fragment proteins thereof which are considered to be the cause of Parkinson's disease And peptides, α-fodrin considered to be the cause of Sjogren's syndrome and its fragment proteins and peptides, thyroid hormone receptor and its fragment proteins and peptides considered to be the cause of Passow's disease, and causes of Guillain-Barre syndrome Gangliosides considered and fragmented proteins and peptides thereof, DNA and fragments thereof considered to cause systemic lupus erythematosus, cholesterol ester transfer protein considered to cause arteriosclerosis, apolipoprotein, and LDL and their fragment proteins and peptides, angiotensin I / II which is considered to cause hypertension and its fragment proteins and peptides, insulin, GAD and IL-1β which are considered to cause type 1 diabetes, and Fragment proteins and peptides, acetylcholine receptor and fragment proteins and peptides thereof considered to be the cause of myasthenia gravis, TNFα and IL-6 and fragment proteins and peptides thereof that are considered to be the cause of rheumatoid arthritis, Examples include TRANCE and RANKL, which are considered to be caused by osteoporosis, and their fragment proteins and peptides.

Examples of cancer-derived antigens include WT1, PR1, GPC3, HER-2, MAGE-A1, MAGE-A2, MAGE-A3, tyrosinase, gp100, CEA, hTRT, EGF receptor, mTERT, PRAME, PSMA, PSA- 1, cytochrome p450, NY-ESO-1, Survivine, MUC-1, MAGE-A10, and PAP-derived proteins and peptides.

Antigens related to allergies include, for example, allergens derived from trees (Acacia, alder tree, velvet blue-tailed potato, Japanese beech, birch, maple, mountain cedar, red cedar, boxwood, hinoki, American elm, Akinori, Togasawara, rubber, eucalyptus , Enoki, Hickory, American linden, Sugar maple, Mesquite, Casino cypress, Quercus, Olive, Pecan, Pepper, Pine, Squid, Russian olive, American sycamore, Sardine, Black walnut, Black willow, etc., Allergens derived from vegetation , Nagahagusa, Suzumenochahiki, Maize, Hirohoshi nokegusa, Seiban sorghum, Oats, Camouflage, Konukagusa, Barley, Rice, Hurghaya, Oawagaeri, Ayuyu, Akaza, Onamomi, Gishigishi, Allergens (wormworms, mites, honeybees, honeybees, etc.) derived from insects Allergens derived from fungi (Alternaria, Aspergillus, Botulinum, Candida, Cephalosporium, Carbularia, Epicoccum, Epidermis, Fusarium, Helmundosporumium, Chain Cladosporium, Kekabi, Penicillium, Pulluliapururans, Kumonosukabi, etc.) , Animal hair allergens (dogs, cats, birds, etc.), house dust-derived allergens, food-derived allergens, and haptens involved in metal allergy

Diseases affected by the infectious pathogen include, for example, adenovirus, herpes virus (eg, HSV-I, HSV-II, CMV, or VZV), poxvirus (eg, pressure ulcer or vaccinia, or contagious molluscum) Orthopoxvirus), picornavirus (eg rhinovirus or enterovirus), orthomyxovirus (eg influenza virus), paramyxovirus (eg parainfluenza virus, mumps virus, measles virus, respiratory organs) Endoplasmic reticulum virus (RSV)), coronavirus (eg SARS), papovavirus (eg papilloma virus such as those causing genital warts, vulgaris warts or plantar costus), hepadnavirus (eg hepatitis) B virus), Viral diseases, such as diseases resulting from viral infections such as Ravivirus (eg, hepatitis C virus or dengue virus) or retrovirus (eg, lentivirus such as HIV); Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Streptococcus pneumoniae, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Bacterial diseases such as diseases caused by bacterial infections such as Brucella, Yersinia, Haemophilus, or Bordetella; Chlamydia, candidiasis, aspergillosis, histop Suma diseases, fungal diseases such as cryptococcal meningitis; malaria, pneumocystis carinii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection, and the like.

Examples of particularly suitable antigens include ovalbumin (OVA), pneumococci, influenza vaccine, Cryj1 (a major allergen of cedar pollen), and HPV16 recombinant protein.

Only one type of antigen may be used, or two or more types may be used in combination. The content of the antigen in the vaccine composition is, for example, in the range of 1 × 10 ⁻⁷ to 1 × 10 ⁻¹ mass%, preferably in the range of 1 × 10 ⁻⁶ to 1 × 10 ⁻² mass%, More preferably, it is in the range of 2 × 10 ⁻⁶ to 2 × 10 ⁻³ mass%.

As the gas component described above, for example, one that can function as an immune signal transmitting substance can be used. Here, “immune signaling substance” means any substance used for transmitting an immune signal for inducing activation or differentiation of immune cells.

Immune signaling substances include, for example, lymphocytes (T cells, B cells, NK cells, etc.), monocytes (macrophages, Langerhans cells, dendritic cells, etc.), granulocytes (neutrophils, eosinophils, basophils). And / or factors that act on keratinocytes. Immune signaling substances are used for, for example, inducing differentiation of helper T cells, which are a type of lymphocyte, into various lineages such as Th1 cells, Th2 cells, Treg cells, Th17 cells, Tfh cells, and memory T cells. It may be a factor. When the immune signaling substance induces Th1 cells, the vaccine composition according to the present invention can be used, for example, for cancer vaccines and infectious disease vaccines. When the immune signaling substance induces Th2 cells, the vaccine composition according to the present invention can be used, for example, for infectious disease vaccines and lifestyle-related disease vaccines. When the immune signaling substance induces Treg cells, the vaccine composition according to the present invention can be used, for example, for an allergy vaccine. When the immune signaling substance induces Th17 cells, the vaccine composition according to the present invention can be used, for example, for an infectious disease vaccine. When the immune signaling substance induces Tfh cells, the vaccine composition according to the present invention can be used, for example, for an infectious disease vaccine. When the immune signaling substance induces memory T cells, the vaccine composition according to the present invention can be used, for example, for an infectious disease vaccine or a cancer vaccine.

The type of gas component described above is not particularly limited, and examples thereof include compounds shown in Table 1 below. These are non-limiting lists, and other gas molecules may be used. Of these, it is particularly preferable to use nitrogen monoxide, carbon monoxide, carbon dioxide, hydrogen sulfide or methane as the gas component.

Only one type of gas component may be used, or two or more types may be used in combination. The content of the gas component in the vaccine composition is, for example, in the range of 1 × 10 ⁻⁷ to 40% by mass, preferably in the range of 1 × 10 ⁻⁶ to 30% by mass, and more preferably 5 × 10 ^{− It is} within the range of ⁵ to 25% by mass.

The vaccine composition according to one embodiment of the present invention may further include a metal organic structure. When such a configuration is adopted, the performance of the vaccine composition can be improved by the action of the metal and the ligand in the metal organic structure. In addition, by including at least a part of the gas component in the pores of the metal organic structure, the gas component can be more stably and quantitatively administered into the living body.

The metal organic structure is composed of a combination of a metal and a multidentate ligand. Here, “multidentate ligand” means a bidentate or higher ligand.

There is no particular limitation on the type of the metal organic structure. By appropriately combining the type and coordination number of the metal ion with the type and topology of the polydentate ligand, a metal organic structure having a desired structure can be produced. The metal organic structure may be crystalline or amorphous.

Examples of the metal elements constituting the metal organic structure include arbitrary elements belonging to alkali metals (Group 1), alkaline earth metals (Group 2), and transition metals (Groups 3 to 12). Can be mentioned. Among these, from the viewpoint of biocompatibility, it is particularly preferable to use at least one metal element selected from the group consisting of calcium, magnesium, iron, zinc, aluminum, potassium, and sodium. However, even when a metal element other than these is used, there is no problem as long as biocompatibility as a metal organic structure is ensured.

The polydentate ligand constituting the metal organic structure is typically an organic ligand, and examples thereof include a carboxylate anion and a heterocyclic compound. Examples of the carboxylic acid anion include dicarboxylic acid and tricarboxylic acid. Specific examples include anions of citric acid, malic acid, terephthalic acid, isophthalic acid, trimesic acid, and derivatives thereof. Examples of the heterocyclic compound include bipyridine, imidazole, adenine, and derivatives thereof. Alternatively, the ligand may be an amine compound, a sulfonate anion, or a phosphate anion. The metal organic structure may further contain a monodentate ligand.

The combination of the metal and the ligand constituting the metal organic structure can be appropriately determined according to the function and the desired pore size. In addition, the metal organic structure may contain 2 or more types of metal elements, and may contain 2 or more types of ligands. The metal organic structure may be surface-modified with a polymer or the like.

As specific examples of the metal organic structure, for example, those listed in Table 1 of non-patent literature (Yabing He et al., Methane Storage in Metal-Organic Frameworks, Chem Soc Rev, 2014) can be used. . Or you may use the thing shown to the following Tables 2 thru | or 4 as a metal organic structure. These are non-limiting lists, and other metal organic structures may be used.

Particularly preferable metal organic structures include the following.

Only one type of metal organic structure may be used, or two or more types may be used in combination. The content of the metal organic structure in the vaccine composition is, for example, in the range of 1 × 10 ⁻⁷ to 99.99999 mass%, preferably in the range of 1 × 10 ⁻⁶ to 99.99999999 mass%, and more Preferably, it is within the range of 5 × 10 ⁻⁶ to 99.99999 mass%.

As described above, it is preferable that at least a part of the gas component is contained in the pores of the metal organic structure. Thereby, more stable and quantitative administration of the gas component becomes possible. The other part of the gas component may be attached to the surface of the antigen or metal organic structure. Further, almost all of the gas component may be contained in the pores of the metal organic structure.

When at least a part of the gas component is contained in the pores of the metal organic structure, the metal organic structure preferably has an irreversible adsorption / desorption profile. That is, the metal organic structure preferably has a larger amount of adsorption at the time of desorption than the amount of adsorption at the time of adsorption at the same pressure. In particular, it is preferable that the residual amount of adsorption of the metal organic structure is not zero when desorption from the pressurized state to the vacuum state is performed after the adsorption from the vacuum state to the pressurized state. In such a case, the gas component is easily retained in the pores of the metal organic structure even under low pressure conditions (for example, under atmospheric pressure).

Further, when at least a part of the gas component is contained in the pores of the metal organic structure, the metal organic structure is configured to decompose in the living body and release at least a part of the gas component. It may be. Thereby, fine adjustments, such as a dosage of a gas component and a release rate, can be performed.

In addition, when at least a part of the gas component is contained in the pores of the metal organic structure, the method is not limited. For example, a metal organic structure solution or dispersion and a gas component solution or dispersion may be mixed. Alternatively, the solid metal organic structure may be exposed to a gas component or a solution or dispersion thereof.

The vaccine composition according to one embodiment of the present invention may further contain a known adjuvant. The vaccine composition may further contain an immunostimulatory agent such as a TLR ligand, an RLR ligand, an NLR ligand, and a cyclic dinucleotide.

The vaccine composition according to one embodiment of the present invention can be used, for example, in a state of being dissolved or dispersed in a solvent. As the solvent, for example, physiological saline, phosphate buffered saline (PBS), glycerin, propylene glycol, polyethylene glycol, or fats and oils can be used.

The vaccine composition according to the present invention can be administered to a subject by any method. As used herein, “subject” refers to any animal, typically a mammal including humans, such as mice, rats, dogs, cats, rabbits, horses, which can induce an immune response upon administration of the vaccine composition in the practical stage. Meaning cow, sheep, pig, goat, monkey, chimpanzee, ferret, mole, etc. A particularly preferred subject is a human.

The vaccine composition according to one embodiment of the present invention is configured to be administered onto the skin and / or mucous membrane, for example.

In the case of transdermal administration, the vaccine composition may be any preparation usually used for transdermal administration, for example, liniments or lotions, external sprays such as aerosols, ointments, plasters, creams. , Gels, or patches such as tapes or poultices. The classification, definition, properties, production method and the like of these compositions are well known in the art, and refer to, for example, the Japanese Pharmacopoeia 16th edition.

When performing mucosal administration, the vaccine composition may be any formulation commonly used for mucosal administration, eg sublingual, nasal, buccal, rectal or vaginal administration, eg gel (jelly), cream, ointment. Semi-solid preparations such as plasters and plasters, liquid preparations, powders, fine granules, granules, solid preparations such as films and tablets, orally disintegrating tablets, sprays for mucous membranes such as aerosols, inhalants, etc. It's okay. The classification, definition, properties, production method and the like of these compositions are well known in the art, and refer to, for example, the Japanese Pharmacopoeia 16th edition.

The vaccine composition according to one embodiment of the present invention is configured to be administered, for example, by intradermal injection, subcutaneous injection, or intramuscular injection. When intradermal, subcutaneous, or intramuscular administration is performed, the pharmaceutical composition may be in a form that has a certain fluidity that can be administered by injection, such as a liquid, suspension, cream, and the like. The classification, definition, properties, production method and the like of these compositions are well known in the art, and refer to, for example, the Japanese Pharmacopoeia 16th edition.

The vaccine composition may further contain an additive as necessary. Depending on the main component of the base, compatibility with the antigen and gas components, the intended administration regimen, etc., for example, skin permeability enhancer, tonicity agent, antiseptic / disinfectant, antioxidant, Solubilizers, solubilizers, suspending agents, fillers, pH adjusters, stabilizers, absorption enhancers, release rate control agents, colorants, plasticizers, adhesives, etc., or combinations of two or more thereof Can be selected.

[Sample preparation]
Example 1
NO (nitrogen monoxide, Kyoto Teijin) was bubbled into 100 mL of physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical) at room temperature for 6 hours to prepare NO saturated physiological saline. To 10 mL of the solution, 1 mg of ZIF-8 (Basolite Z1200, SIGMA-ALDRICH) and 1 mg of OVA (egg-derived albumin, Wako) were added and mixed to obtain a sample solution.

(Example 2)
NO (nitrogen monoxide, Kyoto Teijin) was bubbled in 100 mL of physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical) at room temperature for 6 hours to prepare NO saturated physiological saline. To 10 mL of the solution, 1 mg of OVA (egg-derived albumin, Wako) was added and mixed to obtain a sample solution.

(Comparative Example 1)
Saline (Otsuka raw food injection, Otsuka Pharmaceutical) was used as a sample solution.

(Comparative Example 2)
1 mg of OVA (egg-derived albumin, Wako) was added to and mixed with 10 mL of physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical) to obtain a sample solution.

(Reference Example 1)
ZIF-8 (Basolite Z1200, SIGMA-ALDRICH) 1 mg was added to and mixed with 10 mL of physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical) to obtain a sample solution.

(Reference Example 2)
NO (nitrogen monoxide, Kyoto Teijin) was bubbled in 100 mL of physiological saline (Otsuka raw food injection, Otsuka Pharmaceutical) at room temperature for 6 hours to prepare NO saturated physiological saline. To 10 mL of the solution, 1 mg of ZIF-8 (Basolite Z1200, SIGMA-ALDRICH) was added and mixed to obtain a sample solution.

The above configuration is shown in Table 11 below.

(Examples 3 to 6)
A sample solution was prepared in the same manner as in Example 1 except that the antigen shown in Table 12 below was used.

(Examples 7 to 35)
A sample solution was prepared in the same manner as in Example 1 except that the substances shown in Table 13 below were used as immune signaling substances.

(Examples 36 to 145)
A sample solution was prepared in the same manner as in Example 1 except that the metal organic structures shown in Tables 14 to 16 below were used. Abbreviations in Tables 14 to 16 are the same as those described in Tables 2 to 4, respectively.

[Collection of intraperitoneal cells (PEC cells)]
A mouse was intraperitoneally administered with 2 mL of a 4 wt% thioglycolic acid solution, and cells in the peritoneal cavity were taken out 3 days later. This was washed with PBS (Phosphate Buffered Saline).

[Sample stimulation]
PEC cells were dispensed at 1 × 10 ⁶ cells / well in a 24-well plate, and each sample was added and incubated for 24 hours.

[Cytokine measurement]
Using an ELISA kit (Quantikine ELISA kit, R & D Systems) corresponding to each cytokine (TNF-α, IL-6, IFN-γ, IL-12p40, IL-10), 50 μL / well of cell culture supernatant was used. Evaluation was performed. The results are shown in Table 17 below.

−: Cytokine release amount less than 2 times that of Comparative Example 1 +: Cytokine release amount that is 2 times or more and less than 3 times that of Comparative Example 1 ++: Cytokine release amount that is 3 times or more that of Comparative Example 1

[Measurement of OVA-specific IgG titer in mouse serum (ELISA method)]
100 μL of OVA-containing solution (100 μg / mL) diluted with carbonate buffer was added to a 96-well plate for ELISA, and allowed to stand overnight. The wells were washed three times with a preliminarily prepared washing solution (PBS containing Tween 20), and 200 μL of a blocking solution obtained by diluting a blocking agent (Block Ace, Sumitomo Dainippon Pharma Co., Ltd.) with purified water to 4 g / 100 mL was added at room temperature for 2 hours. I left it alone. Thereafter, the wells were washed three times with a washing solution.

The serum collected from the mouse in advance was centrifuged at 3000 g for 10 minutes at 4 ° C., and the supernatant was collected. Using a solution obtained by diluting the blocking agent to 0.4 g / 100 mL with a phosphate buffer (Nacalai Tesque), serially dilute the above-mentioned supernatant or nasal wash 2 times, and add 50 μL each of the solutions. Left at room temperature for hours.

Thereafter, the wells were washed three times with a washing solution, and an HRP-labeled anti-mouse IgG antibody (Goat-anti mouse IgG Fc HRP, BETHYL) with a solution obtained by diluting the blocking agent to 0.4 g / 100 mL with a phosphate buffer (Nacalai Tesque) Was diluted 10,000 times, added in 100 μL aliquots, and allowed to stand at room temperature for 1 hour. Thereafter, the wells were washed three times with a washing solution, 100 μL of TMB solution (ELISA POD TMB kit, Nacalai Tesque) was added and left in the dark for 30 minutes. Thereafter, 100 μL of 1M sulfuric acid solution was added, and the absorbance at 450 nm was measured for the 96-well plate with a microplate reader (SpectraMax, molecular device). Based on the absorbance at serial dilution, the IgG antibody titer in mouse serum was determined by Log2.

[Evaluation of humoral immunity using mice]
Using the liquid prepared as described above, a mouse immunity test was conducted using a model animal for humoral immunity evaluation. 200 μL of an injection was administered subcutaneously to the back of a mouse (BALB / c mouse, female 7 weeks old) prepared in advance. One week after the administration, the same administration was again performed subcutaneously on the back of the mouse. Two weeks after the second administration, mouse serum was collected, and the serum OVA-specific IgG titer was measured by the ELISA method described above.

[OVA antigen-specific CTL measurement (ELISPOT method)]
Splenocytes (3 × 10 ⁶ cells / well) and antigenic peptide (100 μM) or antigenic protein (100 μg / mL) are placed in a well of an ELISPOT plate (R & D Systems) on which an anti-mouse IFN-γ antibody is immobilized, together with a culture solution. The cells were co-cultured at 37 ° C. under 5% CO ₂ for 20 hours, and the number of IFN-γ producing cell spots (spot number / 3 × 10 ⁶ cells) was measured by ELISPOT method.

[Cellular immunity evaluation using mice]
Using the solution prepared as described above, a mouse immunity test was conducted using a model animal for cellular immunity evaluation. 200 μL of an injection was administered subcutaneously to the back of a mouse (C57BL / 6 mouse, female 7 week old) prepared in advance. One week after the administration, the same administration was again performed subcutaneously on the back of the mouse. One week after the second administration, mouse spleen was collected, and OVA antigen-specific CTL was measured by the ELISPOT method described above.

These results are shown in Table 18 below.

-: Less than 4 times the amount of antibody produced in Comparative Example 1, or the number of CTLs less than 30 cells / well +: More than 4 times and less than 8 times the amount of cytokine released in Comparative Example 1, or CTLs greater than 30 cells / well and less than 100 cells / well Number ++: Cytokine release amount of 8 times or more and less than 16 times that of Comparative Example 1, or CTL number of 100 cells / well or more and less than 300 cells / well ++: Cytokine release amount of 16 times or more of Comparative Example 1, or 300 cells / well or more CTL count

[Evaluation of adsorption properties of metal-organic structures]
The amount of adsorption was measured using BELSORP-max12 (manufactured by Microtrack Bell Co., Ltd.). In addition, the metal organic structure was used in a powder state. Some of the results are shown in FIG. 1A, FIG. 1B and FIG. FIG. 1A is a CO adsorption profile of AP004 [MIL-100 (Fe)]. FIG. 1B is a NO adsorption profile of AP004 [MIL-100 (Fe)]. FIG. 2 is a NO adsorption profile of AP104 (BioMIL-3). In these examples, the adsorption / desorption profile was irreversible. That is, at the same pressure, the adsorption amount at the time of desorption was larger than the adsorption amount at the time of adsorption. Further, the amount of residual adsorption when the desorption from the pressurized state to the vacuum state was performed after the adsorption from the vacuum state to the pressurized state was not zero.

[Introduction of gas components into metal organic structures]
In some of the examples below, a compound in which a gas component was introduced into a metal organic structure was used. Specifically, first, the degassing was performed by heating the metal organic structure under a nitrogen flow. The sample returned to room temperature was then exposed to gas flow. Next, a nitrogen flow was performed at room temperature to discharge excess gas components. Thus, the compound which introduce | transduced the gas component into the metal organic structure was obtained.

In addition, about the said compound, when the one part was heated under nitrogen flow, it confirmed that a gas component was detected with a detector tube. In this way, it was confirmed that the gas component was introduced into the metal organic structure.

[Mouse immunity test]
An injection having the composition shown in Table 19 below was prepared. Specifically, the antigen was weighed out in the amount specified in Table 19, and glycerin was added thereto. Further, a saturated glycerin solution of gas component or a gas component adsorbed on a metal organic structure was added. In the table, MOF means a metal organic structure, and Gly means glycerin.

Using the solution prepared as described above, 50 μL of an injection was administered subcutaneously to the back of a mouse (BALB / c mouse, female 7 weeks old) prepared in advance. Two weeks after the administration, the same administration was again performed subcutaneously on the back of the mouse.

Two weeks after the second administration, mouse serum and spleen cells were collected, and serum OVA-specific IgG antibody and IgG2a antibody were measured by ELISA. In addition, spleen cells were used to simultaneously evaluate the production amounts of OVA-specific IFN-γ and IL-4. The specific evaluation method is as follows.

[Measurement of antigen-specific antibody titer in mouse serum (ELISA method)]
As an antigen, an OVA-containing solution (100 μg / mL) diluted with a carbonate buffer was prepared. 100 μL of this was added to a 96-well plate for ELISA and allowed to stand overnight.

Wells were washed three times with a preliminarily prepared washing solution (PBS containing Tween 20). A blocking solution obtained by diluting a blocking agent (Block Ace, Dainippon Sumitomo Pharma Co., Ltd.) to 4 g / 100 mL with purified water was added 200 μL at a time, and the mixture was allowed to stand at room temperature for 2 hours. Thereafter, the wells were washed three times with a washing solution.

The serum collected from the mouse in advance was centrifuged at 3000 g for 10 minutes at 4 ° C., and the supernatant was collected. Using a solution obtained by diluting the blocking agent with a phosphate buffer (Nacalai Tesque) to 0.4 g / 100 mL, the above supernatant was serially diluted two times. 50 μL of each of the obtained diluted solutions was added and left at room temperature for 2 hours.

Thereafter, the wells were washed 3 times with a washing solution. In addition, an HRP-labeled anti-mouse IgG antibody (Goat-anti mouse IgG Fc HRP, BETHYL) or an HRP-labeled anti-mouse IgG2a antibody (in a solution obtained by diluting the blocking agent with phosphate buffer (Nacalai Tesque) to 0.4 g / 100 mL) Goat-anti mouse IgG2a Fc HRP, BETHYL) was diluted 10,000 times. 100 μL of this diluted solution was added and left at room temperature for 1 hour.

Thereafter, the wells were washed three times with a washing solution, 100 μL of TMB solution (ELISA POD TMB kit, Nacalai Tesque) was added and left in the dark for 30 minutes.

Further, 100 μL of 1M sulfuric acid solution was added, and the absorbance at 450 nm was measured for each of the 96-well plates using a microplate reader. Based on the absorbance at the time of serial dilution, IgG antibody titer or IgG2a antibody titer in mouse serum was determined by Log2.

These results are shown in FIG. As shown in FIG. 3, by using the gas component, the immune characteristics could be controlled. Moreover, the immune characteristics could be further changed by combining the gas component and the metal organic structure.

[Measurement of OVA-specific cytokine production (ELISA method)]
100 μL each of 4 × 10 ⁵ cells / well of spleen cells collected in advance from a mouse was added to a 96-well plate for ELISA. To this, 100 μL of an OVA-containing solution (100 μg / mL) diluted in RPMI medium was added and allowed to stand for 72 hours. The culture supernatant was collected, and the amount of each cytokine produced was quantified using a mouse IFN-γ ELISA kit and mouse IL-4 ELISA kit (R & D systems).

These results are shown in FIGS. 4A and 4B. As shown in FIG. 4A and FIG. 4B, the immune characteristics could be controlled by using the gas component. Moreover, the immune characteristics could be further changed by combining the gas component and the metal organic structure.

Claims (7)

A vaccine composition containing an antigen for inducing immunity and a gas component which is a gas at 25 ° C. and 100 kPa. The vaccine composition according to claim 1, wherein the gas component is an immune signal transmission substance. The vaccine composition according to claim 2, wherein the immune signaling substance is a factor that acts on keratinocytes, monocytes, lymphocytes, or granulocytes. The vaccine composition according to any one of claims 1 to 3, further comprising a metal organic structure. The vaccine composition according to claim 4, wherein at least a part of the gas component is contained in pores of the metal organic structure. The vaccine composition according to any one of claims 1 to 5, wherein the vaccine composition is configured to be administered onto the skin and / or mucous membrane. The vaccine composition according to any one of claims 1 to 5, wherein the vaccine composition is configured to be administered by intradermal injection, subcutaneous injection, or intramuscular injection.

Images