JP5259997B2 - Immune adjuvant dispersion - Google Patents

Description

  The present invention relates to an immune adjuvant having an effect of improving the ability to produce an antibody against an antigen in an immune reaction, and more particularly, to an immune adjuvant dispersion containing chitosan microparticles.

  Immunity is the resistance and healing power of diseases that the living body has, and at the molecular level, it distinguishes itself from “self” and foreign material that has entered from the outside, “non-self”. Is a system that processes and removes only

  Immunity includes an innate immune reaction (innate immunity) that immediately removes pathogens that invade the living body in a non-specific manner and an acquired immune reaction that specifically removes pathogens that break through innate immunity and enter the body. is there. In the acquired immune response, the organism counters the pathogen by producing an antibody that specifically interacts with the pathogen (antigen). In order to acquire resistance (immunity) to a pathogen before infecting with such a pathogen, it is necessary to inoculate a living body with a dead fungus or an attenuated pathogen as a vaccine. Here, in order to produce an antibody effectively over a long period of time with an inoculated vaccine, selection of an immune adjuvant that inoculates with the antigen and assists in the production of the antibody is a very important key.

  Currently, vaccination is mostly by intramuscular injection. This method stimulates the immune system by introducing antigens directly into the body. Successful examples include toxoid vaccines (such as tetanus) and blood vaccines (such as measles) that are attenuated by blood antibodies. There are also many.

  On the other hand, the method of inoculating a vaccine through the mucous membrane and establishing immunity locally in the mucosa has many problems to be solved and is under development. Many infectious diseases start when a pathogen contacts the mucosal surface and invades from the mucosa, or colonizes and grows at that part. In order to prevent such infection on the mucosal surface, the living body has an immune system uniquely developed in the mucosal region. However, even when the vaccine is administered transdermally, immunity is hardly induced in the mucosal region, and effective vaccine treatment has not been possible for pathogens that infect through the mucosa.

  In addition, it is known that immunization establishes not only local but also systemic immunity to the mucosa when vaccination is given to the mucosa, and how to establish immunity locally in many infectious diseases It is considered to be the decisive factor for infection prevention.

  For this reason, the development of a vaccine that establishes immunity through the mucous membrane and the method of inoculation thereof are expected, and research on the combined use of a vaccine and an immune adjuvant has been conducted all over the world. However, there are still many unclear points regarding the establishment of mucosal immunity and the induction system of immunity by vaccination. Also known as substances having an action as an adjuvant are alum, cholera toxin, Freund's complete adjuvant, Freund's incomplete adjuvant, oil adjuvant, saponin, dimethyldioctadecyl ammonium bromide, hexadecylamine, abridine, It is limited to Iscom, cell wall skeleton construct, lipopolysaccharide, endotoxin and liposome. Moreover, many of these are highly toxic, and only alum is approved for human inoculation.

In addition to the above, chitin and chitosan have recently been shown to be useful as immune adjuvants. Chitosan is a highly safe polysaccharide derived from natural products, and is industrially produced by deacetylating chitin obtained from crustaceans such as shrimp and crab. For example, Patent Document 1 discloses the use of a water-soluble chitin oligomer and chitosan oligomer as an immune adjuvant by injection. Patent Document 2 discloses that chitin used by oral inoculation acts as an immunostimulant (adjuvant).
However, when chitin and chitosan are used as an immunological adjuvant in this way, there is a problem that the degree of binding to the antigen is not high and a large amount must be inoculated.

  In addition, as properties unique to chitosan, functions such as film-forming properties, antibacterial properties, water retention and aggregating ability are known, and they are practically used in various fields as functional polymers.

  For example, according to the method disclosed in Patent Document 3, chitosan oligosaccharide is produced by hydrolysis of chitosan, which is used in the fields of pharmaceuticals, foods, cosmetics and the like. In the medical field, chitin and chitosan are expected to be applied as infection-protecting substances as disclosed in Patent Document 4.

  When chitosan is used, it is finely divided in advance. The degree of refinement (for example, the average particle diameter) has been determined by the conventional light scattering method, but the measurement results differ greatly depending on the measurement method and the solvent used, and the resulting chitosan properties are not constant. There was a problem. In order to solve such problems, Patent Document 5 expresses the degree of refinement of chitosan by the viscosity of the solution. Chitosan having a certain range of solution viscosity has a constant property and is useful for various applications. It is disclosed.

Japanese Patent Publication No. 7-47546 JP 55-43041 A JP 2003-212889 A JP-A-9-301807 JP 2007-2123 A

  Accordingly, an object of the present invention is to solve the above-mentioned problems of the prior art, and to provide an immune adjuvant that is highly safe to the living body and that can induce immunity locally at the target site and an inoculation method thereof.

The above object is achieved by the present invention described below. That is, the present invention has a degree of deacetylation of 60% or more, a 1% by mass solution viscosity of 1.5 mPa · s or more and 1,000 mPa · s or less , comprising free chitosan that is not an acid salt, and having a particle size of chitosan particles is 0.5μm or more 20μm or less, and a water, pH is 6.5-9.0 der is, immunoadjuvant dispersion wherein the chitosan particulate is dispersed in the water in a state undissolved Provide liquid.

In here, the immunoadjuvant dispersion, it is preferable that the content of key chitosan microparticles is 0.1 to 10 mass%.

  The present invention further provides a method for inoculating an immunoadjuvant characterized by spraying the above-mentioned immunoadjuvant dispersion liquid into a lung of a non-human animal as a fine mist.

According to the present invention, safety for the living body is high, mainly not only IgG involved in systemic immunity, such as in regard to infection defense mucosa, excellent in ability to produce secretory IgA having antiviral activity inoculation method immune adjuvants dispersion and the dispersion is provided.

Next, the present invention will be described in more detail with reference to preferred embodiments.
In the present invention, “part” or “%” is based on mass unless otherwise specified. In the present invention, the term “solution” may include “dispersion”.
First, chitosan fine particles constituting the immune adjuvant of the present invention will be described.

  The chitosan fine particles used in the present invention are obtained by obtaining chitin from a natural product, deacetylating it to obtain chitosan, and making this chitosan into fine particles. The chitin is obtained from crustacean exoskeletons such as crabs and shrimps, squid, krill and insects, and fungi such as mushrooms, and the origin thereof is not particularly limited and is used in the present invention. be able to.

  Chitin is a natural polymer having 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) as a structural unit. On the other hand, chitosan is a deacetylated product of chitin and is a basic polysaccharide having 2-amino-2-deoxy-D-glucose (glucosamine) as a structural unit. Chitosan has already been industrially produced, and various grades are available. The chitosan used in the present invention is not particularly limited in its origin and production method, and chitosan that has been conventionally industrially produced can be used.

  The chitosan used in the present invention preferably has a degree of deacetylation of 60% or more, and more preferably has a degree of deacetylation of 80% to 100%. If the degree of deacetylation is less than 60%, there is a problem in that the original characteristics of chitosan are not expressed, such as not being easily dissolved in dilute acetic acid aqueous solution.

(Deacetylation degree measuring method)
The degree of deacetylation of chitosan can be calculated from the titration amount after colloid titration. Specifically, a toluidine blue solution is used as an indicator and colloidal titration with an aqueous polyvinyl potassium sulfate solution to quantify the free amino groups in the chitosan molecule and determine the degree of deacetylation of chitosan. An example of a method for measuring the degree of deacetylation is shown below.

1. Titration test Add chitosan to 0.5% acetic acid aqueous solution so that the concentration of pure chitosan is 0.5%, stir and dissolve chitosan to prepare 100 g of 0.5% chitosan / 0.5% acetic acid aqueous solution. To do. Next, 10 g of this solution and 90 g of ion-exchanged water are mixed with stirring to prepare a 0.05% chitosan solution. Further, 50 ml of ion exchange water and about 0.2 ml of toluidine blue solution are added to 10 g of this 0.05% chitosan solution to prepare a sample solution, and titrated with a polyvinyl potassium sulfate solution (N / 400 PVSK). The titration rate is 2 ml / min to 5 ml / min, and the point at which the sample solution is held from 30 to 30 seconds after the color changes from blue to reddish purple is used as the end point titration.
The pure chitosan content means the mass of chitosan in the raw material chitosan sample. Specifically, it is the solid content mass obtained by drying the chitosan sample at 105 ° C. for 2 hours.

2. Blank test A similar titration test is performed using ion-exchanged water instead of the 0.5% chitosan / 0.5% acetic acid aqueous solution used in the above titration test.

3. Calculation of degree of acetylation X = 1/400 × 161 × f × (V−B) / 1000
= 0.4025 * f * (V-B) / 1000
Y = 0.5 / 100-X
X: Mass of free amino group in chitosan (equivalent to glucosamine residue mass)
Y: Mass of bound amino group in chitosan (corresponding to mass of N-acetylglucosamine residue)
f: titer of N / 400 PVSK V: titration of sample solution (ml)
B: Blank test titration (ml)
Deacetylation degree (%)
= (Free amino group) / {(free amino group) + (bonded amino group)} × 100
= (X / 161) / (X / 161 + Y / 203) × 100
161 is the molecular weight of the glucosamine residue, and 203 is the molecular weight of the N-acetylglucosamine residue.

As the chitosan fine particles constituting the immunoadjuvant of the present invention, those having a 1% by mass solution viscosity of 1.5 mPa · s or more and 1,000 mPa · s or less are used. More preferably, it is 1.5 mPa · s or more and 500 mPa · s or less.
In order for the viscosity to be less than 1.5 mPa · s, the cost for miniaturization increases, which is disadvantageous in production cost. On the other hand, if the viscosity exceeds 1,000 mPa · s, the viscosity becomes too high when used as a solution, which hinders use and limits the inoculation method.

  In the present invention, chitosan fine particles having a particle size of 0.5 μm or more and 20 μm or less are used. When the particle diameter exceeds 20 μm, the surface area becomes relatively small, the mobility is lowered, and the action as an immune adjuvant is weakened. The finer the particle diameter, the better. However, in order to make the particle diameter of chitosan less than 0.5 μm, the cost is remarkably increased, which is not preferable from the viewpoint of economy.

  The viscosity of the chitosan solution is such that, for example, chitosan is dissolved in a 1% acetic acid aqueous solution so that the concentration of pure chitosan is 1%, and this solution is kept at 20 ° C. in a thermostatic bath while maintaining a B-type rotational viscometer. Can be determined by measuring the viscosity using In addition, chitosan pure content means the solid content conversion in a chitosan sample, and is specifically the solid content mass after drying a chitosan sample at 105 degreeC for 2 hours.

  Chitosan fine particles having a 1% by mass solution viscosity of 1.5 mPa · s or more and 1,000 mPa · s or less and a particle diameter of 0.5 μm or more and 20 μm or less can be obtained by the method disclosed in JP-A-2007-2123. . The chitosan fine particles obtained by this method are excellent in that the effect of each administration is constant because the particle diameter is uniform.

The immune adjuvant of the present invention can be used as an immune adjuvant dispersion in addition to water.
The immune adjuvant dispersion of the present invention preferably has a pH of 6.5 to 9.0. When the pH is less than 6.5, the chitosan fine particles are dissolved, and the characteristics as the fine particles are impaired. Moreover, when pH exceeds 9.0, there exists a possibility of having a bad influence on a living organism | raw_food, it is unpreferable.

  The substance used for adjusting the pH is not particularly limited, and substances well known to those skilled in the art can be used. Alkali substances used for pH adjustment include alkali metal hydroxides or carbonates, ammonia and amines, specifically lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate. , Potassium carbonate, ammonia, morpholine, N-methylmorpholine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, propanolamine, dipropanolamine, tripropanolamine, N-methylethanolamine, N, N-dimethylethanolamine N-ethylethanolamine, N, N-diethylethanolamine, N-methyldiethanolamine, N-ethyldiethanolamine, N-methylpropanolamine, N, N-dimethylpropanol , And the like Min and aminomethyl propanol. Particularly preferred are sodium hydroxide and ammonia.

  On the other hand, the acidic substance that can be used for pH adjustment is not particularly limited as long as it is water-soluble, formic acid, acetic acid, propionic acid, butyric acid, taurine, pyrrolidone carboxylic acid, citric acid, malic acid, lactic acid, bidoxymalonic acid, malonic acid, Mention may be made of organic acids such as succinic acid, adipic acid, benzoic acid, salicylic acid, aminobenzoic acid, phthalic acid, cinnamic acid and vitamin C, and inorganic acids such as hydrochloric acid, phosphoric acid, nitric acid and sulfuric acid.

  The content of chitosan fine particles in the immunoadjuvant dispersion is preferably an amount that occupies 0.1 to 10% of the total mass, and more preferably 1 to 5%. If the amount of chitosan fine particles is less than 0.1%, the effect as an immune adjuvant of promoting immune response is not sufficiently obtained, while if the addition amount exceeds 10%, the dispersion loses fluidity, It is inconvenient for inoculation and is not preferable.

  The method of mixing the immunoadjuvant dispersion of the present invention and the antigen is not particularly limited, and a mixing / dispersing machine, a fine dispersing machine, and a fine atomizing machine well known to those skilled in the art can be used. Specific examples include an ultrasonic generator, a ball mill, a dyno mill, a sand mill, a roll mill, a glen mill, a dissolver, a disper, and a paint shaker, and it is preferable to use one that can be refined simultaneously with mixing.

  The immunoadjuvant dispersion liquid of the present invention can be inoculated by various inoculation methods well known to those skilled in the art. However, by spraying the aqueous solution as a fine mist on the mucous membrane, a more appropriate adjuvant effect can be exhibited. Can do. The inoculation site is not particularly limited, but it is preferably inoculated intranasally, in the oral cavity, in the lung, in the vagina and in the rectum, and most preferably inoculated into the lung. When the immunoadjuvant dispersion liquid of the present invention is nasally or orally inoculated, the concentration of chitosan fine particles is preferably 0.5 to 5%. In the case of transpulmonary inoculation, 0.1 to 3% is preferable.

  Moreover, when the immunity adjuvant dispersion liquid of this invention is inoculated nasally and orally, it is preferable that the particle diameter of chitosan microparticles | fine-particles is 3 micrometers-10 micrometers. When the particle diameter is larger than 10 μm, the surface area of the particles is relatively small, and the effect as an adjuvant is insufficient. When the particle diameter is less than 3 μm, the production cost is increased, which is uneconomical. On the other hand, in the case of transpulmonary inoculation, the particle diameter is preferably 1 μm to 4 μm. If the particle size is larger than 4 μm, clogging may occur inside the nebulizer syringe. When the thickness is less than 1 μm, the production cost increases, which is uneconomical.

  Animals that can be inoculated with the immune adjuvant of the present invention and dispersions thereof are not particularly limited, and examples include domestic animals such as chickens, pigs, horses, goats, sheep, and pets such as cats, dogs, hamsters, rabbits, and birds. , Pets, fish, marine organisms and mice, monkeys and humans.

  As conditions for an immunoadjuvant with high antibody-producing ability, it is first necessary to easily bind to a target antigenic substance. In addition, it is necessary to be easily taken up into cells and to release the antigenic substance over a long period of time in the cells. Although the mechanism of action of the immunoadjuvant of the present invention is unclear, chitosan microparticles used in the immunoadjuvant of the present invention satisfy these conditions, so that a long-term immunity effect can be exhibited with a small amount of vaccination. Can be considered.

  EXAMPLES Next, although an Example and a comparative example are given and this invention is demonstrated concretely, this invention is not limited to these.

[Example 1]
(Preparation of chitosan fine particles)
One part of the coarsely crushed crab shell that passed through a sieve with a mesh opening of 4 mm was dispersed in 30 parts of water, 1 part of hydrochloric acid was added thereto, and the mixture was stirred at 30 ° C. for 6 hours, and the crab shell was filtered off. Next, this treated crab shell was redispersed in 30 parts of water and filtered off 10 times to obtain a decalcified crab shell. Next, this decalcified crab shell is redispersed in 30 parts of water, 3 parts of sodium hydroxide is added, heated to 70 ° C. and stirred for 3 hours, then chitin is filtered off and redispersed in 30 parts of water. The operation of filtering again was repeated 10 times. Next, the chitin was dried with hot air at 50 ° C. for 20 hours to obtain raw material chitin.

  100 parts of this chitin was dispersed in 2,000 parts of water, 2.5 parts of sodium carbonate was added, 0.15 part of sodium bromite was added, and the mixture was stirred at room temperature for 6 hours. After the treatment, the chitin is filtered off, redispersed in 30 parts of water and then filtered off. This operation was repeated 5 times, dried with hot air at 50 ° C. for 20 hours, and then deacetylated to obtain chitosan.

  The chitosan was pulverized with a hammer mill, passed through a sieve having an opening of 400 μm, further pulverized with a ball mill for 48 hours, and further pulverized with a jet mill to obtain chitosan fine particles. The chitosan fine particles had a particle size distribution center of 4.5 μm and fine particles of 10 μm or less were 100%. When the solution viscosity of 1% of this chitosan was measured, it was 4 mPa · s. Further, the degree of deacetylation of the chitosan fine particles was 87%. In addition, the particle diameter of chitosan microparticles | fine-particles is a measured value by an electron microscope (Hitachi Ltd. high-resolution scanning electron microscope S-5200 type | mold).

(Immune immunization of mice with OVA antigen)
99 parts of purified water was added to 1 part of the chitosan fine particles to obtain a 1% chitosan fine particle dispersion. 2 parts of a 0.25% OVA (egg white albumin) aqueous solution was added to 5 parts of the dispersion and the pH was adjusted to 8.0 using hydrochloric acid and sodium hydroxide to obtain a chitosan fine particle-OVA mixed liquid. .
7 μL of the mixed solution was intranasally inoculated into five 6-week-old female BALB / c mice anesthetized with diethyl ether. From the day of this nasal inoculation, 7 μL of the above mixture was similarly inoculated nasally three times every week. The third day of nasal inoculation was designated as week 0, and once a week thereafter, blood, feces and vaginal lavage fluid were obtained as follows. Moreover, in order to confirm the booster effect, the second nasal inoculation was similarly performed after 2-3 months from the day of the third nasal inoculation.

Blood: Blood was collected from the tail vein after anesthesia with diethyl ether.
Stool: Each individual was left in a resin cage for several minutes, and the stool was collected in an Eppendorf tube.
Vaginal lavage fluid: 50 μL of 1% BSA (bovine serum albumin) was pipetted into the vagina. This operation was performed twice to obtain a vaginal wash.

(Sample preparation)
The blood, stool and vaginal lavage fluid obtained by the above procedure were processed as follows to prepare samples for antibody titer measurement.
Serum sample: 15,000 r. p. m. Was centrifuged for 15 minutes, and the supernatant was centrifuged again under the same conditions to obtain a serum sample. The serum sample was stored frozen at −30 ° C. until use.
Stool sample: The above stool was dispersed in a 1% BSA solution at 4 ° C, and this dispersion was dispersed at 15,000 r. p. m. The supernatant was used as a stool sample. The stool sample was stored frozen at −30 ° C. until use.
Vaginal washing liquid sample: The above-mentioned vaginal washing liquid was mixed at room temperature at 15,000 r. p. m. For 15 minutes, and the supernatant was used as a vaginal wash sample. The sample was stored frozen at −30 ° C. until use.

Using the ELISA method, IgG antibody titer and IgA antibody titer were measured for each sample. The measurement was performed according to the following procedure.
(Measurement of IgG antibody titer)
OVA antigen (manufactured by Seikagaku Corporation) with a coating buffer (containing 1 g of purified water containing Na ₂ CO ₃ : 1.6 g, NaHCO ₃ : 2.82 g, NaN ₃ : 0.1 g; pH 9.0) at 5 μg / The solution was diluted to mL and dispensed at 50 μL per well into a 96-well ELISA plate (Costar 3690 A / 2, Corning) and allowed to stand overnight at 4 ° C. to adsorb (coat) the antigen. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying.

After the above coating is completed, the coating buffer is discarded, and then blocking buffer (containing 10 × Dulbecco's PBS: 100 mL, albumin (Fr.V): 10 g, NaN ₃ : 1 g in 1 L of purified water) is added to the ELISA plate at 100 μL per well. Aliquoted and left for 1 hour.
A U-bottom plate was prepared separately from the ELISA plate, and a 10-fold two-fold dilution series was prepared by diluting the above samples by 2 ⁶ to 2 ¹⁵ times. For the dilution, a blocking buffer was used, and the final dilution measurement method (end point dilution method) was performed.

After the blocking, the blocking buffer was discarded from the ELISA plate, and the sample prepared on the U-bottom plate was dispensed at 50 μL per well and incubated at 37 ° C. for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying.
As a positive control, a 10-fold 2-fold dilution series in which a monoclonal antibody anti-hen egg albumin (MONOCLONAL ANTI-CHICKEN EGG ALBUMIN CLONE OVA) (SIGMA) was diluted 10,000 to 512 thousand times was used. Use As the negative control, the mice not immunized with OVA antigen, a 2-fold dilution series of 10 stages of 2 ^{6-2 15} prepared by diluting the serum sample prepared in the same manner as above in blocking buffer It was.

  During incubation, a plate seal was applied to the plate surface to prevent the solution from drying. After the incubation is complete, discard the sample solution and use an ELISA plate washer (Biotech, MW-96F) to dispense 200 μL of the washing solution (1 × PBS, 0.2% Tween20) per well and then discard it. Was repeated 4 times to wash the plate.

  As a secondary antibody, biotin-labeled goat anti-mouse IgG (Jackson ImmunoResearch) was diluted 1,000 times with a diluent (1 × PBS, 0.2% Tween20), and dispensed at 50 μL per well. Incubated for 1 hour at ° C. At this time, a plate seal was put on the plate surface to prevent the solution from drying. After the incubation was completed, the solution was discarded, and 200 μL of a washing solution (1 × PBS, 0.2% Tween20) was dispensed per well, and then the plate was washed by repeating discarding 4 times.

  As a tertiary antibody, alkaline phosphatase-labeled streptavidin (Invitrogen) was diluted 2,000 times with a diluent (1 × PBS, 0.2% Tween20), and 50 μL was dispensed per well at 37 ° C. Incubated for 1 hour. At this time, a plate seal was put on the plate surface to prevent the solution from drying. After the incubation was completed, the solution was discarded, and 200 μL of a washing solution (1 × PBS, 0.2% Tween20) was dispensed per well, and then the plate was washed by repeating discarding 4 times.

One phosphatase chromogenic substrate p-nitrophenyl phosphate (KPL) was added to a chromogenic substrate buffer solution (diethanolamine: 0.97 mL in 1 L of purified water, MgCl ₂ .6H ₂ O: 0.1 g, NaN ₃ : 0. 1 g containing; pH 9.8) Dissolving in 5 mL to prepare a coloring solution, dispensing 100 μL of this coloring solution per well and incubating at 37 ° C. for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After the incubation, the reaction was stopped by dispensing 50 μL of 1N NaOH per well as a reaction stop solution, and the absorbance of each well was measured at wavelengths of 405 nm and 630 nm (main wavelength 405 nm, subwavelength 630 nm). For the measurement of absorbance, ELISA plate reader Multiskan EX (Thermo Labsystems) was used.

  From the measured absorbance, the average absorbance of the negative control and its standard deviation (SD) were determined. Next, the absorbance of the sample was plotted against the dilution rate to prepare a standard curve, the intersection of the negative control absorbance average value + 2SD standard curve was determined, and the multiplier of 2 at this intersection was obtained as the IgG antibody titer. The IgG antibody titer of the serum sample plotted against the time course from the day of nasal inoculation, with the third day of nasal inoculation as week 0, is shown in FIG.

(Measurement of IgA antibody titer)
OVA antigen (manufactured by Seikagaku Corporation) with a coating buffer (containing 1 g of purified water containing Na ₂ CO ₃ : 1.6 g, NaHCO ₃ : 2.82 g, NaN ₃ : 0.1 g; pH 9.0) at 5 μg / Dilute to mL and dispense 100 μL of this solution per well into a 96-well ELISA plate (SUMILON ELISA plate SMS-8496F, Sumitomo Bakelite Co., Ltd.) and allow to stand overnight at 4 ° C. to adsorb the antigen. It was. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying.

After completion of the above coating, the coating buffer is discarded, and then the blocking buffer (containing 10 × Dulbecco's PBS: 100 mL, albumin (Fr.V): 50 g, NaN ₃ : 1 g in 1 L of purified water) per well is added to the ELISA plate. 200 μL was dispensed and allowed to stand for 1 hour.

  A U-bottom plate was prepared separately from the ELISA plate, and 5-fold and 10-fold dilutions were prepared for each sample. Blocking buffer was used for dilution.

  After the incubation, the blocking buffer was discarded from the ELISA plate, and the sample prepared on the U-bottom plate was dispensed at 100 μL per well and incubated at 37 ° C. for 1 hour. During incubation, a plate seal was applied to the plate surface to prevent the solution from drying.

  As a positive control, cholera toxin (hereinafter abbreviated as CT) -OVA mixed solution (2 parts of 0.25% OVA aqueous solution was added to 1 part of CT solution prepared to 2 mg / mL with 1 × PBS buffer). , Prepared by mixing well by pipetting), and a serum sample prepared in the same manner as described above from a mouse inoculated with a large amount of IgA antibody and diluted 5 times and 10 times with a blocking buffer. Using. Further, as a negative control, a serum sample prepared in the same manner as described above from a mouse not immunized with the OVA antigen was diluted 5 times and 10 times with a blocking buffer.

  After the incubation, the sample solution is discarded, and the washing solution (1 × PBS, 0.2% Tween20) is dispensed at 400 μL per well using an ELISA plate washer (Biotech, MW-96F) and discarded. This was repeated 4 times to wash the plate.

  As a secondary antibody, biotin-labeled goat anti-mouse IgA (KPL) was diluted 1,000 times with a diluent (1 × PBS, 0.2% Tween20), and dispensed at 100 μL per well at 37 ° C. And incubated for 1 hour. At this time, a plate seal was put on the plate surface to prevent the solution from drying. After the incubation was completed, the solution was discarded, and the plate was washed by repeating 400 times of washing solution (1 × PBS, 0.2% Tween20) dispensed and then discarded 4 times.

  As a tertiary antibody, alkaline phosphatase-labeled streptavidin (Invitrogen) was diluted 2,000 times with a diluent (1 × PBS, 0.2% Tween20), and dispensed at 100 μL per well at 37 ° C. Incubated for 1 hour. At this time, a plate seal was put on the plate surface to prevent the solution from drying. After completion of the incubation, the solution was discarded from the ELISA plate, and then the plate was washed by repeating the discarding 4 times after dispensing 400 μL of a washing solution (1 × PBS, 0.2% Tween20) per well.

One phosphatase chromogenic substrate p-nitrophenyl phosphate (KPL) was added to a chromogenic substrate buffer solution (diethanolamine: 0.97 mL in 1 L of purified water, MgCl ₂ .6H ₂ O: 0.1 g, NaN ₃ : 0. 1 g containing; pH 9.8) Dissolving in 5 mL to prepare a coloring solution, dispensing 100 μL of this coloring solution per well and incubating at 37 ° C. for 1 hour. At this time, a plate seal was stuck on the plate surface to prevent the solution from drying. After completion of the incubation, 50 μL of 1N NaOH was dispensed per well as a reaction stop solution to stop the reaction, and the absorbance of each well was measured at wavelengths of 405 nm and 630 nm. For the measurement of absorbance, ELISA plate reader Multiskan EX (Thermo Labsystems) was used.
Each average value of the absorbance of the 5-fold and 10-fold diluted samples was obtained as an IgA antibody titer. The IgA antibody titer of the serum sample plotted against the time course from the day of nasal inoculation, with the third day of nasal inoculation as week 0, is shown in FIG.

[Comparative Example 1]
In place of the chitosan microparticles used in Example 1, cholera toxin (CT), which has a well-established mucosal immune adjuvant, was used for the same experiment as in Example 1. Specifically, a CT-OVA mixed solution was prepared in the same manner as in Example 1, and 3 μL of the mixed solution was passed through five 6-week-old, female BALB / c mice anesthetized with diethyl ether as in Example 1. Samples were prepared by inoculating the nose, blood, feces and vaginal lavage fluid, and the antibody titers of IgG and IgA were determined using the ELISA method.

The IgG antibody titer and IgA antibody titer of a serum sample obtained in the same manner as in Example 1 are indicated by Δ in FIGS. 1 and 2, respectively.
FIG. 1 and FIG. 2 revealed that the antibody titer against OVA was significantly higher in mice immunized with chitosan-OVA mixture than mice immunized with CT-OVA mixture. . Similar to the results for this serum sample, for stool samples and vaginal lavage samples, mice immunized with the chitosan-OVA mixture were more effective against OVA than mice immunized with the CT-OVA mixture. The result showed that the antibody titer was significantly high.

[Example 2]
7 μL of chitosan-OVA mixed solution prepared in the same manner as in Example 1 was intranasally inoculated into five 6-week-old female multimeric immunoglobulin receptor knockout mice anesthetized with diethyl ether. Blood was obtained by the same procedure and method as in Example 1 to prepare a serum sample. The IgG and IgA antibody titers were determined using the ELISA method in the same procedure and manner as in Example 1, except that the serum samples were diluted 100-fold and 200-fold for measurement of IgA antibody titers. The antibody titers of IgG and IgA are indicated by ♦ in FIGS. 3 and 4, respectively.

[Comparative Example 2]
An experiment similar to that of Example 2 was performed using cholera toxin (CT) instead of the chitosan fine particles used in Example 2. That is, 3 μL of a CT-OVA mixed solution prepared in the same manner as in Comparative Example 1 was intranasally inoculated into 5 female multimeric immunoglobulin receptor knockout mice anesthetized with diethyl ether as in Example 2. Then, blood was obtained by the same procedure and method as in Example 2, serum samples were prepared, and antibody titers of IgG and IgA were determined using the ELISA method. Antibody titers of IgG and IgA are indicated by Δ in FIGS. 3 and 4, respectively.

  FIG. 3 and FIG. 4 revealed that the antibody titer against OVA was significantly higher in mice immunized with chitosan-OVA mixture than mice immunized with CT-OVA mixture. .

[Example 3]
A 1% chitosan fine particle dispersion was prepared in the same manner as in Example 1, 2 parts of a 0.25% OVA aqueous solution and 18 parts of 1 × PBS were added to 5 parts of the dispersion, and mixed well by pipetting. A -OVA mixture was obtained. 25 μL of the mixed solution was inoculated into 5 6-week-old, female BALB / c mice anesthetized by intraperitoneal injection with 5 mg / mL Nembutal (use of about 0.01 mL per 1 g of mouse body weight). In addition, as a nebulizer for inoculating the lung, model 1A-1C manufactured by PENNCENTURY was used.

  From the day of this pulmonary inoculation, pulmonary inoculation of the above mixed solution was similarly carried out three times every week. The day of this third pulmonary inoculation was set as week 0, and then blood, feces and vaginal lavage fluid were obtained once a week by the same procedure and method as in Example 1, and samples were prepared by ELISA. And the antibody titer of IgA was calculated | required. In addition, in order to confirm the booster effect, the fourth transpulmonary inoculation was similarly performed after 2-3 months from the day of the third transpulmonary inoculation. The IgG and IgA antibody titers determined for the serum samples are indicated by ♦ in FIGS. 5 and 6, respectively.

[Comparative Example 3]
The same experiment as in Example 3 was performed using cholera toxin (CT) instead of the chitosan fine particles used in Example 3. That is, 2 parts of 0.25% OVA solution was added to 1 part of CT solution adjusted to 2 mg / mL with 1 × PBS, and further diluted with 22 parts of 1 × PBS, and mixed well by pipetting. In the same manner as in Example 3, 25 μL of this CT-OVA mixed solution was inoculated into 5 female BALB / c mice via lung. Thereafter, transpulmonary inoculation and sample preparation were performed in the same procedure and manner as in Example 3, and antibody titers of IgG and IgA were determined by ELISA. The IgG and IgA antibody titers determined for the blood samples are indicated by Δ in FIGS. 5 and 6, respectively.

  FIG. 5 and FIG. 6 revealed that the antibody titer against OVA was significantly higher in mice immunized with chitosan-OVA mixture than mice immunized with CT-OVA mixture. . Similar to the results for this serum sample, for stool samples and vaginal lavage samples, mice immunized with the chitosan-OVA mixture were more effective against OVA than mice immunized with the CT-OVA mixture. The result showed that the antibody titer was significantly high.

[Example 4]
A chitosan-OVA mixture was prepared by the same procedure and method as in Example 3. 25 μL of the mixture was transpulmonarily inoculated into 5 week-old female multimeric immunoglobulin receptor knockout mice anesthetized by intraperitoneal injection of 5 mg / mL Nembutal (about 0.01 mL per g of mouse body weight). The lung was inoculated in the same manner as in Example 3.

  According to the same procedure and method as in Example 3, the mixture was pulmonary inoculated three times every week from the day of pulmonary inoculation. The day of this third pulmonary inoculation was set as week 0, and then blood was obtained once a week by the same procedure and method as in Example 3, and serum samples were prepared. Antibody titers of IgG and IgA were determined by ELISA. Asked. In addition, in order to confirm the booster effect, the fourth transpulmonary inoculation was performed 2 to 3 months after the third transpulmonary inoculation day. The antibody titers of IgG and IgA determined for serum samples are shown by ♦ in FIGS. 7 and 8, respectively.

[Comparative Example 4]
The same experiment as in Example 4 was performed using cholera toxin (CT) instead of the chitosan fine particles used in Example 4. That is, 25 μL of the CT-OVA mixed solution prepared in the same manner as in Comparative Example 3 was subjected to a total of 4 times of transpulmonary administration to 5 female multimeric immunoglobulin receptor knockout mice anesthetized with Nembutal in the same manner as in Example 4. Vaccinated. Serum samples were prepared once a week by the same procedure and method as in Example 4, and the antibody titers of IgG and IgA were determined by ELISA. The IgG and IgA antibody titers determined for the serum samples are indicated by Δ in FIGS. 7 and 8, respectively.

  FIG. 7 and FIG. 8 revealed that the antibody titer against OVA was significantly higher in mice immunized with chitosan-OVA mixture than mice immunized with CT-OVA mixture. .

  In all the examples, the antibody titer against OVA was significantly higher in the example using the chitosan fine particle dispersion than in the comparative example using cholera toxin as an immune adjuvant for OVA. Moreover, since the antibody titer in the 14th week in the figure was increased, a booster effect in which the antibody producing ability was rapidly increased was confirmed by performing the fourth immunization.

  According to the present invention, immunity that is highly safe to the living body, is involved not only in IgG related to systemic immunity, but also locally in the mucous membrane, etc., and has excellent ability to produce secretory IgA having antiviral activity. Adjuvants, immune adjuvant dispersions and methods for inoculating the dispersions are provided. By inoculating the mucosa with the immune adjuvant dispersion of the present invention by the inoculation method of the present invention, immunity can be locally induced at the site of inoculation.

It is a graph which shows the comparison of IgG antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of an IgA antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of IgG antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of an IgA antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of IgG antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of an IgA antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of IgG antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant. It is a graph which shows the comparison of an IgA antibody titer at the time of using chitosan microparticles | fine-particles and CT as an immune adjuvant.

Claims (3)

Deacetylation degree of 60% or more, 1% by mass Solution viscosity of 1.5 mPa · s or more and 1,000 mPa · s or less , consisting of free chitosan which is not acid salt, and particle size of 0.5 μm or more and 20 μm or less chitosan particles is, and a water, pH is 6.5-9.0 der is, immunoadjuvant dispersion characterized in that it is dispersed in the water in a state in which the chitosan particles are not dissolved .   The immune adjuvant dispersion according to claim 1, wherein the content of chitosan fine particles is 0.1 to 10% by mass.   A method for inoculating an immunoadjuvant comprising spraying the immunoadjuvant dispersion according to claim 1 or 2 into a lung of a non-human animal as a fine mist.

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