KR101622738B1 - Microneedle device and method for increasing the response of japanese encephalitis virus antigen with the microneedle device - Google Patents

Abstract

A method for enhancing immunogenicity by a micro needle device that enhances the immunogenicity of a Japanese encephalitis virus antigen. The method for enhancing immunogenicity by the present microneedle device comprises transdermally administering a Japanese encephalitis virus antigen using a micro needle coated with a Japanese encephalitis virus antigen comprising an antigen derived from monkey kidney cells (Vero cells) The antibody of the virus antigen is elevated. In addition, transdermal microinjections coated with Japanese encephalitis virus antigens are transdermally applied and patches containing lauryl alcohol are applied.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for enhancing the efficacy of a Japanese encephalitis virus antigen by a micro needle device and a micro needle device. BACKGROUND OF THE INVENTION < RTI ID = 0.0 >

The present invention relates to a method for enhancing immunogenicity by a micro needle device.

The skin consists of the stratum corneum, epidermis, dermis, and subcutaneous connective tissue in the outermost layer. In general, the stratum corneum layer composed of a sacrificial layer and a lipid bilayer exhibits a strong barrier function against many substances. In the dermal layer, an antigen presenting cell called Langerhans cell exists and is responsible for the immune function. Langerhans cells supplement the protein antigen that enters the skin, decompose internally, and display peptide fragments on MHC molecules. The MHC-peptide complex migrates from the imported lymphatic duct to the subcortical layer of the lymph node to which it belongs, and contacts the T cell with the dendritic cells. By this transfer of Langerhans cells, the antigen is transferred from the skin to the T _H cells present in the lymph nodes. Langerhans cells have MHC class II molecules required to present antigens to T _H cells.

As described above, it has been known that the viral antigen against the dermal layer is effective due to the strong barrier function of the skin's stratum corneum. However, administration by the injection needle into the dermal layer limited to 300 to 2000 m has a problem in precision due to technical difficulties.

A micro needle has been developed as a means to solve this problem. They are designed to puncture the stratum corneum, which is the outermost layer, and are designed as noninvasive viral antigen administration methods in various sizes and shapes (very small projections of several tens to several hundreds of micrometers in height).

In addition, various methods have been devised for application of a drug when a device equipped with a micro needle is used, and a method of coating a drug on the surface of a micro needle and administering it, a method of applying a drug to a needle, A method of forming a needle or a groove, and a method of mixing an agent on the needle itself have been proposed. Since these microneedle devices are provided with extremely small protrusions (microneedles) each having a height of several tens to several hundreds of microns in height, the transdermal absorption and absorption efficiency of a drug are considerably different depending on the application method of the drug.

For example, as a method of effectively promoting the percutaneous absorption property of an antigen (vaccine) using a micro needle, there is a method of coating a part of the surface of the micro needle with a drug, for example, as disclosed in Non-Patent Document 1 . This indicates that all or most of the applied antigen (vaccine) migrates into the body when the antigen (vaccine) is coated on a part of the micro needle (particularly, only the needle part) and is useful as a means for administration to the correct dermis.

However, the importance of efficiently and safely administering medicinal substances such as diagnostic drugs and medicines has recently been recognized. Especially recently, as the problem of acute disseminated encephalomyelitis (ADEM) in Japanese encephalitis virus antigen has been pointed out (Non-Patent Document 2), in order to reduce the risk, Japanese encephalitis virus Development of an antigen has been carried out, and development of an efficient and simple administration method of a virus antigen that requires a small number of virus antigens to cause immunity to many people is desired.

Patent Document 1 discloses administration of a micro needle having a hollow needle for the purpose of achieving a therapeutic effect by reducing the amount of a therapeutic substance, but there is no description about the cause of immunity.

Japan Specification No. 2006-506103

Pharma. Res. 19 (1), 63-70 (2002) Virus 55 Volume 2, 307-312 (2005)

As described above, although the Japanese encephalitis virus antigen is coated on the microneedle to perform accurate administration to the dermis, there is a possibility that the Japanese encephalitis virus antigen can be administered efficiently and easily. However, Specific administration has not been studied.

It is an object of the present invention to provide a method for enhancing immunogenicity by a micro needle device that enhances immunogenicity of a Japanese encephalitis virus antigen.

As a result of repeated studies by the inventors on the above background, the present inventors have found that, by transdermally administering a Japanese encephalitis virus antigen using a micro needle coated with a Japanese encephalitis virus antigen comprising an antigen derived from monkey kidney cells (Vero cells) I found that sex rose. In addition, transdermal administration of microneedles coated with Japanese encephalitis virus antigen followed by application of a patch containing lauryl alcohol confirmed an increase in the effective antibody value.

That is, the method for enhancing immunogenicity by a micro needle device according to the present invention is a method for increasing the immunogenicity of a micro needle device comprising a microneedle comprising polylactic acid coated with a Japanese encephalitis virus antigen comprising an antigen derived from monkey kidney cells (Vero cells) And the Japanese encephalitis virus antigen is transdermally administered. Here, after transdermal administration of the Japanese encephalitis virus antigen, the immunogenicity is further enhanced by the attachment of the patch containing lauryl alcohol having an adjuvant effect. In addition, application of a substance having an adjuvant activity capable of penetrating the skin can be expected to enhance the immunogenicity.

The micro needle device according to the present invention comprises a micro needle made of polylactic acid coated with a Japanese encephalitis virus antigen comprising an antigen derived from monkey kidney cells (Vero cells). Here, it is preferable that the coating contains pullulan as a coating carrier.

According to the present invention, it is possible to raise the immunogenicity of a Japanese encephalitis virus antigen composed of an antigen derived from monkey kidney cells (Vero cells) by a simple operation than an injection. By using a percutaneous immunodeficient microneedle device, it is possible to stimulate the immune response induced by the Japanese encephalitis virus antigen, thereby reducing the effective dose of the antigen in the viral antigen.

Fig. 1 is a view showing an example of a micro needle device according to the present invention, wherein (a) is a perspective view and (b) is a cross-sectional view taken along the line AB of Fig.
2 is a graph showing an example of the measurement result of the Japanese encephalitis virus antigen-specific IgG antibody value.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing an example of a microneedle device according to the present invention, wherein (a) is a perspective view and (b) is a cross-sectional view taken along the line A-B of FIG. 1 (a), a microneedle device (interface) 5 according to the present invention includes a microneedle substrate 8 and a plurality of microneedles 6). The microneedle substrate 8 has a plurality of openings 7 arranged corresponding to the respective microneedles 6. In this example, the shape of the microneedle 6 is conical, but the present invention is not limited to this, and may be a polygonal shape such as a tetragon or other shape. Further, a plurality of micro needles 6 and a plurality of openings 7 are alternately arranged in a tetragonal lattice shape, but the present invention is not limited to this. The numbers of the micro needles 6 and the openings 7 are 1: 1 in the drawing, but the present invention is not limited to this and includes those not including the openings 7.

In this example, a part or the entire surface of the micro needle 6 is coated with a Japanese encephalitis virus antigen comprising an antigen derived from monkey kidney cells (Vero cells). The coating 1 is arranged on the surface of each micro needle 6, for example, as shown in Fig. 1 (b). Although the coating 1 is disposed on the entirety of the micro needle 6, it may be disposed on a part thereof. In use, when the surface of the microneedle substrate on which the microneedle 6 shown in Fig. 1 (a) is placed is brought into contact with the skin and the solution of the medicine is flowed from the opposite surface, liquid flows from each of the openings 7 And transmitted to each micro needle 6, and the Japanese encephalitis virus antigen is percutaneously absorbed. Here, the opening 7 is not essential, and the liquid may be supplied to the microneedle 6 by other means without using the opening 7. In addition, the coating (1) can dissolve the Japanese encephalitis virus antigen into the skin by dissolving the micro needle into the skin when perforating the skin without applying a liquid from the outside.

The microneedles in the microneedle device consist of microneedles (needles) punctured to the skin or mucous membranes and a substrate for supporting the needles, and a plurality of microneedles are arranged on the substrate. The micro needle has a microstructure, and the height (length) h of the micro needle is preferably 50 mu m to 700 mu m, more preferably 100 mu m to 600 mu m, and still more preferably 200 mu m to 500 mu m. Here, the length of the microneedles is preferably 50 占 퐉 or more in order to ensure the administration of the Japanese encephalitis virus antigen from the transdermal route. Setting the length to 700 占 퐉 or less avoids contact with the nerve of the microneedle, And it is possible to reliably avoid the possibility of bleeding. When the length is 700 mu m or less, the amount of Japanese encephalitis virus antigen that enters the skin can be efficiently administered.

Here, the micro needle is a convex shape structure and means a structure including a needle shape or a needle shape in a broad sense. In the case of a cone structure, the diameter at the base is usually about 50 to 200 mu m. The microneedles are not limited to needle-shaped ones having sharp ends, but also shapes having no sharp ends. The micro needles are preferably made of a synthetic resin of a base metal or a natural resin material. The shape of the microneedles is conical in the present embodiment, but the present invention is not limited to this, and may be a polygonal pyramid such as a tetragon or another shape.

The microneedle substrate is a base for supporting the microneedles, and the shape is not limited. For example, as shown in Fig. 1, the microneedle substrate may be a substrate having a hole (opening) penetrating therethrough. In this case, The solution of the Japanese encephalitis virus antigen coated on the micro needle can be flowed, and the Japanese encephalitis virus antigen can be administered through the opening and the micro needle. Examples of the material of the micro needle or the substrate include silicon, silicon dioxide, ceramic, metal (stainless steel, titanium, nickel, molybdenum, chromium, cobalt) and synthetic or natural resin materials. A biodegradable polymer such as polylactic acid, polyglycolide, polylactic acid-co-polyglycolide, pullulan, caprolactone, polyurethane or polyanhydride, polycarbonate as non-degradable polymer, Particularly preferred are synthetic or natural resin materials such as acid, ethylene vinyl acetate, polytetrafluoroethylene and polyoxymethylene. Also suitable are polysaccharides such as hyaluronic acid, pullulan, dextran, dextrin or chondroitin sulfate. Particularly, since polylactic acid is a biodegradable resin and has been used as an implant preparation (Japanese Patent Publication No. 2002-517300 or Journal of Controlled Release 104 (2005) 51-66), the most suitable micro- It is one of the needle materials.

The density of the microneedles (needles) is typically set such that the rows of needles are provided with a density of about 1 to 10 per millimeter (mm). Generally, the rows are spaced at substantially the same distance relative to the space of the needles in the row and have a needle density of 100 to 10000 per cm ² , preferably 100 to 5000, more preferably 200 to 2000, Preferably 400 to 1,000. With more than 100 needle densities, it is possible to perforate the skin efficiently, and with more than 10,000 needle densities, it becomes difficult to impart the puncturable strength to the micro needle.

Examples of the manufacturing method of the micro needle include wet etching or dry etching using a silicon substrate, precision machining (discharge machining, laser machining, dicing, hot embossing, injection molding, etc.) using metal or resin, And the like. By these processing methods, the needle portion and the support portion are integrally formed. As a method of hollowing the needle part, there is a method of making the needle part and then performing secondary processing by laser processing or the like.

The temperature and humidity of the installation environment of the apparatus can be controlled to be minimized in order to minimize changes in concentration and physical properties of the drug due to solvent volatilization of the coating liquid when coating the micro needles. In order to prevent the evaporation of the solvent, it is desirable to control either the temperature lowering or the humidity increasing or both. The humidity at room temperature when the temperature is not controlled is 50 to 100% RH as relative humidity, and preferably 70.0 to 100% RH. If it is 50% RH or lower, evaporation of the solvent may occur, and the physical properties of the coating liquid may change. The humidification method is not particularly limited as long as a desired humidity state can be secured, but there are vaporization type, vapor type, and water-insoluble type.

The preparation of the Japanese encephalitis virus antigen used in the present invention can be carried out, for example, with reference to WO 01/076624.

The coating liquid to be coated on the micro needle can include, in addition to the Japanese encephalitis virus antigen, a coating carrier and a liquid composition. The coating of the present invention is preferably a state in which a coating solution is fixed to a micro needle (needle) and fixed. In this case, a drying process is added to the coating solution to fix it.

As the coating carrier, a carrier of a polysaccharide having relatively comparable compatibility (uniformly crossing property) with a Japanese encephalitis virus antigen is preferable, and a carrier such as polyhydroxymethylcellulose, hydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, Tran, polyethylene glycol, pullulan, carmellose sodium, chondroitin sulfate, hyaluronic acid, dextran, and gum arabic are preferable, and hydroxypropylcellulose, pullulan and gum arabic are more preferable. (Molecular weight: 30,000 to 50,000), HPC-L (molecular weight: 55,000 to 70,000), HPC-M (molecular weight: 110,000 to 150,000), and hydroxypropylcellulose , HPC-H (molecular weight: 250,000 to 400,000)), pullulan, and hyaluronic acid are more preferable. Particularly, in view of compatibility with the Japanese encephalitis virus antigen, pullulan is most preferable.

The content of the coating carrier in the whole coating liquid is 1 to 70% by weight, preferably 1 to 40% by weight, and particularly preferably 3 to 25% by weight. Further, the coating carrier may need a certain viscosity to prevent the liquid from flowing down, and it is necessary that the viscosity is 100 to 100000 cps. A more preferable viscosity is 500 to 60000 cps. When the viscosity is within this range, it becomes possible to apply a desired amount of the coating liquid at once, without depending on the material of the micro needle. In general, the higher the viscosity is, the more the amount of the coating liquid tends to increase.

The liquid composition used to coat the micro needles is prepared by mixing the biocompatible carrier, the Japanese encephalitis virus antigen to be delivered, and optionally one of the coating aids with a volatile liquid. Volatile liquids can be water, dimethylsulfoxide, dimethylformamide, ethanol, isopropyl alcohol and mixtures thereof. Of these, water is most preferred. The liquid coating solution or suspension may typically have a concentration of Japanese encephalitis virus antigen of 0.1 to 65% by weight, preferably 1 to 30% by weight, more preferably 3 to 20% by weight.

Other known formulation adjunct materials may be added to the coating, provided they do not adversely affect the solubility and viscosity characteristics required of the coating and the properties and physical properties of the dried coating.

The thickness of the coating of the micro needle is less than 50 mu m, preferably less than 25 mu m, more preferably 1 to 10 mu m. In general, the thickness of the coating is the average thickness measured across the surface of the micro needle after drying. The thickness of the coating can generally be increased by applying a plurality of coatings of the coating carrier, i. E. By repeating the coating process after adhesion of the coating carrier.

The height (length) h of the microneedles is preferably 50 m to 700 m as described above. The height H of the coating of the micro needle varies depending on the height h of the micro needle, and can be in the range of 0 mu m to 700 mu m, usually in the range of 10 mu m to 700 mu m, To about 500 mu m.

In order to enhance the immunity caused by the Japanese encephalitis virus antigen using the micro needle device of the present invention, for example, aliphatic alcohols having an adjuvant effect can be applied to a site administered by the micro needle device of the present invention have. In such aliphatic alcohols, straight-chain or branched aliphatic alcohols are preferred. In such aliphatic alcohols, the number of carbon atoms and the molecular weight are not particularly limited, but from the viewpoint of skin permeability, the number of carbon atoms is more preferably 8 to 20. These aliphatic alcohols may be either saturated or unsaturated.

Among these aliphatic alcohols, there are many cases in which they are used as an absorption promoting agent in percutaneous absorption. The aliphatic alcohols in the present invention can be expected to exhibit an adjuvant effect in addition to the absorption promoting action with reference to WO 2007/015441.

Such aliphatic alcohols are, for example, octyldodecanol, lauryl alcohol, oleyl alcohol, isostearyl alcohol, decanol, etc. Among them, lauryl alcohol, oleyl alcohol and isostearyl alcohol are particularly preferable , And lauryl alcohol are most preferable.

When mixed with a virus antigen, the amount of the aliphatic alcohol of the present invention is preferably 0.1 to 99% by weight, more preferably 5 to 90% by weight, and even more preferably 10 to 80% by weight. The content of the aliphatic alcohol in the most preferable composition is from 15 to 75% by weight.

The form of the pharmaceutical preparation containing the adjuvant is not particularly limited as long as it is a form that can be percutaneously administered. The form of the pharmaceutical preparation containing the adjuvant may be selected from a patch such as a papain or patch product, an ointment preparation, a cream preparation, a liquid preparation, But a patch formulation is particularly preferable.

The site of administration of the patch preparation is not particularly limited, but it is preferable that it is close to the site of administration of the antigen, and it is more preferable to attach it to the site of the site of administration of the antigen.

This transdermal preparation can be prepared by using a combination of an optional component such as a solubilizer, a dissolution aid, a pH adjuster, an antiseptic, an absorption promoter, a stabilizer, a filler, a thickener, a pressure sensitive adhesive and a wetting agent in combination with the adjuvant can do.

The composition of the medicinal preparation containing this adjuvant is preferably blended at 0.1 to 99 wt%, more preferably 5 to 90 wt%, particularly preferably 10 to 80 wt%. The content of the aliphatic alcohol in the most preferable composition is from 15 to 75% by weight.

Example

(Experimental Example 1)

Japanese encephalitis virus antigens containing antigens derived from monkey kidney cells (Vero cells) adjusted as described below were concentrated by centrifugation using BIOMAX-10K (manufactured by Millipore), mixed with polymeric polymer (pullulan) Thereafter, it was coated on a polylactic acid micro needle (height: about 300 μm, density: 841 pieces / cm ² , tetragonal shape) of a microneedle device while maintaining a relative humidity of 90 to 100% HR with a humidifier. Coating content was 2 μg / patch of antigen, 4 weeks of ddY mice (female) under abdominal hysterectomy under anesthesia, and the coated microneedles were administered to the skin for 2 hours (4 cases). In the adjuvant group (5 cases), the adjuvant (lauryl alcohol) patch was attached to the upper part of the area where the micro needle was administered. After one week, the mice were boosted under the same conditions. One week later, blood samples were collected and the Japanese encephalitis virus antigen-specific IgG antibody was measured. The results are shown in Fig.

(Measurement of Antibody Value)

The collected blood was allowed to stand at 4 占 폚 overnight, and the serum was separated by centrifugation and immobilized (treated at 56 占 폚 for 30 minutes). The viral antigen diluted to 5 μg / mL with the coating buffer was added to the wells at 100 μL / well and allowed to stand at 4 ° C. overnight. The solidified plate was washed three times with 400 μL / well of washing buffer, and 250 μL / well of blocking buffer was added, followed by reaction at 37 ° C. for 1 hour. Thereafter, the sample was washed three times with 400 μL / well of washing buffer, and the diluted buffer was diluted 100-fold with a dilution buffer. Each diluted sample was diluted twice with 100 μL / well of each sample and incubated at 37 ° C. for 2 hours .

Next, the wells were washed three times with 400 μL / well of the washing buffer, and 100 μL / well of the diluted HRP-labeled antibody was added, followed by reaction at 37 ° C. for 90 minutes. The buffer was washed three times with 400 μL / well, and 100 μL / well of TMBZ solution was added. After reacting at room temperature for 30 minutes in a dark place, the reaction was stopped by addition of 100 μL / well of 0.3 N sulfuric acid and the absorbance at 450 nm was measured.

Coating buffer; 0.05 M carbonate buffer (pH 9.5)

Cleaning buffer; PBS (PBS-T) containing 0.05% Tween 20

Blocking buffer; PBS containing 0.5% BSA

Dilution buffer; PBS-T containing 0.5% BSA

(MN + LAtape) in which a Japanese encephalitis virus antigen-specific IgG antibody value was obtained in the micro needle device MN alone, and an adjuvant (lauryl alcohol) patch was also used as shown in Fig. 2, , A higher level of Japanese encephalitis virus antigen-specific IgG antibody can be obtained.

The present invention includes the following.

(1) A method for producing a microarray comprising the steps of: (1) providing a plurality of micro needles, which are arranged on a substrate in a two-dimensional shape and made of polylactic acid capable of perforating the skin, wherein the micro needles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells Micro needle device.

(2) The microneedle device according to (1), wherein the microneedles are conical or square.

(3) The microneedle device according to (1) or (2) above, wherein the coating comprises pullulan as a coating carrier.

(4) The microneedle device according to any one of (1) to (3) above, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.

(5) The microneedle device according to any one of (1) to (4) above, wherein the coating comprises a substance having an adjuvant activity.

(6) The microneedle device according to (5), wherein the substance having the adjuvant activity is lauryl alcohol.

(7) The microneedle device according to any one of (1) to (6) above, wherein the substrate of the microneedle device has a plurality of openings capable of delivering Japanese encephalitis virus antigen solution or Japanese encephalitis virus antigen solution.

(8) The microneedle device according to any one of (1) to (7), wherein the height of the microneedles is 200 to 500 mu m.

(9) The microneedle device according to any one of (1) to (8), wherein the microneedles are arranged at a density of 400 to 1000 / cm ² .

(10) In a method in which a Japanese encephalitis virus antigen is administered by a micro needle device, and then a patch containing a substance having an adjuvant activity is stuck and the main efficacy of a Japanese encephalitis virus antigen is elevated, A microneedle device comprising a plurality of microneedles arranged in a two-dimensional shape on a substrate and made of polylactic acid capable of perforating the skin, wherein the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) Wherein the micro needle device is a micro needle device.

(11) A pharmaceutical composition comprising a plurality of micro needles, which are arranged in a two-dimensional shape on a substrate and made of polylactic acid capable of perforating the skin, the micro needles being coated with a Japanese encephalitis virus antigen derived from monkey kidney cells A method for raising the main efficacy of a Japanese encephalitis virus antigen by a micro needle device.

(12) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the micro needle device according to (11), wherein the micro needle is conical or squared.

(13) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the micro needle device according to (11) or (12), wherein the coating comprises pullulan as a coating carrier.

(14) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the microneedle device according to any one of claims 11 to 13, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.

(15) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (14), wherein the coating comprises a substance having an adjuvant activity.

(16) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the microneedle device according to (15), wherein the substance having the adjuvant activity is lauryl alcohol.

(17) The microneedle device according to any one of (11) to (16) above, wherein the substrate of the microneedle device has a plurality of openings capable of delivering Japanese encephalitis virus antigen solution or Japanese encephalitis virus antigen solution A method for raising the main efficacy of a Japanese encephalitis virus antigen.

(18) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (17), wherein the height of the micro needle is 200 to 500 mu m.

(19) A method for raising the main efficacy of a Japanese encephalitis virus antigen by the micro needle device according to any one of (11) to (18), wherein the micro needles are arranged at a density of 400 to 1000 pieces / cm ² .

(20) A method of enhancing the efficacy of a Japanese encephalitis virus antigen by the micro needle device according to any one of (11) to (19) above, And the main effect of the Japanese encephalitis virus antigen by the micro needle device is further elevated.

(21) A method for further enhancing the main efficacy of a Japanese encephalitis virus antigen by the micro needle device according to (20), wherein the substance having the adjuvant activity of the patch is lauryl alcohol.

The present invention makes it possible to raise the antigenicity of a Japanese encephalitis virus antigen composed of a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) by using an efficient and easy operation by using a micro needle device, There is a possibility of the above.

Claims (21)

A microneedle device comprising a plurality of microneedles arranged in a two-dimensional shape on a substrate and made of a polylactic acid capable of perforating the skin, the microneedles being coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) ,
The micro-needles are arranged in a conical or pyramidal, and a height 200~500㎛ of the micro-needle, and the density of the micro-needles are 400-1000 / cm ^2,
The coating is a state in which the coating solution is stuck to the microneedle with a coating thickness of less than 50 탆 and a height (H) of 30 탆 to 500 탆,
Wherein the coating is performed at a relative humidity of from 70.0 to 100% RH at room temperature. The microneedle device according to claim 1, wherein the coating comprises pullulan as a coating carrier. The microneedle device according to claim 1, wherein the coating comprises a substance having an adjuvant activity. The microneedle device according to claim 3, wherein the substance having the adjuvant activity is lauryl alcohol. The microneedle device according to any one of claims 1 to 4, wherein the substrate of the microneedle device has a plurality of openings capable of delivering Japanese encephalitis virus antigen solution or Japanese encephalitis virus antigen solution. In a method of applying a Japanese encephalitis virus antigen by a micro needle device and then pasting a patch containing a substance having an adjuvant activity and raising the main efficacy of a Japanese encephalitis virus antigen, as,
A plurality of micro-needles made of polylactic acid that are arranged in a two-dimensional shape on a substrate and capable of perforating the skin, wherein said micro-needles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells)
The micro-needles are arranged in a conical or pyramidal, and a height 200~500㎛ of the micro-needle, and the density of the micro-needles are 400-1000 / cm ^2,
The coating is a state in which the coating solution is stuck to the microneedle with a coating thickness of less than 50 탆 and a height (H) of 30 탆 to 500 탆,
Wherein the coating is performed at a relative humidity of from 70.0 to 100% RH at room temperature. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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