JPWO2010013601A1 - Microneedle device and method for increasing the efficacy of Japanese encephalitis virus antigen by microneedle device - Google Patents

Abstract

Provided is a method for enhancing immunogenicity using a microneedle device that enhances the immunogenicity of a Japanese encephalitis virus antigen. The immunogenicity enhancement method using this microneedle device is an efficient method in which Japanese encephalitis virus antigen is transdermally administered using a microneedle coated with Japanese encephalitis virus antigen consisting of antigen derived from monkey kidney cells (Vero cells). The antibody of Japanese encephalitis virus antigen rises. Further, after transdermal administration of the microneedles coated with Japanese encephalitis virus antigen, a patch containing lauryl alcohol is pasted.

Description

  The present invention relates to a method for enhancing immunogenicity using a microneedle device.

The skin consists of the outermost stratum corneum, epidermis, dermis, and subcutaneous connective tissue. Usually, the stratum corneum consisting of a dead cell layer and a lipid bilayer exhibits a strong barrier function against many substances. In the dermis layer, antigen-presenting cells called Langerhans cells exist and have an immune function. Langerhans cells capture protein antigens that have entered the skin, break down inside, and express peptide fragments on MHC molecules. The MHC-peptide complex moves from the imported lymphatic vessel to the subcortical layer of the regional lymph node, and contacts through the T cell and the finger process cell. Langerhans cells, by moving in this manner, is transmitted to the T _H cells antigen is present in the lymph nodes from the skin. Langerhans cells have MHC class II molecules necessary to present antigen to the T _H cells.

  Thus, it was known that the virus antigen to the dermis layer is effective due to the strong barrier function by the stratum corneum of the skin, but the administration by the injection needle to the dermis layer limited to 300 to 2000 μm is technical. There was a problem in accuracy due to difficulty.

  Microneedles have been developed as means for solving this problem. These are intended to puncture the outermost stratum corneum, and have been devised in various sizes and shapes (very small protrusions of several tens to several hundreds of micrometers), especially non-invasive It is expected as a new virus antigen administration method.

  In addition, various methods have been devised for applying drugs when using a device equipped with microneedles, such as a method of coating a drug on the surface of the microneedle and administering the drug or a biological component through the needle. A method of forming a hole (hollow needle) or a groove for making the needle, a method of mixing a drug in the needle itself, and the like have been proposed. These microneedle devices are all provided with very small protrusions (microneedles) having a height of about several tens to several hundreds of micrometers. Therefore, depending on the method of applying the drug, Absorption efficiency is also considered to differ greatly.

  For example, as a method of efficiently promoting transdermal absorbability of an antigen (vaccine) using a microneedle, there is a method of coating a drug on a part of the surface of the microneedle, which is disclosed in Non-Patent Document 1, for example. This is useful when a part of the microneedle (especially only the needle part) is coated with an antigen (vaccine), and all or most of the applied antigen (vaccine) is transferred into the body, which is useful as an accurate dermis administration means. It shows that there is.

  Meanwhile, the importance of efficiently and safely administering pharmaceutical substances such as diagnostic agents and drugs has recently been recognized. In particular, as the problem of acute disseminated encephalomyelitis (ADEM) has recently been pointed out in Japanese encephalitis virus antigens (Non-patent Document 2), in order to reduce the risk, Vero cells derived from monkey kidney cells Japanese encephalitis virus antigens have been developed using, and it is desired to develop an efficient and simple administration method of virus antigens, such as how many humans can immunize with less virus antigens.

  Patent Document 1 discloses administration with a microneedle having a hollow needle for the purpose of reducing the amount of a therapeutic substance and achieving a therapeutic effect, but there is no description of inducing immunity.

JP 2006-506103 A

Pharma. Res. 19 (1), 63-70 (2002) Virus Vol. 55, No. 2, 307-312 (2005)

Despite the possibility of efficient and simple administration of Japanese encephalitis virus antigen by coating Japanese encephalitis virus antigen on microneedles and performing accurate administration to the dermis as described above, No specific study has been made on the administration of encephalitis virus antigens using microneedles.
An object of the present invention is to provide a method for enhancing immunogenicity using a microneedle device that enhances the immunogenicity of a Japanese encephalitis virus antigen.

  The present inventor has conducted daily studies with the above as a technical background, and as a result, transdermally administers Japanese encephalitis virus antigens using microneedles coated with Japanese encephalitis virus antigens composed of antigens derived from monkey kidney cells (Vero cells). And found that the antibody property increased. Furthermore, after the transdermal administration of microneedles coated with Japanese encephalitis virus antigen, a patch containing lauryl alcohol was affixed, whereby a further efficient increase in antibody titer could be confirmed.

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

  The microneedle device according to the present invention includes a microneedle made of polylactic acid coated with a Japanese encephalitis virus antigen made of an antigen derived from monkey kidney cells (Vero cells). Here, the coating preferably includes pullulan as a coating carrier.

  According to the present invention, immunogenicity of Japanese encephalitis virus antigen consisting of an antigen derived from monkey kidney cells (Vero cells) can be increased by a simpler operation than injection. By using a microneedle device for transcutaneous immunostimulation, the immune response induced by Japanese encephalitis virus antigens can be stimulated, and the effective dose of antigens in the virus antigens can be reduced.

It is a figure which shows an example of the microneedle device which concerns on this invention, (a) is a perspective view, (b) is AB sectional drawing of (a). It is a graph which shows an example of the measurement result of Japanese encephalitis virus antigen specific IgG antibody titer.

  1A and 1B are diagrams showing an example of a microneedle device according to the present invention, in which FIG. 1A is a perspective view and FIG. 1B is a cross-sectional view taken along line AB of FIG. As shown in FIG. 1 (a), a microneedle device (interface) 5 according to the present invention comprises a microneedle substrate 8 and a plurality of microneedles 6 arranged in a two-dimensional shape capable of perforating the skin or mucous membrane. Have. The microneedle substrate 8 includes a plurality of openings 7 arranged corresponding to the microneedles 6. In this example, the shape of the microneedle 6 is a conical shape, but the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape. Further, although the plurality of microneedles 6 and the plurality of openings 7 are alternately arranged in a square lattice shape, the present invention is not limited to this. Furthermore, although the number of the microneedles 6 and the openings 7 is 1: 1 in the drawing, the present invention is not limited to this, and includes those that do not include the openings 7.

  In this example, a part or the whole surface of the microneedle 6 is coated with a Japanese encephalitis virus antigen composed of an antigen derived from monkey kidney cells (Vero cells). The coating 1 is arrange | positioned on the surface of each microneedle 6, for example, as shown in FIG.1 (b). The coating 1 is disposed on the entire microneedle 6, but may be disposed on a part thereof. At the time of use, when the microneedle substrate surface on which the microneedles 6 shown in FIG. 1 (a) are disposed is brought into contact with the skin and a drug solution is allowed to flow from the opposite surface, the liquid flows out from each opening 7 and each micro It is transmitted to the 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 another means without using the opening 7. In addition, the coating 1 can release the Japanese encephalitis virus antigen into the skin by dissolving with a body fluid when the microneedle is perforated in the skin without applying a liquid from the outside.

  The microneedle in the microneedle device includes a microneedle (needle) that is punctured into the skin or mucous membrane and a substrate that supports the needle portion, and a plurality of microneedles are arranged on the substrate. The microneedle has a microstructure, and the height (length) h of the microneedle is preferably 50 μm to 700 μm, more preferably 100 μm to 600 μm, and further preferably 200 μm to 500 μm. Here, the length of the microneedle is set to 50 μm or more to ensure administration of the Japanese encephalitis virus antigen from the skin, and the length of 700 μm or less avoids contact of the microneedle with the nerve, This is because the possibility of pain can be surely reduced and at the same time the possibility of bleeding can be surely avoided. If the length is 700 μm or less, the amount of Japanese encephalitis virus antigen that enters the skin can be efficiently administered.

  Here, the microneedle means a convex structure and means a needle shape in a broad sense or a structure including a needle shape. In the case of a conical structure, the diameter at the base is usually about 50 to 200 μm. . Further, the microneedle is not limited to a needle shape having a sharp tip, and includes a shape having no sharp tip. The microneedles are preferably made using a non-metallic synthetic or natural resin material. Further, the shape of the microneedle is a conical shape in this example, but the present invention is not limited to this, and may be a polygonal pyramid such as a quadrangular pyramid or another shape.

The microneedle substrate is a base for supporting the microneedle, and the form thereof is not limited. For example, the substrate may be a substrate having a through hole (opening) as shown in FIG. In addition to allowing the solution of Japanese encephalitis virus antigen coated on the microneedle to flow from the back of the substrate, the Japanese encephalitis virus antigen can also be administered by flowing through the opening and the microneedle. Examples of the material of the microneedle or the substrate include silicon, silicon dioxide, ceramic, metal (stainless steel, titanium, nickel, molybdenum, chromium, cobalt, etc.) and synthetic or natural resin materials. Considering the unit price of the material, biodegradable polymers such as polylactic acid, polyglycolide, polylactic acid-co-polyglycolide, pullulan, capronolactone, polyurethane, polyanhydride, polycarbonates that are non-degradable polymers, polymethacrylic acid, A synthetic or natural resin material such as ethylene vinyl acetate, polytetrafluoroethylene, or polyoxymethylene is particularly preferable. Also suitable are polysaccharides such as hyaluronic acid, pullulan, dextran, dextrin or chondroitin sulfate. In particular, 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). It is one of the most suitable microneedle materials in view of the above.

The density of microneedles (needles) is typically spaced between the rows such that the rows of needles provide a density of about 1 to 10 per millimeter (mm). In general, the rows are separated by a substantially equal distance from the needle space in the row and have a needle density of 100-10000 per cm ² , preferably 100-5000, more preferably 200-2000, and even more Preferably it is 400-1000. When there is a needle density of 100 or more, the skin can be efficiently perforated, and when the needle density exceeds 10,000, it is difficult to give the microneedles strength capable of perforating the skin.

  Microneedle manufacturing methods include wet etching or dry etching using a silicon substrate, precision machining using metal or resin (electric discharge machining, laser machining, dicing, hot embossing, injection molding, etc.), machinery Examples include cutting. By these processing methods, the needle portion and the support portion are integrally molded. As a method for making the needle part hollow, there is a method of performing secondary processing by laser processing or the like after producing the needle part.

  When coating the microneedles, the temperature and humidity of the installation environment of the apparatus can be controlled to be constant in order to minimize changes in the concentration and physical properties of the drug due to solvent evaporation of the coating solution. In order to prevent transpiration of the solvent, it is preferable to control either the temperature or the humidity or both. When the temperature is not controlled, the humidity at room temperature is 50 to 100% RH as relative humidity, and preferably 70.0 to 100% RH. If it is 50% RH or less, the solvent may evaporate and the physical properties of the coating solution may change. The humidification method is not particularly limited as long as the target humidity state can be secured, but there are a vaporization method, a steam method, a water spray method, and the like.

  Preparation of the Japanese encephalitis virus antigen used in the present invention can be performed with reference to, for example, WO01 / 076664.

  The coating liquid that coats the microneedles can contain a coating carrier and a liquid composition in addition to the Japanese encephalitis virus antigen. The coating of the present invention is preferably a state in which the coating liquid stays and adheres to the microneedles (needles). For this purpose, the coating liquid may be fixed by applying a drying step.

  As the coating carrier, a polysaccharide carrier that is relatively compatible with the Japanese encephalitis virus antigen (a property that uniformly crosses) is preferable. Polyhydroxymethylcellulose, hydroxypropylcellulose, polyhydroxypropylmethylcellulose, polymethylcellulose, dextran, polyethylene glycol, Pullulan, carmellose sodium, chondroitin sulfate, hyaluronic acid, dextran, gum arabic and the like are preferred, and hydroxypropyl cellulose, pullulan and gum arabic are more preferred. Further, hydroxypropylcellulose (HPC-SSL (molecular weight: 15,000 to 30,000), HPC-SL (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), HPC-H (molecular weight: 250,000 to 400,000)), pullulan and hyaluronic acid are more preferable. In particular, pullulan is most preferable in terms of compatibility with the Japanese encephalitis virus antigen.

  The content of the coating carrier in the whole coating liquid is 1 to 70% by weight, preferably 1 to 40% by weight, particularly preferably 3 to 25% by weight. In addition, this coating carrier may require a certain degree of viscosity so that it does not drip, and the viscosity is required to be about 100 to 100,000 cps. A more preferable viscosity is 500 to 60000 cps. When the viscosity is in this range, it is possible to apply a desired amount of the coating liquid at a time without depending on the material of the microneedle. In general, the higher the viscosity, the greater the amount of coating solution.

The liquid composition used to coat the microneedles is prepared by mixing a biocompatible carrier, the Japanese encephalitis virus antigen to be delivered, and possibly any coating aids with a volatile liquid. To do. The volatile liquid can be water, dimethyl sulfoxide, dimethylformamide, ethanol, isopropyl alcohol, mixtures thereof, and the like. Of these, water is most preferred. The liquid coating solution or suspension can typically have a Japanese encephalitis virus antigen concentration of 0.1 to 65 wt%, preferably 1 to 30 wt%, more preferably 3 to 20 wt%. %.
Other known formulation adjuvants may be added to the coating as long as they do not adversely affect the required solubility and viscosity characteristics of the coating and the properties and physical properties of the dried coating.

  The thickness of the microneedle coating is less than 50 μm, preferably less than 25 μm, more preferably 1 to 10 μm. In general, the coating thickness is the average thickness measured across the surface of the microneedle after drying. The thickness of the coating can generally be increased by applying multiple coatings of the coating carrier, i.e. by repeating the coating process after fixing the coating carrier.

  As described above, the height (length) h of the microneedles is preferably 50 μm to 700 μm. The coating height H of the microneedle varies depending on the height h of the microneedle, but can be in the range of 0 μm to 700 μm, usually in the range of 10 μm to 700 μm, preferably about 30 μm to 500 μm. is there.

In order to enhance the induction of immunity by Japanese encephalitis virus antigen using the microneedle device of the present invention, for example, an aliphatic alcohol having an adjuvant effect can be applied to the site administered by the microneedle device of the present invention. . Of these aliphatic alcohols, linear or branched aliphatic alcohols are preferred. In such aliphatic alcohols, the carbon number and molecular weight are not particularly limited, but considering the skin permeability, it is more preferably 8 to 20 carbon atoms. Such aliphatic alcohols may be saturated or unsaturated.
Among these fatty alcohols, they are often used as absorption enhancers in percutaneous absorption. However, referring to WO2007 / 015441, the aliphatic alcohols in the present invention have an adjuvant effect in addition to the absorption promoting action. You can expect.

  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 is most preferable. .

  When mixed with a viral antigen, the aliphatic alcohols of the present invention are preferably blended at 0.1 to 99% by weight, more preferably 5 to 90% by weight, particularly 10 to 80% by weight. More preferably. The content of aliphatic alcohols in the most preferred composition is 15 to 75% by weight.

The form of the pharmaceutical preparation containing the adjuvant is not particularly limited as long as it can be administered transdermally. However, patches, ointments, creams, solutions, gels, lotions, etc. However, a patch preparation is particularly preferable.
The administration site of the patch preparation is not particularly limited, but is preferably closer to the antigen administration site, and more preferably applied to the upper part of the antigen administration site.
The preparation for transdermal administration contains, as a base, optional components such as a solubilizer, a solubilizer, a pH adjuster, an antiseptic, an absorption accelerator, a stabilizer, a filler, a thickener, an adhesive, and a wetting agent. By using the adjuvant in combination, it can be produced by a conventional method.

  The composition of the pharmaceutical preparation containing the adjuvant is preferably blended at 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 aliphatic alcohols in the most preferred composition is 15 to 75% by weight.

(Experimental example 1)
A Japanese encephalitis virus antigen containing an antigen derived from monkey kidney cells (Vero cells) prepared as follows was concentrated by centrifugation using BIOMAX-10K (Millipore), mixed with a polymer (pullulan), While maintaining a relative humidity of 90 to 100% HR with a humidifier, the microneedle made of polylactic acid (height of about 300 μm, density of 841 pieces / cm ² , quadrangular pyramid shape) of a microneedle device was coated. The coating content was 2 μg / patch of antigen, and a 4-week-old ddY mouse (female) was shaved from the abdomen under anesthesia, and then the coated microneedle was punctured for 2 hours into the skin (4 cases). In the adjuvant administration group (5 cases), an adjuvant (lauryl alcohol) patch was affixed to the upper part of the microneedle administration site. One week later, boosting was performed under the same conditions. After one week of blood collection, the Japanese encephalitis virus antigen-specific IgG antibody titer was measured. The results are shown in FIG.

(Measurement of antibody titer)
The collected blood was allowed to stand overnight at 4 ° C., and then the serum was separated by centrifugation and inactivated (treated at 56 ° C. for 30 minutes). The virus antigen diluted to 5 μg / mL with the coating buffer was added to the well at 100 μL / well and allowed to stand at 4 ° C. overnight. The solid-phased plate was washed 3 times with 400 μL / well of washing buffer, 250 μL / well of blocking buffer was added, and reacted at 37 ° C. for 1 hour. Thereafter, the sample was washed 3 times with a washing buffer of 400 μL / well, sufficiently drained, then diluted 100-fold with a dilution buffer, and 100 μL / well of each sample diluted serially in a 2-fold series was added at 37 ° C. for 2 hours. Reacted.
Next, the plate was washed 3 times with a washing buffer of 400 μL / well, diluted HRP-labeled antibody 100 μL / well was added, and reacted at 37 ° C. for 90 minutes. Wash 3 times with 400 μL / well of buffer, add 100 μL / well of TMBZ solution, react at room temperature for 30 minutes in the dark, then add 100 μL / well of 0.3 N sulfuric acid to stop the reaction, and absorb the absorbance at 450 nm. It was measured.
Coating buffer; 0.05M carbonate buffer (pH 9.5)
Washing buffer; PBS containing 0.05% Tween 20 (PBS-T)
Blocking buffer; PBS containing 0.5% BSA
Dilution buffer: PBS-T containing 0.5% BSA

  As shown in FIG. 2, a sufficient Japanese encephalitis virus antigen-specific IgG antibody titer can be obtained with the microneedle device (MN) alone, and even higher when the adjuvant (lauryl alcohol) patch is used together (MN + LAtape). Encephalitis virus antigen-specific IgG antibody titers can be obtained.

The present invention includes the following.
(1) A plurality of microneedles that are two-dimensionally arranged on a substrate and are made of polylactic acid capable of perforating the skin, and coated with Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). Microneedle device.
(2) The microneedle device according to (1) above, wherein the microneedle is conical or pyramidal.
(3) The microneedle device according to (1) or (2), wherein the coating contains pullulan as a coating carrier.
(4) The microneedle device according to any one of (1) to (3), 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), wherein the coating contains a substance having an adjuvant activity.
(6) The microneedle device according to (5) above, wherein the substance having adjuvant activity is lauryl alcohol.
(7) The substrate of the microneedle device according to any one of (1) to (6), wherein the substrate has a plurality of openings through which a Japanese encephalitis virus antigen solution or a Japanese encephalitis virus antigen solution can be transmitted. Microneedle device.
(8) The microneedle device according to any one of (1) to (7), wherein a height of the microneedle is 200 to 500 μm.
(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) A method for increasing the response of Japanese encephalitis virus antigen by administering a patch containing a substance having an adjuvant activity after administration of Japanese encephalitis virus antigen using a microneedle device. A needle device having a plurality of microneedles made of polylactic acid that is two-dimensionally arranged on a substrate and can perforate the skin, and the microneedles are Japanese encephalitis virus antigens derived from monkey kidney cells (Vero cells) Is coated with a microneedle device.
(11) A plurality of microneedles made of polylactic acid that is two-dimensionally arranged on a substrate and capable of perforating the skin, and the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). A method for increasing the response of Japanese encephalitis virus antigens using the microneedle device.
(12) The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to (11) above, wherein the microneedle is a cone type or a pyramid type.
(13) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to (11) or (12), wherein the coating contains pullulan as a coating carrier.
(14) The method for increasing the response of 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) The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (14), wherein the coating contains a substance having an adjuvant activity.
(16) The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to (15), wherein the substance having an adjuvant activity is lauryl alcohol.
(17) The substrate of the microneedle device according to any one of (11) to (16), wherein the substrate has a plurality of openings capable of transmitting Japanese encephalitis virus antigen solution or Japanese encephalitis virus antigen solution. To increase the response of Japanese encephalitis virus antigens using the microneedle device.
(18) A method for increasing the response of a Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (17), wherein the height of the microneedle is 200 to 500 μm.
(19) The method for increasing the efficacy of Japanese encephalitis virus antigen by the microneedle device according to any one of (11) to (18), wherein the microneedles are arranged at a density of 400 to 1000 / cm ^2. .
(20) A patch having further adjuvant activity after transdermal administration of Japanese encephalitis virus antigen by the method for increasing the efficacy of Japanese encephalitis virus antigen by the microneedle device according to any of (11) to (19) above A method of increasing the response of Japanese encephalitis virus antigens with a further microneedle device.
(21) The method for increasing the efficacy of a Japanese encephalitis virus antigen by the further microneedle device according to (20), wherein the substance having the adjuvant activity of the patch is lauryl alcohol.

  The present invention makes it possible to increase the antigenicity of a Japanese encephalitis virus antigen comprising a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) by an efficient and simple operation by using a microneedle device. And has industrial applicability.

Claims (21)

  A microneedle comprising a plurality of microneedles that are two-dimensionally arranged on a substrate and made of polylactic acid capable of perforating the skin, and which is coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) device.   The microneedle device according to claim 1, wherein the microneedle is conical or pyramidal.   The microneedle device according to claim 1 or 2, wherein the coating comprises pullulan as a coating carrier.   The microneedle device according to any one of claims 1 to 3, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.   The microneedle device according to any one of claims 1 to 4, wherein the coating contains a substance having an adjuvant activity.   The microneedle device according to claim 5, wherein the substance having adjuvant activity is lauryl alcohol.   The microneedle device according to any one of claims 1 to 6, wherein a substrate of the microneedle device has a plurality of openings through which a Japanese encephalitis virus antigen solution or a Japanese encephalitis virus antigen solution can be transmitted.   The microneedle device according to any one of claims 1 to 7, wherein a height of the microneedle is 200 to 500 µm. The microneedles are arranged in a density of 400 to 1000 present / cm ^2, the microneedle device according to any of claims 1 to 8.   After administering Japanese encephalitis virus antigen using a microneedle device, a patch containing a substance having an adjuvant activity is further applied to increase the response of Japanese encephalitis virus antigen. A plurality of microneedles made of polylactic acid that are two-dimensionally arranged on a substrate and can perforate the skin, and the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells). A microneedle device characterized by that.   A microneedle comprising a plurality of microneedles that are two-dimensionally arranged on a substrate and made of polylactic acid capable of perforating the skin, and the microneedles are coated with a Japanese encephalitis virus antigen derived from monkey kidney cells (Vero cells) A method of increasing the response of Japanese encephalitis virus antigens using a device.   The method for increasing the responsiveness of a Japanese encephalitis virus antigen by the microneedle device according to claim 11, wherein the microneedle is conical or pyramidal.   The method according to claim 11 or 12, wherein the coating comprises pullulan as a coating carrier to increase the response of the Japanese encephalitis virus antigen by the microneedle device.   The method for increasing the response of Japanese encephalitis virus antigen by the microneedle device according to claim 11, wherein the coating is performed at a relative humidity of 70.0 to 100% RH at room temperature.   The method according to claim 11, wherein the coating contains a substance having an adjuvant activity, and the response of the Japanese encephalitis virus antigen is increased by the microneedle device according to claim 11.   The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to claim 15, wherein the substance having an adjuvant activity is lauryl alcohol.   The substrate of the microneedle device has a plurality of openings through which the Japanese encephalitis virus antigen solution or the Japanese encephalitis virus antigen solution can be transmitted. A method for increasing the response of encephalitis virus antigens.   The method for increasing the efficacy of a Japanese encephalitis virus antigen by the microneedle device according to claim 11, wherein the height of the microneedle is 200 to 500 μm. The method of raising the response of the Japanese encephalitis virus antigen by the microneedle device in any one of Claim 11 to 18 with which the said microneedle is arrange | positioned with the density of 400-1000 piece / cm < ² >.   After the transdermal administration of the Japanese encephalitis virus antigen by the method for increasing the response of the Japanese encephalitis virus antigen using the microneedle device according to any one of claims 11 to 19, a patch having an adjuvant activity is further applied. A method of increasing the response of Japanese encephalitis virus antigens using a microneedle device.   The method for increasing the efficacy of a Japanese encephalitis virus antigen using a further microneedle device according to claim 20, wherein the substance having the adjuvant activity of the patch is lauryl alcohol.

Images