Microneedle patch incorporated with drug or vaccine and method for producing same

Abstract

[Problem] To provide a new method for producing a microneedle patch incorporated with a drug or a vaccine such as a live virus vaccine. [Solution] A method for producing a microneedle patch, the method comprising: (a) a step for injecting a solution containing a biosoluble material and a drug or vaccine into a female part of a mold; (b) a step for disposing, above the mold, a backing member in which a pillar part is provided on a backing layer; (c) a step for inserting the leading end portion of the pillar part into the solution injected into the mold; and (d) a step for drying the solution and the backing member and then removing the backing member to which a microneedle has adhered from the mold.

Description

Microneedle patch incorporating a drug or vaccine and method for manufacturing the same

[0001] This invention relates to a novel method for manufacturing microneedle patches incorporating a drug or vaccine. More specifically, it relates to a method for manufacturing a microneedle patch containing a drug or vaccine that can achieve a reduction in the amount of drug or vaccine used and an improvement in manufacturing efficiency. The invention also relates to a novel microneedle patch incorporating a drug or vaccine.

[0002] The COVID-19 pandemic highlighted the critical need for rapid and accessible vaccine solutions. COVID-19 vaccines were developed to mitigate the severity and global spread of the pandemic, and currently there are several platforms for COVID-19 vaccines, including mRNA vaccines, adenovirus vector vaccines, and inactivated whole virus preparations.

[0003] While current platforms have significantly reduced COVID-19-related morbidity and mortality, challenges remain. These include a decline in immunity over time, limited cross-protection against new variants, and logistical obstacles in repeated dosing.

[0004] To address this problem and acquire sustained immunity, novel vaccines using recombinant vaccinia virus have been developed (see, for example, Patent Document 1).

[0005] Ohara et al., co-inventors of the present invention, have developed a recombinant vaccinia virus strain (hereinafter also referred to as "r-DIs-S") derived from a highly attenuated Dairen I minute-pock variant (DIs) engineered to express the SARS-CoV-2 spike (S) protein. Attenuated vaccinia virus strains like DIs are compelling vaccine vectors due to their safety, immunogenicity, and previous success in smallpox eradication. The DIs strain is characterized by a genomic deletion of approximately 15.4 kb compared to the Copenhagen strain of the vaccinia virus vaccine, exhibits a limited host range, and lacks the ability to replicate in most mammalian cells. Ohara et al. have shown that r-DIs-S induces potent humoral and cellular immune responses in both mice and macaques, providing broad protection from early SARS-CoV-2 strains to the Omicron BA.1 variant (TY38-873) (Non-Patent Literature 1). Importantly, the immunity was persistent, lasting at least six months after vaccination in mouse models. Furthermore, intradermal (ID) delivery of r-DIs-S has been shown to circumvent pre-existing immunity to vaccinia vectors, potentially limiting the effectiveness of viral vector vaccines. Recombinant vaccinia vaccines can be administered multiple times. Vaccinia virus vaccines have been reported to be most effective when administered by epidermal or intradermal inoculation.

[0006] Thus, recombinant vaccinia vaccines can provide long-term immunity, but until now they have been administered via a highly invasive, painful bifurcated needle.

[0007] Microneedle array patches (hereinafter also abbreviated as "MAP") are considered a promising alternative to address the above problems. Microneedle array patches cause less pain when applied (Non-Patent Documents 2 and 3), have improved thermal stability, allow for dose savings of vaccines, and can be self-administered. Microneedle designs include hollow, coated, porous, hydrogel, and soluble types, but among these, dissolving microneedles (hereinafter also referred to as "dMN") are the most advanced toward clinical adoption due to their safety and efficacy profile (Non-Patent Documents 4 and 5).

[0008] However, incorporating live viral vaccines into soluble microneedles (dMNs) remains technically challenging due to issues such as inaccuracy in dosage, antigen instability, and viral loss during the dMN manufacturing process.

[0009] International Publication No. 2022-025298

[0010] Ishigaki, H., Yasui, F., Nakayama, M., et al. An attenuated vaccinia vaccine encoding the severe acute respiratory syndrome coronavirus-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human angiotensin-converting enzyme 2 transgenic mice from severe acute respiratory syndrome coronavirus-2 and its variants. Frontiers in Microbiology 13 (2022).Hegde, N. R., Kaveri, S. V. & Bayry, J. Recent advances in the administration of vaccines for infectious diseases: microneedles as painless delivery devices for mass vaccination. Drug Discovery Today 16, 1041-1049 (2011).Mistilis, M. J., Joyce, J. C., Esser, E. S., et al. Long-term stability of influenza vaccine in a dissolving microneedle patch. Drug Delivery and Translational Research 7, 195-205 (2017).Larraneta, E., McCrudden, M. T. C., Courtenay, A. J., et al. Microneedles: a new frontier in nanomedicine delivery. Pharmaceutical Research 33, 1055-1073 (2016).Tuan-Mahmood, T.-M., McCrudden, M. T.C., Torrisi, BM, et al. Microneedles for intradermal and transdermal drug delivery. European Journal of Pharmaceutical Sciences 50, 623-637 (2013).

[0011] The present invention aims to provide a novel method for manufacturing microneedle patches incorporating vaccines or drugs, such as live viral vaccines. Furthermore, the present invention also aims to provide novel microneedle patches incorporating vaccines or drugs, such as live viral vaccines.

[0012] Conventional methods for manufacturing soluble microneedles employ a two-step process using separate materials for the microneedle and backing layer (support layer). In this method, a solution containing the vaccine or drug is first poured into a cavity corresponding to the shape of the microneedle mold to form the needle portion. Then, the material for the backing layer is poured in and dried again to obtain a soluble microneedle patch (see Figure 1). However, because the material used for the backing layer is easily soluble in water, there is a problem that the vaccine or drug solution in the needle portion dissolves again during drying, spreading the vaccine or drug to the backing layer. Furthermore, because the needle portion and backing layer are formed in two stages as described above, the drying time is long, and there is also the problem that the activity of the vaccine or drug, especially live vaccines, decreases during drying.

[0013] Based on the problems in conventional methods for manufacturing soluble microneedle patches, the inventors conducted thorough research and found that by designing a manufacturing process in which the backing layer and the needle portion are separated and the vaccine or drug is filled only into the needle portion, a new manufacturing method that can solve the problems of the present invention can be provided, thus completing the present invention.

[0014] In other words, the present invention has the following configuration: [1] A method for manufacturing a microneedle patch, comprising the steps of: (a) injecting a solution containing a biosoluble material and a drug or vaccine into a female mold; (b) arranging a support member having a support column on a support layer above the mold; (c) inserting the tip portion of the support column into the solution injected into the mold; and (d) drying the solution and the support member, and then removing the support member to which the microneedles are attached from the mold. [2] The method according to [1], wherein the support member is manufactured in advance by 3D printing. [3] The method according to [1] or [2], wherein the vaccine is a coronavirus vaccine, a novel coronavirus vaccine, an influenza virus vaccine, a dengue virus vaccine, or a poliovirus vaccine. [4] The method according to any one of [1] to [3], wherein the vaccine is a recombinant vaccinia virus used as a vaccine for SARS-CoV-2. [5] The manufacturing method according to any one of [1] to [4], wherein the biosoluble material is at least one selected from the group consisting of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose, a copolymer of methyl vinyl ether and maleic anhydride, trehalose, sucrose, maltose, and silk fibroin. [6] The manufacturing method according to any one of [1] to [5], further comprising the step of plasma treatment of the mold before injecting the solution into the female mold in step (a). [7] The manufacturing method according to any one of [1] to [6], wherein in step (c), the support portion is inserted into the mold shape such that the ratio of the height of the tip portion of the support portion inserted into the microneedle to the height of the obtained microneedle is 8:1 to 2:1. [8] A microneedle patch obtained by the manufacturing method according to any one of [1] to [7]. [9] A microneedle patch comprising a biosoluble material, a microneedle containing a drug or vaccine, and a support member having a support column on a support layer, wherein the tip portion of the support column is inserted into the microneedle.

[10] The microneedle patch according to [9], wherein the support layer is manufactured by 3D printing.

[11] The microneedle patch according to [9] or

[10] , wherein the vaccine is a coronavirus vaccine, a novel coronavirus vaccine, an influenza virus vaccine, a dengue virus vaccine, or a poliovirus vaccine.

[12] The microneedle patch according to any one of [9] to

[11] , wherein the vaccine is a recombinant vaccinia virus used as a vaccine for SARS-CoV-2.

[13] The microneedle patch according to any one of [9] to

[12] , wherein the biosoluble material is at least one selected from the group consisting of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose, a copolymer of methyl vinyl ether and maleic anhydride, trehalose, sucrose, maltose, and silk fibroin.

[14] The microneedle patch according to any one of [9] to

[13] , wherein the ratio of the height of the tip portion to the height of the microneedle is 8:1 to 2:1.

[0015] In the manufacturing method of the present invention, since the vaccine or drug is used only in the needle portion, the amount of vaccine or drug used can be significantly reduced. Furthermore, in the manufacturing method of the present invention, only the drying time of the solution dedicated to the needle portion is required, so the drying time can be significantly shortened, and the activity of the vaccine or drug can be maintained. Therefore, by using the manufacturing method of the present invention, it is possible to reduce costs by reducing the amount of vaccine or drug used, as well as maintain the activity of the vaccine or drug by shortening the drying time. In addition, in the manufacturing method of the present invention, it is possible to localize the vaccine payload (or drug payload) only in the microneedle, thereby improving both drying efficiency and viral stability (or drug stability).

[0016] In addition, in the microneedle patch incorporating the agent or vaccine according to the present invention, the microneedles can deliver the agent or vaccine to the epidermis. Therefore, it is possible to target the epidermis where a large number of Langerhans cells that induce an immune response are distributed, and it can be expected to prevent a decrease in the effect of immune induction by existing immunity. As a characteristic of vaccinia virus (recombinant vaccine of smallpox vaccine), it has been shown that r-DIs-S can prevent a decrease in the vaccine effect by existing immunity when administered intradermally. Therefore, in the microneedle patch of the present invention, a vaccine using vaccinia virus can be preferably used. In addition, the microneedle patch of the present invention has less invasiveness, does not cause bleeding, and can deliver an agent or vaccine as compared with existing bifurcated needles.

[0017] A schematic diagram comparing the production method of the present invention with the conventional production method is shown. A schematic diagram (side view) of the microneedle patch of the present invention is shown. A schematic diagram of the administration method of the microneedle patch of the present invention is shown. The results of an optimization test of the conditions for vaccine formulation are shown. The microneedle patch of the present invention (pillar guide MAP) produced in Example 1 is shown. A schematic diagram for producing PG-MAP and the results of the characterization of PG-MAP are shown. In vivo delivery and immunogenicity of r-DIs-S-loaded PG-MAP are shown. The protective effect of r-Dis-S-loaded MAP against lethal infection by SARS-CoV-2 B.1.351 is shown. Aspects for Carrying Out the Invention

[0018] I. Micro-needle Patch One embodiment of the present invention is a micro-needle patch comprising a bio-dissolvable material, one or more micro-needles containing a drug or a vaccine, and a support member provided with one or more support columns on a support layer, wherein a tip portion of the support column is inserted into the micro-needle (hereinafter also referred to as "the micro-needle patch of the present invention"). Although details will be described later, the micro-needle patch of the present invention has a structure in which a tip portion of a support column is inserted into a micro-needle. The micro-needle patch of the present invention is also referred to as "the micro-needle patch with a support column of the present invention" or "the micro-needle patch with a pillar of the present invention". Hereinafter, the micro-needle patch of the present invention will be described in detail for each component.

[0019] 1. Micro-needle The micro-needle comprised in the micro-needle patch of the present invention contains a bio-dissolvable material and a drug or a vaccine.

[0020] (1) Bio-dissolvable material In this specification, a bio-dissolvable material means a material that, when in contact with body fluids in the body, dissolves into the body (dissolves) by a chemical reaction (mainly hydrolysis) and is finally completely absorbed by surrounding tissues and the like. The bio-dissolvable material used in the micro-needle of the present invention preferably also has biocompatibility, that is, the property that no side reactions, generation of foreign substances, or reactions occur when the material comes into contact with tissues in the body. Such bio-dissolvable materials include at least one selected from the group consisting of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, a copolymer of methyl vinyl ether and maleic anhydride, trehalose, sucrose, maltose, and silk fibroin.

[0021] The molecular weight of the bio-dissolvable material is preferably 1 kDa to 500 kDa, more preferably 4 to 200 kDa. For example, the molecular weight of hyaluronic acid is preferably 4 to 50 kDa.

[0022] In the microneedles of the microneedle patch of the present invention, the biosoluble material functions as a matrix material on which a drug or virus is dissolved or dispersed, and the microneedles are designed to be inserted into living tissue such as human skin, and all or part of the microneedles dissolve. In one preferred embodiment of the microneedle patch of the present invention, the microneedles are soluble microneedles.

[0023] (2) The vaccines contained in the vaccine microneedles include vaccines for infectious diseases, cancer, neurological disorders, allergies, and therapeutic vaccines for smoking cessation or other addictions. In one preferred embodiment of the microneedle patch of the present invention, the vaccine contained in the microneedles is a vaccine for infectious diseases.

[0024] Examples of vaccines for infectious diseases include, but are not limited to, coronavirus vaccines, COVID-19 vaccines, influenza virus vaccines, dengue virus vaccines, polio virus vaccines, herpes simplex virus vaccines, varicella virus vaccines, Sendai virus vaccines, Sindbis virus vaccines, smallpox virus vaccines, vaccinia virus vaccines, and seasonal influenza virus vaccines.

[0025] In one preferred embodiment of the present invention, the vaccine contained in the microneedle is a coronavirus vaccine, a COVID-19 vaccine, an influenza virus vaccine, a dengue virus vaccine, or a poliovirus vaccine. In another preferred embodiment of the present invention, the vaccine contained in the microneedle is a coronavirus vaccine or a COVID-19 vaccine.

[0026] Furthermore, the types of vaccines contained in the microneedles may include attenuated vaccines, inactivated vaccines, virus-like particles (VLPs), purified subunit antigens, recombinant antigens, synthetic peptides, recombinant vectors, DNA vaccines, nucleic acid vaccines, mucosal immunization vaccines, or combination vaccines.

[0027] If the vaccine contained in the microneedle is a coronavirus vaccine, a COVID-19 vaccine, or a dengue virus vaccine, an inactivated virus vaccine, a weakened virus vaccine (live virus vaccine), a recombinant protein vaccine, a vectorized vaccine, an RNA vaccine, or a DNA vaccine can be used, but it is preferable to use a weakened virus vaccine (live virus vaccine). Also, if the vaccine contained in the microneedle is an influenza virus vaccine or a polio virus vaccine, it is preferable to use a weakened virus vaccine (live virus vaccine).

[0028] In one preferred embodiment of the microneedle patch of the present invention, the vaccine contained in the microneedle is a recombinant vaccinia virus used as a vaccine for SARS-CoV-2. In another preferred embodiment of the microneedle patch of the present invention, the vaccine contained in the microneedle is a recombinant vaccinia virus strain (r-DIs-S) derived from a highly attenuated Dairen I minute-pock variant (DIs) that has been engineered to express the SARS-CoV-2 spike (S) protein.

[0029] In another preferred embodiment of the microneedle patch of the present invention, the vaccine contained in the microneedle is a recombinant vaccinia virus used as a vaccine for dengue virus.

[0030] Details of recombinant vaccinia virus used as a vaccine for SARS-CoV-2 are described in the above-mentioned Patent Document 1 (International Publication 2022-025298), and the entire contents of said document are incorporated herein by reference. The preparation of recombinant vaccinia virus is described in paragraphs

[0021] to

[0033] of Patent Document 1. Therefore, recombinant vaccinia virus used as a vaccine for SARS-CoV-2 can be prepared by referring to these descriptions in Patent Document 1. A part of it is described below.

[0031] Information on all genes encoding SARS-CoV-2 proteins, genes encoding the outer shell protein region, and genes encoding the non-structural protein region involved in replication can be found specifically in the gene sequence information of the SARS-CoV-2 virus strain: hCoV-19 / Japan / AI / I-004 / 2020 (shown as Sequence ID No. 9 in Patent Document 1), which is registered on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) as GenBank accession number: LC521925. Therefore, genes contained in recombinant vaccinia virus that can be used in the microneedle patch of the present invention, i.e., genes containing the non-structural protein region or structural protein region of SARS-CoV-2, can be obtained by conventional genetic engineering methods. For example, nucleic acid synthesis methods using DNA synthesizers, which are commonly used as genetic engineering methods, can be used. Furthermore, after isolating or synthesizing a template gene sequence, specific primers can be designed for each gene, and the gene sequence can be amplified using PCR or a cloning vector. Those skilled in the art can perform the above methods according to "Molecular Cloning 4th Edt. Cold Spring Harbor Laboratory Press (2012)" or similar standards. Known methods can be used to purify the obtained PCR product.

[0032] Gene DNA encoding either a non-structural protein region or a structural protein region of the entire SARS-CoV-2 gene region can be used to produce recombinant vaccinia virus. The non-structural protein region consists of ORF1a, ORF1b, ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 regions, and it is preferable to select and use, for example, the ORF1b region. The structural protein region consists of the spike (S), envelope (E), integral membrane (M), and nucleoprotein (N) regions, and it is preferable to select and use, for example, the spike (S) protein region.

[0033] The DNA sequence encoding the ORF1b region in the non-structural protein region derived from SARS-CoV-2 is shown as Sequence ID No. 1 in Patent Document 1, and the DNA sequence encoding the S protein region in the structural protein region derived from SARS-CoV-2 is shown as Sequence ID No. 5 in Patent Document 1. Furthermore, a variant DNA of the DNA sequence encoding the ORF1b region (in which six bases in the DNA sequence encoding the ORF1b region are substituted with other bases to improve gene expression efficiency within recombinant vaccinia virus) is shown as Sequence ID No. 3 in Patent Document 1, and a variant DNA of the DNA sequence encoding the S protein region (in which eight bases in the DNA sequence encoding the S protein region are substituted with other bases to improve gene expression efficiency within recombinant vaccinia virus) is shown as Sequence ID No. 7 in Patent Document 1. However, in addition to the DNA sequences shown as Sequence IDs No. 1, 3, 5, and 7 in Patent Document 1, the following DNAs can also be used in the present invention. Hereinafter, the nucleotide sequences shown as SEQ ID NOs: 1, 3, 5, and 7 in Patent Document 1 are shown as SEQ ID NOs: 1, 2, 3, and 4 in this application, respectively.

[0034] DNA that has 80% or more identity (homology) to DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 1, and that encodes a non-structural protein derived from SARS-CoV-2 (mutant DNA of the ORF1b region). DNA that has 80% or more identity (homology) to DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 2, and that encodes a non-structural protein derived from SARS-CoV-2 (mutant DNA of the region in which the aforementioned base substitution mutation has been added to the ORF1b region). DNA that has 80% or more identity (homology) to DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 3, and that encodes a structural protein derived from SARS-CoV-2 (mutant DNA of the S protein region). DNA having 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity (homology) with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 4, and encoding a structural protein derived from SARS-CoV-2 (mutant DNA of the region in which the aforementioned base substitution mutation has been added to the S protein region). DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 1, and encoding a non-structural protein derived from SARS-CoV-2 (mutant DNA of the ORF1b region). DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 2, and encoding a non-structural protein derived from SARS-CoV-2 (mutant DNA of the region in which the aforementioned base substitution mutation has been added to the ORF1b region). DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 3, and encodes a structural protein derived from SARS-CoV-2 (mutant DNA of the S protein region). DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 4, and encodes a structural protein derived from SARS-CoV-2 (mutant DNA of the region in which the aforementioned base substitution mutation is added to the S protein region).

[0035] Here, "encoding non-structural proteins derived from SARS-CoV-2" means encoding proteins produced in cells when the virus replicates. Furthermore, the genes encoding these non-structural proteins include not only the full sequence but also partial sequences. Similarly, "encoding structural proteins derived from SARS-CoV-2" means encoding proteins that make up the virus's outer shell. Furthermore, the genes encoding these structural proteins include not only the full sequence but also partial sequences.

[0036] The above-mentioned mutant DNA can be obtained by chemical synthesis, or by using DNA consisting of the base sequence represented by SEQ ID NOs: 1, 2, 3, or 4, or a fragment thereof, as a probe, and obtaining it from cDNA libraries and genome libraries by known hybridization methods such as colony hybridization, plaque hybridization, and Southern blotting. Stringent conditions for the above hybridization include, for example, 0.1×SSC to 10×SSC, 0.1% to 1.0% SDS, and a temperature of 20°C to 80°C. More specifically, conditions include pre-hybridization at 37°C to 56°C for 30 minutes or more, followed by washing in 0.1×SSC and 0.1% SDS at room temperature for 10 to 20 minutes, 1 to 3 times. For detailed procedures regarding hybridization, refer to "Molecular Cloning 4th Edt. Cold Spring Harbor Laboratory Press (2012)," etc.

[0037] In producing recombinant vaccinia virus for use in the microneedle patch of the present invention, there are no particular limitations, and any known method can be employed. For example, first, a nucleotide sequence DNA encoding a non-structural protein region (such as the ORF1b region) or structural protein region (such as the S protein region) of SARS-CoV-2 is inserted into a desired expression vector (plasmid) (e.g., DIs strain homologous recombination vector (plasmid) (Koji Ishii et al. Virology 2006)). Then, by transfecting cells infected with a desired vaccinia virus strain (e.g., attenuated vaccinia virus DIs strain) with this plasmid vector, homologous recombination is induced within the vaccinia virus genome, and recombinant vaccinia virus expressing a desired region of the non-structural or structural protein of SARS-CoV-2 can be produced. The expression vector is not particularly limited, but for example, a pSMART® vector having a DIs strain homologous gene sequence region can be used, and the expression promoter contained in the recombinant vaccinia virus used in the microneedle patch of the present invention can be any expression promoter that has been used for vaccinia virus gene expression in the past (for example, the mH5 promoter).

[0038] The aforementioned attenuated vaccinia virus DIs strain is a highly attenuated host-range gene-deficient strain established from the Dalian strain (DIE), which was used as a smallpox vaccine, using the one-day oocyte passage method. It can only proliferate in Chick Embryo Fibroblast (CEF) cells and was developed by Dr. Isamu Tagaya of the National Institute of Preventive Health (now the National Institute of Infectious Diseases) (Tagaya et al. Nature, 192:381-382, 1961). Due to the large-scale gene deletion, it cannot proliferate in most mammalian cells, including those of mice, guinea pigs, rabbits, and humans. For this reason, its safety is guaranteed even if administered to immunocompromised or immunosuppressed patients.

[0039] The recombinant vaccinia virus produced can be used as a template for PCR with primers specific to the non-structural or structural protein region genes of SARS-CoV-2, allowing for confirmation of gene transfer to the desired non-structural or structural protein region.

[0040] Therefore, in another preferred embodiment of the microneedle patch of the present invention, the vaccine contained in the microneedle is a recombinant vaccinia virus (hereinafter also referred to as "recombinant vaccinia virus 1") comprising all or part of a cDNA encoding a non-structural protein or structural protein derived from SARS-CoV-2 and an expression promoter. In recombinant vaccinia virus 1, the vaccinia virus is preferably a DIs strain. In recombinant vaccinia virus 1, the cDNA encoding the non-structural protein preferably includes a cDNA encoding the ORF1b region in a non-structural protein derived from SARS-CoV-2.

[0041] Furthermore, in recombinant vaccinia virus 1, preferably, the cDNA encoding the non-structural protein is any of the following DNAs (a) to (d): (a) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1; (b) DNA having 80% or more identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a non-structural protein derived from SARS-CoV-2; (c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2; (d) DNA having 80% or more identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2, and encoding a non-structural protein derived from SARS-CoV-2.

[0042] Furthermore, in recombinant vaccinia virus 1, preferably, the cDNA encoding the structural protein includes the cDNA encoding the spike protein in the structural protein derived from SARS-CoV-2.

[0043] Furthermore, in recombinant vaccinia virus 1, preferably, the cDNA encoding the structural protein is one of the following DNAs (a) to (d): (a) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3; (b) DNA having 80% or more identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3, and encoding a structural protein derived from SARS-CoV-2; (c) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4; (d) DNA having 80% or more identity with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 4, and encoding a structural protein derived from SARS-CoV-2.

[0044] (3) The drugs contained in the drug microneedles include therapeutic agents other than the vaccines described above. The therapeutic agents may be selected from small molecules and larger biotechnologies that produce or purify molecules (e.g., peptides, proteins, antibodies, fusion proteins, DNA, RNA, aptamers).

[0045] The types of drugs are not particularly limited, but may include, for example, protein-based drugs (insulin, immunoglobulins (e.g., IgG, IgM, IgA, IgE), TNF-α, antiviral drugs, etc.), polynucleotide drugs (plasmids, siRNA, RNAi, nucleoside anticancer drugs, etc.), and small molecule drugs (alkaloids, glycosides, phenols, etc.). These drugs may also include anti-infective drugs, hormones, inotropic or blood flow modulating drugs, and pain management drugs. Other drugs that can be included in microneedles include those useful for the prevention, diagnosis, relief, treatment, or cure of diseases. Non-limiting examples of the aforementioned drugs include anti-angiogenic drugs, antidepressants, antidiabetic drugs, antihistamines, anti-inflammatory drugs, butorphanol, calcitonin and its analogues, COX-II inhibitors, dermatological drugs, dopamine agonists and antagonists, enkephalins and other opioid peptides, epidermal growth factor, erythropoetin and its analogues, follicle-stimulating hormone, glucagon, growth hormone and its analogues (such as growth hormone-releasing hormone), growth hormone antagonists, heparin, hirudin and hirudin analogues (such as Hirulog), IgE inhibitors and other protein inhibitors, immunosuppressants, insulin, insulinotropin and its analogues, and interferon. This includes interleukins, luteinizing hormone, luteinizing hormone-releasing hormone and its analogues, monoclonal or polyclonal antibodies, motion sickness medications, muscle relaxants, narcotic analgesics, nicotine, nonsteroidal anti-inflammatory drugs, oligosaccharides, parathyroid hormone and its analogues, parathyroid hormone antagonists, prostaglandin antagonists, prostaglandins, scopolamine, sedatives, serotonin agonists and antagonists, sexual dysfunction agents, tissue plasminogen activator, tranquilizers, carrier / adjuvant-containing or non-carrier vaccines, vasodilators, and major diagnostic agents (such as tuberculin and other hypersensitivity agents as described in U.S. Patent No. 6,569,143, titled "Method of Intradermally Injecting Substances" (as incorporated herein by reference)).

[0046] (4) Other components: Microneedles may contain carbohydrates selected from the group consisting of trehalose, glucose, galactose, sucrose, maltose, lactose, dextrin, maltodextrin, β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, glucan, amylose, amylopectin, chitin, carboxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, gelatin, chitosan, and any combination thereof, as components other than biosoluble materials and vaccines.

[0047] (5) Composition of the microneedle The content of biosoluble material in the microneedle is 25 to 75%, more preferably 25 to 50%. The content of other components in the microneedle is, for example, trehalose, preferably at a concentration of 5 to 20% (w / v), more preferably 5 to 10% (w / v). If the microneedle contains a vaccine, the vaccine content can be appropriately determined depending on the type of vaccine, but preferably 1 × 10 ４ ~1 x 10 １０ It is PFU, more preferably 1 × 10 ８ ~1 x 10 １０ This is a PFU. If the microneedle contains drugs other than vaccines, the drug content can be appropriately determined depending on the type of drug, but is preferably 5-20% (w / v), more preferably 10-15% (w / v).

[0048] (6) Shape of the microneedle The microneedle can have a shape such as a roughly conical or roughly pyramidal shape, but a polygonal shape (for example, a roughly pyramidal shape) is preferable because it is easier to penetrate the skin than a roughly conical shape.

[0049] The diameter of the tip of the microneedle is typically 10 μm to 60 μm. The diameter or maximum dimension of the base is, for example, about 50 μm to 800 μm. The height of the microneedle determines the depth of penetration into the skin. In the microneedle of the present invention, considering that it reaches the dermis and does not stimulate pain receptors, it is preferable that the height be between 300 μm and 1500 μm.

[0050] When multiple microneedles are provided, a smaller spacing is preferable for uniform administration of the vaccine into the body, but a spacing of 500 to 5000 μm is preferable.

[0051] While a larger tip angle of a microneedle increases its mechanical strength, a larger tip angle also increases the force required for penetration. A tip angle of 15 to 30° is preferable, as it reduces the force required for microneedle penetration to less than 0.2 N.

[0052] In the microneedle patch of the present invention, the tip portion of each support column provided on the support layer described later may be directly inserted into each individual microneedle. Furthermore, in the microneedle patch of the present invention, the microneedles can also be used as a microneedle array in which multiple microneedles are erected on a microneedle substrate. In this case, the tip portion of each support column provided on the support layer described later is inserted into each individual microneedle via the microneedle substrate. It is preferable that the microneedle substrate of the microneedle array is composed of the same material components as the microneedles described above.

[0053] 2. Support Member (1) Shape of the Support Member The support member in the microneedle patch of the present invention comprises a support layer and one or more support columns, the one or more support columns being erected on the support layer. In this specification, the terms "support layer," "base material," or "backing" can be used interchangeably. In this specification, the terms "support column" or "pillar" can be used interchangeably.

[0054] In the microneedle patch of the present invention, the tip portion of a support column provided on the support layer is inserted in the height direction of the microneedle, and the microneedle partially adheres to the support column, thereby integrating the microneedle and the support member (see Figures 1 and 2). Here, the form in which one support column is inserted into one microneedle (one support column is partially attached) and the form in which two or more support columns are inserted (two or more support columns are partially attached) are also included in the scope of the microneedle patch of the present invention. However, the form in which one support column is inserted into one microneedle (one support column is partially attached to one microneedle) is preferred in terms of overall shape stability when applying the microneedle patch to the skin, etc., and efficiency when manufacturing the microneedle patch. More specifically, during puncture, if multiple support structures are inserted into a single microneedle, or vice versa, or if multiple microneedles are connected to support structures, it is conceivable that the microneedle portion cannot be pressed perpendicularly against the skin due to interference between adjacent structures caused by the multiple support structures. Furthermore, the microneedle portion is expected to be tilted due to the multiple support structures, resulting in a significant decrease in puncture efficiency. In addition, if one support structure is inserted into a single microneedle, the dose of drug or vaccine per microneedle can be estimated. Moreover, when manufacturing microneedle patches, if the ratio is not one-to-one, only one or part of the microneedle portion or support structure may be inserted at an angle or partially, weakening the adhesion between the two. Ultimately, it is expected that the needle portion and support structure may separate during demolding, resulting in decreased manufacturing efficiency.

[0055] In the present invention, the "tip portion" of the support column refers to the part of the support column that is inserted into the microneedle and partially attached to the microneedle. Furthermore, if the microneedle is a microneedle array in which multiple microneedles are erected on a microneedle substrate, it refers to the part that is inserted into the microneedle array (i.e., the microneedle and the microneedle substrate) and partially attached to the microneedle array.

[0056] The support column of the support member can have a shape such as a roughly cylindrical, roughly conical, or roughly pyramidal shape, depending on the shape of the microneedle. The support column consists of a tip portion and other parts (also called the "body portion"), and the tip portion and the body portion may have the same shape (in this case, the entire support column is a single shape), or the tip portion and the body portion may have different shapes (see Figure 2). For example, the body portion may be roughly cylindrical and the tip portion may be roughly conical or roughly frustoconical (roughly truncated cone), or the body portion may be roughly prism-shaped (square prism, etc.) and the tip portion may be roughly pyramidal or roughly frustoconical (roughly truncated pyramidal). Furthermore, the tip portion of the support column will be joined to the microneedle through the drying process. Therefore, in order to increase the adhesive strength between the tip portion and the microneedle, it is preferable to increase the contact area between the tip portion and the microneedle, which improves the efficiency of final demolding when manufacturing microneedle patches and allows for more accurate force transmission during puncture. To increase the contact area at the tip of the support column, one could increase the surface roughness of the tip or create a rocking structure at the tip by forming a fine pattern. Furthermore, the body portion may be composed of two or more shapes.

[0057] One example of a non-limiting shape for the tip is a trapezoidal tip, as shown in the embodiment (designed with an upper base of 100 μm, a lower base of 400 μm, and a height of 400 μm, as a non-limiting example).

[0058] The size of the support column is preferably such that the overall height of the support column is 1000 to 2000 μm, more preferably 1500 to 2000 μm. Having the overall height of the support column within this range allows for a balance between moldability and mechanical strength.

[0059] When a microneedle patch is provided with multiple microneedles, the number of support columns in the support member is determined according to the number of microneedles. In the case where one support column is inserted into one microneedle (one support column is partially attached to one microneedle), it is preferable that the number of support columns is the same as the number of microneedles. In the case where two or more support columns are inserted into one microneedle, the total number of support columns in the support member can be appropriately determined according to the number of support columns inserted into one microneedle.

[0060] The spacing between the support columns in the support member is determined appropriately according to the spacing between multiple microneedles, but a smaller spacing is preferable for uniform administration of the vaccine into the body, with a spacing of 500 to 5000 μm being preferred.

[0061] (2) Method for Manufacturing the Support Member In the microneedle patch of the present invention, it is preferable that the support member is manufactured by 3D printing. By manufacturing the support member using a 3D printer, it is possible to achieve precise dose control and poke-and-release delivery of the vaccine. Furthermore, in the microneedle patch of the present invention, by pre-fabricating the support member, the drying time when manufacturing the microneedle patch of the present invention can be shortened and the drug can be prevented from rising to the support member. In prior art studies (for example, European Journal of Pharmaceutical Sciences, vol. 66, January 23, 2015, p. 148-156), a liquid polymer is dried as a method for manufacturing the support member, but there is a problem that the drug diffuses into the support member. However, by pre-fabricating the support member, it is possible to prevent the drug from diffusing into the support member.

[0062] More preferably, the support member is manufactured using a stereolithography (SLA) 3D printer or a fused deposition modeling (FDM) 3D printer, using the designed drawings or data designed using 3D CAD software. Here, considering the accuracy, reproducibility, manufacturing efficiency, surface irregularities, and cost of the tip portion of the support column, which has micro-dimensions and is inserted into the cavity of the female mold, it is even more preferable to use SLA. As a SLA 3D printer, stereolithography (SLA), projector type (DLP), mask type (MSLA), etc., can be used, but stereolithography is preferred because it is easier to form smooth curved surfaces for the shape of the support column, especially the tip portion. Furthermore, after manufacturing with a SLA 3D printer, it is preferable to wash off any residue from the surface and perform UV irradiation as a post-treatment to remove any residue due to incomplete UV curing. This makes it possible to prevent chemical reactions with vaccines or biosoluble materials when the tip portion is immersed in a solution or dispersion containing the vaccine.

[0063] The material used to manufacture the plug of the present invention can be a resin that can be used with a 3D printer, such as an epoxy resin that can be cured at a wavelength of 405 nm, or an acrylate resin or a mixture of both.

[0064] 3. Microneedle Patch In the microneedle patch of the present invention, the tip portion of the support column provided on the support layer is inserted in the height direction of the microneedle, and the microneedle partially adheres to the support column, thereby integrating the microneedle and the support member. Here, the size of the tip portion of the support column (i.e., the portion of the support column that is inserted into the microneedle and partially adheres to it) can be appropriately determined according to the size of the microneedle. However, if the ratio of the height of the tip portion to the height of the microneedle is too large, proper drying during microneedle formation may be hindered, or the formation of the tip portion of the microneedle may be incomplete. Therefore, the ratio of the height of the tip portion of the support column to the height of the microneedle is preferably 8:1 to 2:1, and more preferably 4:1 to 2:1. Furthermore, if the height of the tip portion relative to the overall height of the support column is too large, the distance between the support layer and the mold becomes small during the manufacturing process of the microneedle patch. Due to surface tension, the solution that forms the microneedles rises onto the support layer, and when peeling the support layer from the mold, there is a risk of damaging both the support layer and the mold. Therefore, the ratio of the height of the tip portion to the overall height of the support column is preferably 15:6 to 15:1, and more preferably 15:4 to 15:1.

[0065] II. Method for Manufacturing Microneedle Patches Another embodiment of the present invention is a method for manufacturing microneedle patches (hereinafter also referred to as "the manufacturing method of the present invention"), which includes the steps of: (a) injecting a solution containing a biosoluble material and a drug or vaccine into a female mold; (b) arranging a support member having a support column on a support layer above the mold; (c) inserting the tip portion of the support column into the solution injected into the mold; and (d) drying the solution and the support member, and then removing the support member to which the microneedles are attached from the mold.

[0066] Figure 1 shows a schematic diagram comparing the manufacturing method of the present invention with a conventional manufacturing method. The upper right panel of Figure 1 shows the conventional manufacturing method (two-stage molding method), and the lower panel shows the method of the present invention. Conventional methods for manufacturing soluble microneedles use a two-stage process in which the microneedles and the backing layer (support layer) are made using separate materials. In this method, first, a solution containing vaccine or drug is poured into a cavity corresponding to the mold shape of the microneedle to form the needle portion, and then the material for the backing layer is poured in and dried again to obtain a soluble microneedle array patch (upper right panel of Figure 1). However, because the material used as the backing layer is easily soluble in water, there is a problem that the vaccine solution (or drug solution) in the needle portion dissolves again during drying, and the vaccine or drug spreads to the backing layer. In addition, as described above, since the needle portion and the backing layer are molded in two stages, the drying time is long, and there is also the problem that the activity of the vaccine or drug decreases during drying. In contrast, the manufacturing method of the present invention employs a manufacturing process in which the microneedle portion and the backing layer (support member) are manufactured separately, and the vaccine or drug is filled only into the microneedle portion (lower right diagram in Figure 1). As a result, since the vaccine or drug is used only in the needle portion, it is possible to significantly reduce the amount of vaccine or drug used. In addition, since only the drying time of the solution in the microneedle portion is required, the drying time can be significantly shortened, and the activity of the vaccine or drug can be maintained.

[0067] The biosoluble material used in step (a) is at least one selected from the group consisting of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose, copolymer of methyl vinyl ether and maleic anhydride, trehalose, sucrose, maltose, and silk fibroin. Preferably, it has a molecular weight of 1 kDa to 500 kDa, more preferably 4 to 200 kDa. For example, the molecular weight of hyaluronic acid is preferably 4 to 50 kDa.

[0068] The solution used in step (a) refers to a solution in which the biosoluble material is dissolved in water, an organic solvent, or a mixed solvent of water and an organic solvent. This solution can be obtained by dissolving the biosoluble material in water, an organic solvent, or a mixed solvent of water and an organic solvent. The organic solvent is not particularly limited as long as it is a solvent that dissolves the biosoluble material, but examples include ethanol, dichloromethane, acetone, dimethyl sulfoxide (DMSO), isopropanol, etc.

[0069] The concentration of the biosoluble material in the solution used in step (a) is preferably 5 to 20% (w / v), more preferably 10 to 15% (w / v). When the concentration of the biosoluble material is within this range, the moldability and mechanical strength of the needle can be ensured.

[0070] Details of the vaccine and drug used in the manufacturing method of the present invention are the same as those described in detail for the micropatch of the present invention.

[0071] In one preferred embodiment of the manufacturing method of the present invention, the vaccine used in step (a) is a coronavirus vaccine, a novel coronavirus vaccine, an influenza virus vaccine, a dengue virus vaccine, or a poliovirus vaccine. In another preferred embodiment of the manufacturing method of the present invention, the vaccine used in step (a) is a coronavirus vaccine or a novel coronavirus vaccine.

[0072] If the vaccine used in step (a) is a coronavirus vaccine or a novel coronavirus vaccine, an inactivated virus vaccine, a weakened vaccine (live virus vaccine), a recombinant protein vaccine, a vectorized vaccine, an RNA vaccine, or a DNA vaccine can be used, but it is preferable to use a weakened vaccine (live virus vaccine). Also, if the vaccine used in step (a) is an influenza virus vaccine or a polio virus vaccine, it is preferable to use a weakened vaccine (live virus vaccine).

[0073] In one preferred embodiment of the manufacturing method of the present invention, the vaccine used in step (a) is a recombinant vaccinia virus used as a SARS-CoV-2 vaccine. In another preferred embodiment of the manufacturing method of the present invention, the vaccine used in step (a) is a recombinant vaccinia virus strain (r-DIs-S) derived from a highly attenuated Dairen I minute-pock variant (DIs) that has been engineered to express the SARS-CoV-2 spike (S) protein.

[0074] The viral titer of the vaccine in the solution used in step (a) is preferably 1 × 10⁻⁶. ４ ~1 x 10 １０ PFU, more preferably 1 × 10 ８ ~1 x 10 １０ This is PFU (Proprioceptive Fluid). When the vaccine concentration is within the above range, the necessary viral titer for confirming the immune effect can be ensured.

[0075] The solution used in step (a) may contain carbohydrates selected from the group consisting of trehalose, glucose, galactose, sucrose, maltose, lactose, dextrin, maltodextrin, β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, glucan, amylose, amylopectin, chitin, carboxymethylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, gelatin, chitosan, and any combination thereof, as components other than the biosoluble material and vaccine.

[0076] The solution used in step (a) can be obtained by mixing a biosoluble material, vaccine or drug, other components and a solvent such as water, and stirring at room temperature at a concentration of 500 to 1500 ppm using a magnetic stirrer or the like.

[0077] In step (a), a solution containing a biosoluble material and a vaccine is injected into a female mold. The mold used here is a female micromold prepared from a metal master mold consisting of numerous microneedles, and the material used is preferably polydimethylsiloxane (PDMS), SUS, etc. The shape and size of the microneedles of the metal master mold can be appropriately determined according to the shape and size of the desired microneedles.

[0078] The mold may only have the template shape of the microneedle to be prepared. The female micromold may have a desired number of cavities corresponding to the template shape of the microneedle. Furthermore, cavities may be appropriately provided in the female micromold, for example, vertically and horizontally. The spacing between cavities is usually 500 to 5000 μm, preferably 1000 to 3000 μm.

[0079] Furthermore, the cavity can have a shape in which a microneedle substrate and multiple microneedles are joined together. In the manufacturing method of the present invention, since a microneedle patch in which the microneedle and the support member are integrated is manufactured, it is preferable to provide a cavity (empty space) that includes a space for placing the support member on the mold shape of the microneedle. In this case, when a solution containing a biosoluble material and a drug or vaccine is injected in a volume exceeding that of the mold shape of each microneedle, a microneedle array can be obtained in which multiple microneedles are joined to the microneedle substrate and erected upright. The micromold itself can have any number of cavities as desired. Furthermore, the cavities of the micromold itself can be appropriately provided vertically and horizontally. The spacing between the cavities is preferably 500 to 5000 μm.

[0080] Step (a) may include a step of plasma-treating the mold before injecting the biosoluble material and the drug or vaccine solution into the female mold. The surface of PDMS, a suitable material for the female mold, is inherently hydrophobic and therefore has low affinity for water, which is a non-solvent. Plasma treatment improves the hydrophilicity of the PDMS surface, allowing for smoother formation of microneedles.

[0081] That is, in one preferred embodiment of the manufacturing method of the present invention, the female mold used in step (a) is plasma treated. In another preferred embodiment of the manufacturing method of the present invention, step (a) includes a step of plasma treating the female mold. The plasma treatment conditions are 47W and 30s to 120s.

[0082] In step (b), a support member having a support column on a support layer is placed above the female mold (i.e., on the upper surface side of the female mold). Details of the support member used in step (b) are the same as those described in detail for the microneedle patch of the present invention.

[0083] In one preferred embodiment of the manufacturing method of the present invention, the support member is pre-fabricated by 3D printing. Details of the 3D printing are the same as those described for the microneedle patch of the present invention.

[0084] In step (b), when positioning support members, which have support columns provided on a support layer, above the female mold, the support members are aligned and positioned so that the tip portions of the support columns to be inserted are properly inserted into each cavity corresponding to the mold shape of the microneedles.

[0085] In step (c), the tip of the support column is inserted into the solution injected into the female mold, and the tip is immersed in the solution. In the manufacturing method of the present invention, by inserting the pre-manufactured support member into the solution as a separate component from the microneedle, the drying time can be significantly reduced, and the activity of vaccines and drugs can be maintained.

[0086] Here, the size of the tip portion of the support column (i.e., the portion of the support column that partially adheres to the microneedle obtained by insertion into the solution or suspension) can be appropriately determined according to the size of the microneedle. However, if the ratio of the height of the tip portion to the height of the microneedle to be obtained (or the depth of the cavity) is too large, proper drying during microneedle formation may be hindered, or the formation of the microneedle tip portion may be incomplete. Therefore, the tip portion of the support column is inserted into the female mold such that the ratio of the height of the tip portion of the support column to the height of the microneedle is preferably 8:1 to 2:1, more preferably 4:1 to 2:1. In addition, to avoid the above problems, a height adjustment portion can be provided at the corner of the support member.

[0087] Furthermore, if the height of the tip portion relative to the entire support portion is too large, the distance between the support layer and the mold will decrease during the manufacturing process of the microneedle patch. Due to surface tension, the solution that forms the microneedles will rise to the support layer, and when the support layer is peeled from the mold, there is a risk of damaging both the support layer and the mold. Therefore, the ratio of the height of the tip portion to the entire support portion is preferably 15:6 to 15:1, more preferably 15:4 to 15:1, when the tip portion of the support portion is inserted into the female mold.

[0088] In step (d), the solution injected into the female mold and the support member, whose tip is inserted into and immersed in the solution, are dried. The drying temperature is preferably 0 to 25°C, more preferably 0 to 4°C. Within this temperature range, if a live virus vaccine is used as the vaccine, it is possible to maintain a high survival rate of the virus after the production of the microneedle patch. Drying is preferably carried out by natural drying, and is usually carried out for 8 to 12 hours.

[0089] In step (d), after the solution injected into the female mold and the support member are dried, a support member with microneedles attached can be obtained, which is then removed from the mold. This allows for the creation of a microneedle patch in which the support member and microneedles are integrated. Furthermore, if a microneedle array is used as the microneedle, a microneedle array patch in which the support member and microneedle array are integrated can be obtained.

[0090] III. Use of the Microneedle Patch of the Present Invention Another aspect of the present invention is a method of administering the microneedle patch of the present invention to the skin surface (hereinafter also referred to as "the administration method of the present invention"). The microneedle patch of the present invention can be administered to the skin surface, and the microneedles can be applied to the epidermis or intradermally to administer a vaccine to a tissue site of a human or other mammal. A schematic diagram of the administration method of the present invention is shown in Figure 3.

[0091] Microneedle patches can be self-administered or administered by another person (e.g., a parent, guardian, or healthcare professional).

[0092] The administration method of the present invention further includes a simple and effective method for administering a vaccine to a patient using a microneedle patch. The administration method of the present invention may include identifying the application site and, preferably, disinfecting the area (e.g., using an alcohol wipe) before applying the microneedle patch. The microneedle patch is then applied to the patient's skin / tissue and pressed into the patient's skin / tissue manually (e.g., using a thumb or finger) or using a device to facilitate the application of the patch so that the microneedles penetrate the tissue surface.

[0093] The microneedle patch of the present invention preferably has soluble microneedles, and after administration, the support member (and the remaining microneedle patch structure) can be removed from the patient's skin / tissue.

[0094] The present invention will be described below using examples, but the scope of the present invention is not limited to these examples.

[0095] 1. Materials and Methods (1) Preparation of Vaccine Solution A recombinant vaccinia virus r-Dis-S having the SARS-CoV-2 spike gene was prepared at the Tokyo Metropolitan Institute of Medical Science (Tokyo, Japan). Specifically, a synthetic gene of the gene (SEQ ID NO: 4) represented by the spike protein gene region mnCoV-Japan-S (3926 bp) was inserted into a recombinant vector for producing a Dis recombinant vaccine: pSMART-Dis-L3 (SEQ ID NO: 5), and a recombinant vector (pSMART-Dis-L3-mnCoV-Japan-S-GPTF) (SEQ ID NO: 6) was prepared. Using these recombinant vectors, the establishment of recombinant vaccinia virus (rDis-S) was advanced. "rDis-S" is a recombinant vaccinia virus obtained using the vector (pSMART-Dis-L3-mnCoV-Japan-S-GPTF) into which the gene shown in Sequence List 1 was inserted, and is sometimes referred to as "rDis-mnCoV-S". For the evaluation of the SARS-CoV-2 virus structural protein gene recombinant vaccinia vaccine, after confirming the gene sequence inserted into the recombinant Dis (rDis-S), it was confirmed by the Real-Time Detection polymerase chain reaction (RTD-PCR) method that mRNA was expressed, and it was confirmed by the Western blot method that the spike protein was expressed.

[0096] To prepare the vaccine solution, 30 wt% hyaluronic acid with a molecular weight of 14 kDa (Contipro a.s., Dolni Dobrouc, CZECH REPUBLIC) and 10% trehalose (T9531-10G, Sigma-Aldrich, St. Louis, USA) were added to a suspension of r-Dis-S virus (1.3 × 10 ９ PFU / mL) in DMEM (10569-010, Thermo Fisher (Waltham, USA)), and the mixture was gently dissolved by pipetting at 4°C.

[0097] To optimize the hyaluronic acid (HA) concentration, four formulations containing 5%, 10%, 15%, and 20% HA (w / v) were prepared. Trehalose was maintained at a constant concentration of 5% (w / v) in all formulations. The HA powder was dissolved in Dulbecco's modified Eagle medium (DMEM) at 25°C for 6 hours, and the virus solution was prepared at 1 × 10⁻⁶. ７ The solution was added until the final titer of PFU / mL was reached. Each 500 μL sample was transferred to a sterile 1.5 mL microcentrifuge tube and stored at 4°C for 6 hours. Viral titer was measured using a plaque assay.

[0098] To evaluate the effect of temperature on viral stability during microneedle array patch (MAP) fabrication, r-Dis-S (initial titer: 1.0 × 10⁻¹⁰) was used. ５ A 15% HA solution containing PFU / mL was prepared and stored at 4°C or 25°C for 6 hours to simulate the dispensing and drying conditions during MAP preparation. After this incubation period, the samples were subjected to plaque assays.

[0099] (2) Fabrication of Pillar-Adhered Microneedle Array Patches (Pilar-Guided-MAP (hereinafter also referred to as "PG-MAP")) A 14 x 14 microneedle master mold was fabricated from tungsten carbide using electrical discharge machining (EDM). The mold shape was confirmed by optical microscopy (VH-5500; Keyence (Osaka, Japan)) and image analysis (Zeiss ZEN, Carl Zeiss AG, Oberkochen, Germany). A polydimethylsiloxane (PDMS) negative mold was cast from the master mold. The support members for the MAP were designed using 3D CAD software (Fusion 360, Autodesk, Inc., San Francisco, USA) and fabricated using a stereolithography (SLA) 3D printer (Saturn 2, Elegoo, Shenzhen, China) with a water-washable resin (8K, Elegoo, Shenzhen, China). The components of the above resin are as follows: 1,2-Ethanediol bisacrylate: 56% 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid: 21% Propyldimethylol ethoxylated, esters with acrylic acid: 15% Ethylphenyl (2,4,6-trimethylbenzoyl) phosphinate: 6% Titanium dioxide: 1.3% Carbon black: 0.7%

[0100] After printing, the backing was rinsed with deionized water and ethanol, UV cured (Mercury Plus V2.0, Elegoo, Shenzhen, China), and oven-dried at 40°C for 1 hour to remove residual photoinitiators. The dimensions were determined so that the MAP backing layer thickness ranged from 500 to 550 μm and the top cover fit inside the bottom mold. First, the PDMS negative mold prepared from the master mold was plasma-treated to enhance adhesion between the viscous hyaluronic acid (molecular weight 14 kDa) r-Dis-S solution and the PDMS mold surface. This plasma treatment was used to improve the wettability between the substrate and the vaccine solution. Next, 15 wt% HA and vaccinia virus (1.0 × 10⁻¹⁶) were added. ８ PFUmL －１ 50 μl of a mixed formulation containing the r-Dis-S solution was dispensed into a plasma-treated PDMS mold. After aligning the pre-fabricated backing layer with the PDMS mold filled with r-Dis-S solution, the assembled patch was placed in a sealed container with a portable dehumidifier and dried in a refrigerator at 4°C and 10% relative RH for 8 hours to ensure complete microneedle drying. After complete drying, the backing layer was carefully removed from the PDMS mold. All fabrication was performed in a biosafety cabinet (MHE-131AJ, Sanyo (Osaka, Japan)) at 25°C. Before casting, the PDMS mold was plasma-treated (YHS-R, Sakigake (Tokyo, Japan)) to improve HA adhesion. The MAP was dried in a sealed container with a portable dehumidifier (MD-3, Toyo Living (Tokyo, Japan)) at 4°C for 8 hours to ensure complete microneedle drying.

[0101] Drying speed: To evaluate the drying speed of the one-step molding method and the pillar guide method, MAPs were prepared using both the conventional one-step molding method and the pillar guide method, which is the manufacturing method of the present invention. A 15% HA solution was used. In the one-step molding method, 1.0 mL of the solution was added to each PDMS mold, but in the pillar guide method, only 50 μL was used. Initial mass (m ０To obtain the following, the support members with microneedles still inside the PDMS mold were weighed at one-hour intervals using an analytical balance (SECURA125-1SJP, Sartorius AG, Göttingen, Germany). The drying time was defined as the time required to obtain three consecutive weights with a weight change of less than 1 mg. Time to 90% mass loss (t ９０ The values ​​were estimated using linear interpolation. The samples (n=2) were dried in a refrigerator at 4°C (10% RH) or at room temperature (25°C, 10% RH).

[0102] Theoretical estimation of tip-loading efficiency (TLE) Tip-loading efficiency (TLE) is determined by the geometric parameters of the microneedle array, i.e., the microneedle volume (V ｎｅｅｄｌｅ ) and total distribution volume (V ｔｏｔａｌ ) was calculated from. Single microneedle (V ｎｅｅｄｌｅ The internal volume of ) was obtained from the master mold parameters. V ｔｉｐｓ This is the total volume of 14 × 14 microneedle cavities, and is therefore expressed by the following formula:

[0103]

[0104] TLE is expressed by the following formula.

[0105]

[0106] Here, V ｔｏｔａｌ This is the volume of solution dispensed into the mold (1 mL for the one-step method, 50 μL for the pillar-guided method). This estimate assumes complete cavity filling and no bridging. Deviations are discussed qualitatively in the discussion section.

[0107] To evaluate viral titer and virus retention after MAP preparation, microneedles were completely dissolved in 1 mL of DMEM and incubated at room temperature for 5 minutes. The resulting solution was immediately used in a plaque assay.

[0108] The mechanical strength of the microneedles was measured using a force gauge (MX2-500N, IMADA (Aichi Prefecture, Japan)). Compressive force was applied until breakage of each individual needle was detected, ensuring penetration into the skin layer without breakage.

[0109] (3) Plaque assay A plaque assay for vaccine stability testing was performed as follows: Chicken embryo cells (CRL-12203 UMNSAH / DF-1 cells, ATCC, Manassas, USA) were 8 × 10⁶ ５ Seeds were seeded in a 6-well plate at a cell / well density and stored in a humid atmosphere at 37°C and 5% CO2. ２ The samples were incubated for 24 hours. For each sample, the virus solution was serially diluted in maintenance medium containing 2% fetal bovine serum (FBS) (A525671, Thermo Fisher Scientific, Waltham, USA), and 100 μL of each virus solution was dispensed into the designated wells and incubated at 37°C in a humidified atmosphere at 5% CO2. ２ The samples were incubated for 1 hour. The overlay medium (0.5% / MC / DMEM + GlutaMAX / 5% FBS) was prepared from DMEM (10569-010, Thermo Fisher Scientific) supplemented with methylcellulose (22224-55, Nacalai Tesque (Kyoto, Japan)), 5% FBS (A525671, Thermo Fisher Scientific (Waltham, USA)), penicillin-streptomycin (PS) (15140-122, Thermo Fisher Scientific), and HEPES (1M) (15630-080, Thermo Fisher Scientific). Add overlay medium (2 mL / well) to each well of the titration plate, then add 5% CO2. ２ The samples were incubated at 37°C for 3 days in an incubator.

[0110] Next, the overlay medium was carefully aspirated, and the cells in each well were gently washed with PBS. Cells were fixed by adding 2 mL of 10% neutral buffered formalin to each well and incubated in a biosafety cabinet for at least 1 hour. After fixation, the formalin was carefully removed, and each well was washed twice with 1 mL of tap water. Then, 2 mL of tap water was added to each well, the plate lid was opened, and the virus was inactivated by exposure to ultraviolet light for 1 hour in a biosafety cabinet. After UV treatment, crystal violet solution (2 mL) was added to each well and incubated at room temperature for 15 minutes to identify the viral dilution factor corresponding to 5 to 100 plaques per well, and the viral titer was calculated by counting the plaques.

[0111] Ethical Statement: All animal experiments complied with the guidelines for the proper conduct of animal research established by the Science Council of Japan and ARRIVE. The protocols were approved by the Animal Management and Use Committee of the Tokyo Metropolitan Institute of Medical Science (authorization number 24-071).

[0112] (4) BALB / c mice (8 weeks old; SLC Japan Co., Ltd.) that were negative for animal vaccination were housed in a 12-hour light / dark cycle and given free access to food and water. The mice were anesthetized with intraperitoneal ketamine / xylazine (150 μL) and their backs were shaved. A blood sample (50 μL) was taken from the retroorbital venous plexus before vaccination, and baseline serum was isolated and stored at -20°C. Fifteen mice were randomly assigned to three groups (n=5 in each group). In the first control group, mice were administered 5 μL of sterile phosphate-buffered saline (PBS) by skin scarring. In the second group, recombinant vaccinia virus DI strain (6.14–6.78 × 10⁻¹⁰) carrying the SARS-CoV-2 S gene (r-Dis-S) was administered. ６ Two MAPs containing PFU were applied to shaved dorsal skin (one patch in the upper right quadrant and one in the lower left quadrant) for 5 minutes. In the third group, mice were given 1 × 10⁶ ７PFU-mediated DIs virus was administered at 5 μL via skin scarring. After MAP application, the MAP was carefully removed. The MAP was observed under a microscope before and after insertion, and the number of microneedles retained in the patch and the number of microneedles inserted into the skin were counted. The puncture rate was calculated, and the dose was estimated based on the initial number of microneedles and viral titer. The viral titer of the lysed microneedles (after patch retrieval) was evaluated using a plaque assay as described above. The second vaccination was performed at 4 weeks using the same procedure. Additional blood samples were collected from the submandibular vein at 0, 2, 4, and 6 weeks for immunological analysis.

[0113] Immunogenicity Evaluation: The following experiments were conducted to evaluate immune induction after vaccination.

[0114] Enzyme-linked immunosorbent assay (ELISA) The IgG response to inactivated r-DI and SARS-CoV-2 spike protein was quantified using a standard ELISA protocol. After coating plates with NP-inactivated DI or recombinant S protein (AcroSPN-C52H8), blocking, incubation with diluted serum, secondary HRP-conjugated antibody, substrate development (OPD), and absorbance measurement at 492 nm were performed. Details are as follows: DI virus was grown in primary chicken fibroblasts and purified using OptiPrep® (iodixanol) density gradient medium and ultracentrifugation. The purified DI was suspended in PBS and inactivated with 0.2% NP40. The inactivated DI solution was spread onto 96-well round-bottom plates and incubated overnight at 4°C. For IgG detection against the SARS-CoV-2 spike protein, 96-well plates were coated with 1 μg / mL of recombinant C-terminal His-labeled spike protein (Acro, #SPN-C52H8) of the early SARS-CoV-2 pandemic strain dissolved in 50 mM carbonate buffer (pH 9.6) and incubated overnight at 4°C. The plates were blocked with 1% BSA in PBS(-) containing 0.5% Tween 20 and 2.5 mM ethylenediaminetetraacetic acid, and incubated with 100-fold or 1000-fold dilutions of serum collected from vaccinated mice. After thorough washing, the plates were incubated with wasabi peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (62-6520, Thermo Fisher Scientific (Waltham, Massachusetts, USA)). Next, o-phenylenediamine dihydrochloride and hydrogen peroxide (H) in citrate-phosphate buffer. ２ O ２ 100 μL of the mixture was added to each well. The reaction was quenched by adding 1 M sulfuric acid, and the absorbance was measured at a wavelength of 492 nm.

[0115] In an in vitro neutralization test of vaccinia virus, to evaluate the neutralizing antibody activity, mouse serum was heated and inactivated at 56°C for 30 minutes. Serial dilutions of the heated and inactivated serum were mixed with an equal volume of 3000 PFU / mL DIs and incubated overnight at 37°C. DF-1 cells were then infected with 30 μL of the virus / serum mixture in a 24-well plate. 96 hours post-infection, the neutralization titer was expressed as the reciprocal of the maximum serum dilution that resulted in a 50% reduction in plaque count compared to wells infected with virus alone (without serum).

[0116] SARS-CoV-2 Challenge Experiment Ten weeks after the second vaccination, mice were intranasally inoculated with SARS-CoV-2 strain TY8-612 (GISAID: EPI_ISL_1123289), lineage B. 1.351 (Reference 1), provided by Dr. Masayuki Saijo, Dr. Mutsuyo Takayama-Ito, and Dr. Masaaki Sato (Department of Virology I, National Institute of Infectious Diseases). Each mouse received 1 × 10⁶ doses in a 50 μL volume. ５ PFU was administered. Body weight was monitored daily, and mice that lost more than 30% of their initial body weight were humanely euthanized and recorded as dead.

[0117] Measurement of Infectious SARS-CoV-2 Titer The infectious SARS-CoV-2 titer (viral load) was measured using a standard plaque assay. Briefly, a left lung lobe was collected, homogenized, and centrifuged. The supernatant was stored at -80°C, and the infective titer was measured by a plaque assay on Vero E6 / TMPRSS2 cells using a 0.6% agarose overlay and crystal violet staining. Details are as follows. The left lung lobe of each necropsy mouse was weighed and homogenized with 9 volumes of Hanks Balance saline (Thermo Fisher Scientific) using a multi-bead shocker (Yasui Kikai Co., Ltd., Osaka, Japan). The homogenate was centrifuged at 3000 x g for 10 minutes at 4°C. The supernatant was collected and stored at -80°C until use. For the plaque assay, stepwise 10-fold dilutions of the supernatant (100 μL / well) were placed in a 6-well plate. E6 / TMPRSS2 cells were inoculated onto a confluent monolayer and incubated at 37°C for 1 hour. Unbound viruses were removed by washing the cells with DMEM. The cells were then overlaid with DMEM containing 10% FBS and 0.6% agarose (Sigma-Aldrich, St. Louis, Missouri, USA). After incubation at 37°C for 72 hours, the cells were fixed with 10% neutral buffered formalin and stained with 1% crystal violet. SARS-CoV-2 titer was defined as plaque-forming units per gram of lung tissue (PFU / g lung), with a detection limit of 100 PFU / g lung.

[0118] Following the instructions of the SARS-CoV-2 RNA quantification manufacturer, total RNA was extracted from 70 μL of supernatant of lung homogenate prepared for plaque assay using the RNeasy Mini kit (Kaiagen, Hilden, Germany). RT-qPCR targeting the SARS-CoV-2 N gene was performed using the CDC primer / probe set (N2-F, N2-R, N2-P). 50 nanograms of total RNA were used to quantify the SARS-CoV-2 N protein gene (Reference 2). The primers and probes used are as follows: Forward primer: 5'-TTACACATTTGCGCAAAAA-3'(2019-SARS-CoV-2_N2-F) (Sequence Listing 7) Reverse primer: 5'-GCGCGACAATTCCGGAAAAA-3'(2019-SARS-CoV-2_N2-R) (Sequence Listing 8) and probe 5'-FAM-ACAATTTGCCCCAGCCCTTCAG-BHQ-3'(2019-SARS-CoV-2_N2-P) (Sequence Listing 9)

[0119] As mentioned above, viral RNA quantification was performed using a one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR). The viral load was measured per 1 μg of total RNA. １０ It was calculated as a copy.

[0120] Histopathological examination of the lungs: The right upper lobe of each autopsy mouse was fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned to a thickness of 4 μm, stained with hematoxylin and eosin (H&E), and subjected to standard histological examination. Images of the H&E-stained sections were obtained using an all-in-one fluorescence microscope (BZ-X710, Keyence (Osaka, Japan)) equipped with a Plan Apo 20× Lambda objective lens (NA 0.75, Nikon (Tokyo, Japan)).

[0121] For statistical analysis, data plotted on a linear scale were expressed as mean ± standard deviation (SD), and data plotted on a logarithmic scale were expressed as geometric mean ± geometric standard deviation (SD). Estimatory statistical analysis was performed using one-way ANOVA, followed by Tukey's test as needed. Statistical significance was set at p < 0.05. All analyses were performed using the Prism software package (version 10.4.2; GraphPad software).

[0122] [Preliminary Experiment] Optimization of Vaccine Formulation Determination of Viral Titer in MN-Based Delivery Neutralizing antibody response is an important indicator of vaccine efficacy and is positively correlated with the dose. In a mouse model, intradermal administration of vaccinia virus was 10 ６ from 10 ８ It has been shown to dose-dependently increase both virus-specific IgG and neutralizing antibodies within the PFU range (Reference 3). Conventional replicated smallpox vaccines are administered via a branched needle and typically contain ~2 × 10⁻⁶ ５ Contains PFU. In particular, clinical dilution studies have shown a 10-fold lower potency (~10 per dose). ４ It has been shown that seroconversion can be induced in over 97% of individuals even at PFU doses. Modified Vaccinia Ankara (MVA-BN) vaccine (Jynneos, Imvamune, Imvanex) is a highly attenuated vaccinia virus, similar to DI. The liquid formulation of the MVA-BN vaccine is administered at a standard dose (1 x 10⁻¹⁰). ８ TCID ５０ When administered subcutaneously at (50% tissue culture infectious dose) versus 1 / 5 of the standard dose (2 x 10) ７ TCID ５０ A comparison was made when the delivery was administered intradermally (References 4, 5). Based on these findings, in order to evaluate the immunogenicity of microneedle-based delivery, the target dose for mouse immunization was set to 1 × 10⁶. ７　 I selected the r-DI-S PFU.

[0123] Effect of HA concentration on viral stability In this example, hyaluronic acid (HA) was selected because it has a wide range of biomedical applications, is FDA approved, and possesses desirable properties as a biocompatible and biodegradable material. First, the effect of hyaluronic acid concentration on viral titer stability was investigated. Formulations containing 5%, 10%, 15%, and 20% HA showed dose-dependent improvement in viral stability, with the 20% HA formulation resulting in the best preservation of infectivity titer (Figure 4a). This result is consistent with previous reports indicating that HA forms a protective matrix around viral particles, reducing titer loss under ambient conditions (Reference 6). In this example, to balance viral preservation with handling advantages such as precise distribution of the HA solution into the PDMS type and to minimize viral loss in the solution, the 15% HA formulation was selected in subsequent experiments.

[0124] On the other hand, increasing the solute concentration in the external liquid increases the osmotic pressure across the entire virus-solvent interface, leading to stress such as surface tension effects. Therefore, to preserve maximum viral activity in dried formulations for industrial applications, rapid drying of the virus formulation is expected to minimize the exposure time of virus particles to harmful osmotic pressure fluctuations. The 20% HA formulation showed the highest stability within the range tested by the inventors, but further increases in HA concentration may exceed the threshold. To evaluate the effect of temperature during MAP preparation, MAP preparation including dispensing and drying steps was considered, and the viral titer was measured after storing the HA-virus solution at 4°C or 25°C for 6 hours. Viral titer by plaque assay showed a significant effect of working temperature on viral stability. Samples stored at 4°C retained approximately 71% of their initial titer, while samples stored at 25°C retained only 41% (Figure 4b). These findings suggest that lower temperatures during the dispensing and drying steps in MAP preparation may enhance the stability of vaccinia virus. These results confirm that minimizing thermal exposure during MAP preparation is crucial for maximizing vaccine efficacy.

[0125] Figure 4 shows the results of optimization tests for vaccine formulation conditions. Figure 4a shows the effect of hyaluronic acid (HA) concentration on the viral viability of DIs after drying. Plaque assay analysis showing the effect of HA concentration on the stability of DIs in the formulation is shown. Increasing the HA concentration from 5% to 20% gradually improved the stability of viral titer. Data are shown as mean ± standard deviation (n=4). P-values ​​were calculated using one-way ANOVA (*p>0.05; **p<0.05). Figure 4b shows the change in viral titer over 6 hours at different storage temperatures (4°C vs. 25°C). Data are shown as mean ± standard deviation (n=4). P-values ​​were calculated using one-way ANOVA (*p>0.05; **p<0.05). Samples stored at 4°C retained approximately 71% of the initial viral titer, while samples stored at 25°C decreased, retaining only 41% of the titer.

[0126] [Example 1] Development and Evaluation of Pillar-Guided MAPs Conventional soluble microneedle array patches (MAPs) often suffer from problems of inaccurate dose control, long drying times, and inefficient payload localization. When water-soluble materials are used for the backing layer, it causes redissolution of the needles and undesirable diffusion of vaccine material into the backing layer. These problems lead to drawbacks such as reduced vaccine activity and prolonged drying times that result in vaccine loss. To address these problems, the inventors have developed a pillar-guided MAP (PG-MAP) system featuring a 3D-printed backing layer with an integrated pillar structure (Figures 5a-b). These pillars can enhance vaccine filling to the tip and minimize vaccine loss into the backing layer.

[0127] (1) Design of PG-MAP The support members fabricated using an SLA printer incorporated a pillar insertion design. The support members were designed with pillars with a base diameter of 300 μm and a length of 1,000 μm arranged in a 14 × 14 array. The tip of the pillar was designed to 400 μm to maximize surface contact with the hyaluronic acid solution when inserted into the microneedle cavity (Figure 5a). Optical microscopy analysis of five independent support members revealed the average pillar tip height (P ｔｉｐ＿ｈｅｉｇｈｔ ) is 368 ± 16 μm, pillar (support section) length (P ｌｅｎｇｔｈ ) is 1003 ± 18 μm, pillar base (P ｂａｓｅ ) was shown to be 312 ± 14 μm (Figure 5b).

[0128] Figure 5 shows the design and characteristics evaluation results of the backing (support member). Figure 5a shows the schematic design of the MAP and the 3D printed backing layer. The same figure shows a cross-sectional view showing the pillar position and the filling of the microneedle tip. Figure 5b shows an optical microscope image of the fabricated backing pillar (support member with a support column).

[0129] (2) Process for fabricating pillar guide MAPs Plasma-treated PDMS molds are filled with 15% HA and r-Dis-S solution (1.0 × 10 ８ The MAP was filled with PFU / mL, aligned with the pre-fabricated backing layer, and dried at 4°C and 10% RH for 8 hours. After complete drying, the backing layer was carefully removed from the PDMS mold (Figure 6a). The fabricated MAP had a needle length (N) of 688 ± 25 μm. ｌｅｎｇｔｈ ) and a base length of 477±31 μm (B ｌｅｎｇｔｈ ) was found (Figure 6b) (n=15). It was confirmed that the tip was properly attached to the pillar. However, the formation of a bridge between the needles was observed, which led to vaccine loss (Figure 6c). It is thought that this bridge formation occurred as a result of dispensing the solution into the space between the cavities of the needles, and it is thought that it is possible to suppress or minimize bridge formation by optimizing the viscosity of the formulation, using a precision dispenser for accurate dispensing, and improving the mold design.

[0130] The drying rate of PG-MAP using the same hyaluronic acid preparation (15% HA) was compared with that of the conventional one-step molding method. Under drying conditions of 4°C and ~10% RH, the total mass loss (t ９０ The time required to reach 90% mass loss was shorter with the pillar-guided (PG) method than with the conventional one-step molding (OS) method. The PG-MAP design using 50 μL of vaccine solution per mold achieved 90% mass loss (t) in 9 hours. ９０ This achieved a rate that was almost half the time required for the 1 mL casting approach (one-step molding method) (Figure 6d).

[0131] Theoretically, the PG method yielded a tip loading efficiency (TLE) of 16.5%, compared to only 2.0% with the conventional OS method. Expressed as a fold change, the PG approach increased the TLE by 8.3 times (Figure 6e).

[0132] Our results demonstrate that the reduction in drying time during MAP preparation achieved by the pillar-guided (PG) method leads to improved preservation of viral titer, with a viability rate of 50.4% compared to 40.4% in the conventional one-step (OS) method (Figure 6f). This improved stability may be due to reduced exposure of the virus to osmotic pressure for extended periods in the liquid phase, minimizing viral degradation during MAP preparation. The difference in viral titer between the two methods suggests that process optimization of live virus-based vaccine formulations is crucial. Therefore, limiting the time the virus remains in the liquid state using the pillar-guided method is essential for preserving viral titer and ensuring the feasibility of vaccine administration using MAPs.

[0133] Next, evaluation of mechanical strength showed that all MAPs exhibited sufficient destructive power for skin penetration, regardless of HA concentration (Figure 6g, red dotted line). This balance between high viral survival rate and mechanical performance is expected to highlight the promising nature of this pillar guide fabrication approach for practical applications.

[0134] Figure 6 shows a schematic diagram of the PG-MAP fabrication process and the results of the PG-MAP characterization. Figure 6a shows a schematic diagram of the PG-MAP mold and the micromolding process. Figure 6b shows an optical microscope image of the fabricated PG-MAP. Figure 6c is an optical microscope image of the PG-MAP showing the needle arrangement and bridge formation. Figure 6d shows the drying dynamics of the PG-MAP and the conventional casting method. Figure 6e shows a comparison of the tip filling efficiency (TLE) of the PG-MAP and the one-step casting method. Figure 6f shows that the retention of viral titer is improved with the PG method when comparing the pillar guide method (survival rate: 50.4%) and the conventional one-step molding method (survival rate: 40.4%), after measuring the viral titer stability after fabrication using a plaque assay (n=4). Figure 6g shows the results of measuring the breaking force of PG-MAPs made with 5%, 10%, and 15% hyaluronic acid (HA) by axial force measurement (n=5). The red dotted line (58 mN) indicates the threshold for skin puncture (Reference 7).

[0135] [Example 2] In vivo immunogenicity and protection against SARS-CoV-2 To evaluate the immunogenicity of r-DIs-S administered via PG-MAP, 9.4 ± 1.8 × 10⁻¹⁶ plaque assays were performed on BALB / c mice. ６ Immunization was performed using two MAPs containing PFU / patch (total dose per mouse: approximately 1.9 × 10⁻⁶). ７ PFU was administered, followed by a booster dose in the fourth week. Each MAP was applied to the skin of the back for 5 minutes.

[0136] Vaccination Protocol and Dosage Management This study demonstrates the effectiveness of rDIsS-equipped PGMAP vaccine in inducing a potent and sustained immune response in a mouse model. By standardizing the vaccination procedure, including administration to the same site on the back, application with consistent pressure, and precise quantification of the dose, the immune response induced by PGMAP could be reliably evaluated. Vaccine efficacy was evaluated in BALB / c mice, comparing a group that received intradermal immunization with rDIsS-equipped PGMAP with a group that received PBS or DIs via skin scarification (Figure 7a). The vaccines were administered separately twice at 4-week intervals, and challenge infection was induced at 16 weeks (Figure 7b).

[0137] The puncture rate was calculated by counting the needles remaining after puncture and was 80% and 88%, respectively (Figure 7c). Next, the exact amount of viral titer loaded into the MAP was evaluated by plaque assay before inoculation. The MAP contained an average of 9.4 ± 1.8 × 10⁶ of viral titer after the first and second inoculations, respectively. ６ PFU / patch and 11.6±1.8×10 ６ The PFU / patch was loaded (Figure 7d).

[0138] After immunization, MAP-inoculated mice showed a stronger anti-DIs IgG response than conventionally swabbed mice, with an average 1.2-fold increase in optical density over six weeks (Figure 7e). Furthermore, MAP inoculation specifically induced spike protein-specific IgG (Figure 7f). In contrast, these antibodies were not detected in mice vaccinated by swabbing or PBS, demonstrating the specific immunogenicity of MAP inoculation.

[0139] Analysis of the relationship between antibody response dose and antibody response to vaccines and SARSCoV2 spikes showed a correlation between administered rDIsS and antibody response. To quantify this relationship, a correlation coefficient (R) was used to determine if a linear relationship was observed. ２ The study was conducted using ). Dose-antibody correlation analysis revealed a strong positive correlation between the amount of rDIsS administered and the early antibody titer at week 2. Total IgG (R ２≈0.83 (Figure 7g) and a significant correlation was observed with spike-specific IgG, suggesting that higher antigen levels greatly promote immune activation in the early stages. However, at 4 and 6 weeks R ２ The values ​​decreased (Figure 7h). This suggests that the booster effect and maturation of the acquired immune response over time may be influencing the results. Further research is needed to clarify the extent to which the maturation of the acquired immune response, including the maturation of antibody avidity (binding strength), contributes. These results indicate that while initial antibody production is closely related to the dose, the immunological memory response and subsequent boosters gradually play a dominant role as vaccination progresses. Overall, these results highlight that PGMAP enables accurate and immunogenically effective rDIsS administration.

[0140] Figure 7 shows the in vivo delivery and immunogenicity of r-DIs-S-loaded PG-MAP. Figure 7a shows a comparison of intradermal r-DIs-S-loaded PG-MAP application and bifurcated needle scarification in BALB / c mice using an immunization protocol. Figure 7b shows the vaccination schedule, including blood collection, and the 16-week viral challenge. Figure 7c shows MAP images before and after application to the dorsal skin of mice, showing microneedle insertion. Figure 7d shows the results of quantitative analysis of r-DIs-S viral titers delivered per patch, measured by plaque assay before each vaccination (mean ± standard deviation, n=4). Figure 7e shows the time course of the anti-DIs IgG response in mice, measured by ELISA (n=5). Mice vaccinated with r-Dis-S-equipped PG-MAP showed significantly higher serum anti-vaccinia IgG titers than mice scarified with native DI or PBS. P values ​​were calculated using one-way ANOVA (*p>0.05; **p<0.05, ***p<0.01). Figure 7f shows the time course of the r-Dis-S specific antibody response in mice measured by ELISA (n=5). Importantly, only the PG-MAP group induced a strong spike protein-specific IgG response, indicating successful antigen expression from the r-Dis-S vector in vivo. P values ​​were calculated using one-way ANOVA (*p>0.05; **p<0.05, ***p<0.01). Figure 7g shows the correlation between the administered r-Dis-S dose and the IgG response at week 2. Figure 7h shows the correlation coefficient (R) between the dose administered over six weeks and the S protein-specific antibody response. ２ This shows the temporal changes in ). It is highly likely that this reflects the effects of immunological memory and secondary responses after booster shots.

[0141] [Example 3] Protection against SARS-CoV-2 Challenge Next, the protective effect of r-Dis-S equipped MAP against lethal challenge infection in BALB / c mice infected with SARS-CoV B. 1.351 mutant strain was investigated (Figure 8). Vaccinated mice were intranasally infected with SARS-CoV-2 TY 8612 strain 10 weeks after the second vaccination. Both PBS and Dis-vaccinated mice rapidly lost weight after SARS-CoV-2 infection, and one Dis-vaccinated mouse died on day 6 post-infection. By day 7 post-infection, three PBS-vaccinated mice and three Dis-vaccinated mice showed a weight loss of more than 30% and decreased activity. These mice met the endpoint cutoff value and were euthanized. In contrast, mice inoculated with r-Dis-S-equipped MAP rapidly recovered from weight loss after SARS-CoV-2 infection, and all PG-MAP-inoculated mice survived and maintained their weight throughout the study. Lung virus titers, measured by plaque assay and RT-qPCR, were significantly lower in the vaccinated group compared to the control group (Figure 8c–d). Seven days post-infection, lung pathology in vaccinated mice was significantly improved compared to control mice inoculated with PBS(-) or DI. Histopathological analysis of lung sections revealed that PG-MAP-inoculated mice showed preserved alveolar structure and minimal inflammation, while control mice showed extensive alveolar destruction and immune cell infiltration (Figure 8e). These results suggest that vaccination with r-DI-S-equipped MAP significantly reduces lung virus titers, thereby mitigating the severity of symptoms caused by infection with the SARS-CoV-2 TY8-612 strain.

[0142] Figure 8 shows the protective effect of r-Dis-S-equipped MAP against lethal infection by SARS-CoV-2 B.1.351. Figure 8a shows the time course of body weight of BALB / c mice with and without vaccination after infection with SARS-CoV-2 TY8-612 strain. Figure 8b shows the survival rate of BALB / c mice after SARS-CoV-2 infection. Figure 8c shows the viral load of left lung homogenate measured by RT-qPCR of the SARS-CoV-2 N protein gene. P values ​​were calculated using one-way ANOVA, followed by Tukey's test (*** p < 0.001; **** p < 0.0001; ns, not important). Figure 8d shows the results of measuring the infectious viral titer in left lung homogenate by plaque assay. The dashed line indicates the limit of detection (100 PFU / g lung). Viral titers below the LOD are expressed as 50 PFU / g lung tissue. P values ​​were calculated using one-way ANOVA, followed by Tukey's test (*: p < 0.05; **: p < 0.01; ns: not significant). Figure 8e shows a representative section of the right upper lung lobe of a BALB / c mouse infected with SARS-CoV-2, 7 days post-infection (hematoxylin-eosin staining; section thickness: 4 μm). Scale bar: 100 μm.

[0143] In the examples, the feasibility of administering recombinant vaccinia virus (r-Dis-S) live vaccine using the pillar-guided microneedle array patch (PG-MAP) of the present invention was demonstrated. The pillar-guided fabrication method enabled precise filling of the virus solution only at the microneedle tip, resulting in a tip filling efficiency of 16.5%, an 8.3-fold increase compared to the conventional one-step molding method. Plaque assays after fabrication confirmed that 50.4% of the initial viral titer was retained using the PG-MAP method, compared to 40.4% using the conventional approach. This improvement was achieved by reducing the drying time to 9.7 hours with PG-MAP compared to 18.1 hours with the one-step method (both at 4°C and 10% RH).

[0144] Furthermore, mechanical evaluation confirmed that PG-MAP has sufficient strength for intradermal application, with an insertion rate into mouse skin ranging from 80% to 88%. In immunogenicity testing, the first vaccination required 9.4 ± 1.8 × 10⁻⁶ doses. ６ PFU / patch, 11.6 ± 1.8 × 10 for the second vaccination. ６ MAPs containing PFU / patch viral titers were used. This experiment demonstrated that r-dis-S MAP vaccination induces a potent anti-DIs IgG and SARS-CoV-2 spike-specific IgG response. Mice immunized with r-dis-S MAP showed a 1.2-fold higher anti-DIs IgG response over six weeks compared to mice immunized by conventional scarification. Importantly, only the MAP-vaccinated group expressed spike-specific antibodies and confirmed in vivo expression of the SARS-CoV-2 antigen.

[0145] Furthermore, in a lethal SARS-CoV-2 challenge model, PG-MAP-vaccinated mice showed 100% survival and complete protection, while all control animals died from infection. These results quantitatively confirm that the pillar-guided MAP platform enables accurate, efficient, and immunogenically effective delivery of live viral vaccines with stable viral stability and reliable protective efficacy in vivo.

[0146] The inventors expect the PG-MAP system according to the present invention to be a promising candidate for decentralized vaccination strategies, particularly under limited cold chain infrastructure and healthcare personnel. They believe the technology of the present invention has the potential to accelerate global immunization efforts against emerging infectious diseases.

[0147] Related information and papers

Claims

1. A method for manufacturing a microneedle patch, comprising the steps of: (a) injecting a solution containing a biosoluble material and a drug or vaccine into a female mold; (b) arranging a support member having a support column on a support layer above the mold; (c) inserting the tip portion of the support column into the solution injected into the mold; and (d) drying the solution and the support member, and then removing the support member with the microneedles attached from the mold.

2. The manufacturing method according to claim 1, wherein the support member is manufactured in advance by 3D printing.

3. The manufacturing method according to claim 1, wherein the vaccine is a coronavirus vaccine, a novel coronavirus vaccine, an influenza virus vaccine, a dengue virus vaccine, or a polio virus vaccine.

4. The method for producing a vaccine according to claim 1, wherein the vaccine is a recombinant vaccinia virus used as a vaccine for SARS-CoV-2.

5. The method for producing a biosoluble material according to claim 1, wherein the biosoluble material is at least one selected from the group consisting of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose, a copolymer of methyl vinyl ether and maleic anhydride, trehalose, sucrose, maltose, and silk fibroin.

6. The manufacturing method according to claim 1, wherein in step (a), the step of plasma treatment is performed on the mold before injecting the solution into the mold.

7. The manufacturing method according to claim 1, wherein in step (c), the support column is inserted into the mold shape such that the ratio of the height of the tip portion of the support column inserted into the microneedle to the height of the obtained microneedle is 8:1 to 2:

1.

8. A microneedle patch obtained by the manufacturing method described in any one of claims 1 to 7.

9. A microneedle patch comprising a biosoluble material, a microneedle containing a drug or vaccine, and a support member having a support column on a support layer, wherein the tip portion of the support column is inserted into the microneedle.

10. The microneedle patch according to claim 9, wherein the support layer is manufactured by 3D printing.

11. The microneedle patch according to claim 9, wherein the vaccine is a coronavirus vaccine, a novel coronavirus vaccine, an influenza virus vaccine, a dengue virus vaccine, or a polio virus vaccine.

12. The microneedle patch according to claim 9, wherein the vaccine is a recombinant vaccinia virus used as a vaccine for SARS-CoV-2.

13. The microneedle patch according to claim 9, wherein the biosoluble material is at least one selected from the group consisting of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose, a copolymer of methyl vinyl ether and maleic anhydride, trehalose, sucrose, maltose, and silk fibroin.

14. The microneedle patch according to claim 9, wherein the ratio of the height of the tip portion to the height of the microneedle is 8:1 to 2:1.

Images