Vaccine composition for sublingual administration

Abstract

Provided are a vaccine composition suitable for sublingual administration, a vaccine production method and a vaccine administration method.　Provided is a vaccine composition that contains an immune antigen and an adjuvant and is to be sublingually administered to a subject.

Description

[Technical Field]

[0001] The present invention relates to a vaccine composition for sublingual administration. [Background technology]

[0002] In addition to the choice of immunizing antigen and adjuvant, the administration route can also affect vaccine efficacy. For viruses such as influenza and coronavirus that infect epithelial cells in the bronchi and lungs, it is more important to induce viral antigen-specific secretory IgA antibodies in the upper respiratory tract mucosa than circulating IgG antibodies (Non-Patent Document 1). Recently, vaccine administration via the nasal or oral route has been developed, which induces secretory IgA antibodies / mucosal immune responses, as opposed to circulating IgG antibodies / systemic immune responses (Non-Patent Document 2). Meanwhile, there has been little progress in practical protein vaccines for sublingual administration. In humans and primates, including monkeys, the sublingual area has ample space, making vaccine administration easier than nasal administration, but the efficacy of sublingually administered vaccines has not been confirmed (Non-Patent Documents 3 and 4). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] A. Ainai, et al. Human immune responses elicited by an intranasal inactivated H5 influenza vaccine, Microbiology and Immunology. 2020; 64:313-325. [Non-patent document 2] R. Mudgal, et al. Prospects for mucosal vaccine: shutting the door on SARS-CoV-2, HUMAN VACCINESIMMUNOTHERAPEUTICS 2020, VOL. 16, NO. 12, 2921-2931. [Non-patent document 3] RS Veazey, et al, Evaluation of mucosal adjuvants and immunization routes for the induction of systemic and mucosal humoral immune responses in macaques, Human Vaccines & Immunotherapeutics 11:12, 2913--2922; December 2015. [Non-patent document 4] Alan D. Curtis II, et al, A simultaneous oral and intramuscular prime / sublingual boost with a DNA / Modified Vaccinia Ankara viral vector-based vaccine induces simian immunodeficiency virus-specific systemic and mucosal immune responses in juvenile rhesus macaques, J Med Primatol. 2018 October; 47(5): 288-297. Summary of the Invention [Problem to be solved by the invention]

[0004] Provided are a vaccine composition suitable for sublingual administration, a method for producing a vaccine, and a method for administering a vaccine. [Means for solving the problem]

[0005] As a result of extensive research, the present inventors have discovered a new vaccine composition suitable for administration via the upper respiratory tract mucosa, particularly sublingual administration.

[0006] The present invention includes the following [1] to "18C". [1] A vaccine composition for sublingual administration to a subject, comprising an immunizing antigen and an adjuvant. [2] The vaccine composition according to [1], further comprising a mucolytic agent. [3] The vaccine composition described in [1], which is administered after administering a mucolytic agent sublingually to a subject. [4] The vaccine composition according to [2] or [3], wherein the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelators. [5] The vaccine composition according to [4], wherein the reducing agent is cysteine ​​or a derivative thereof. [6] The vaccine composition according to [5], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine. [7] The vaccine composition according to [4], wherein the protease is trypsin, papain, or bromelain. [8] The vaccine composition according to [4], wherein the protein denaturing agent is urea or guanidinium hydrochloride. [9] The vaccine composition according to [4], wherein the deoxyribonuclease is human DNase I.

[10] The vaccine composition according to [4], wherein the calcium chelating agent is bicarbonate or ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[11] The vaccine composition according to [1], wherein the immunogen is derived from chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, or HIV.

[12] The vaccine composition described in [1], wherein the immunogen comprises the spike protein receptor-binding domain of SARS-CoV-2.

[13] The vaccine composition according to [1], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene, or an aluminum salt.

[14] The composition according to

[13] , wherein the TLR ligand is double-stranded RNA (dsRNA).

[15] The composition according to

[14] , wherein the dsRNA is Poly(I:C).

[16] The vaccine composition according to [1], wherein the weight ratio of the immunogen to the adjuvant contained therein is 10:400 to 400:400, preferably 20:400 to 200:400, and more preferably 30:400 to 150:400.

[17] The vaccine composition according to [1], wherein the immunogen is contained in an amount of 10 to 400 μg, preferably 20 to 200 μg, and more preferably 30 to 150 μg.

[18] The vaccine composition described in [1], which is administered to a subject three times at four-week intervals.

[0007] [1A] A method for making a subject immunogenic to an immunizing antigen, comprising: 1) A method comprising administering an immunizing antigen and an adjuvant sublingually to a subject. [2A] The method according to [1A], wherein the immunizing antigen and adjuvant are administered sublingually together with a mucolytic agent. [3A] The method according to [1A], wherein the mucolytic agent is administered sublingually to the subject, followed by administration of the immunizing antigen and adjuvant. [4A] The method according to [2A] or [3A], wherein the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturing agents, deoxyribonucleases, reducing agents, and calcium chelating agents. [5A] The method according to [4A], wherein the reducing agent is cysteine ​​or a derivative thereof. [6A] The method according to [5A], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine. [7A] The method according to [4A], wherein the protease is trypsin, papain or bromelain. [8A] The method according to [4A], wherein the protein denaturing agent is urea or guanidinium hydrochloride. [9A] The method according to [4A], wherein the deoxyribonuclease is human DNase I. [10A] The method according to [4A], wherein the calcium chelating agent is bicarbonate or ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). [11A] The method according to [1A], wherein the immunogen is derived from a virus selected from chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, and HIV; wherein preferably, the administration prevents or treats infection with the virus in a subject. [12A] The method described in [1A], wherein the immunizing antigen comprises the spike protein receptor-binding domain of SARS-CoV-2. [13A] The method according to [1A], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene, or an aluminum salt. [14A] The method according to [13A], wherein the TLR ligand is double-stranded RNA (dsRNA). [15A] The method according to [14A], wherein the dsRNA is Poly(I:C). [16A] The method according to [1A], wherein the weight ratio of the immunogen to the adjuvant contained is 10:400 to 400:400, preferably 20:400 to 200:400, more preferably 30:400 to 150:400. [17A] The method according to [1A], wherein the immunogen is contained in an amount of 10 to 400 μg, preferably 20 to 200 μg, more preferably 30 to 150 μg. [18A] The method according to [1A], wherein the immunizing antigen and adjuvant are administered to the subject three times at four-week intervals.

[0008] [1B] A combination of an immunizing antigen and an adjuvant for use in sublingual administration to confer immunogenicity to the immunizing antigen in a subject. [2B] The combination described in [1B], administered sublingually together with a mucolytic agent. [3B] The combination described in [1B], which is administered sublingually after administering a mucolytic agent sublingually to a subject. [4B] The combination according to [2B] or [3B], wherein the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelators. [5B] The combination according to [4B], wherein the reducing agent is cysteine ​​or a derivative thereof. [6B] The combination according to [5B], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine. [7B] The combination according to [4B], wherein the proteolytic enzyme is trypsin, papain or bromelain. [8B] The combination according to [4B], wherein the protein denaturing agent is urea or guanidinium hydrochloride. [9B] The combination according to [4B], wherein the deoxyribonuclease is human DNase I. [10B] The combination according to [4B], wherein the calcium chelating agent is bicarbonate or ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). [11B] The combination according to [1B], wherein the immunogen is derived from a virus selected from chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, and HIV; wherein the combination is preferably for use in the prevention or treatment of infection with the virus. [12B] The combination described in [1B], wherein the immunizing antigen comprises the spike protein receptor-binding domain of SARS-CoV-2. [13B] The combination according to [1B], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene, or an aluminum salt. [14B] The combination according to [13B], wherein the TLR ligand is double-stranded RNA (dsRNA). [15B] The combination according to [14B], wherein the dsRNA is Poly(I:C). [16B] The combination according to [1B], wherein the weight ratio of the immunizing antigen to the adjuvant is 10:400 to 400:400, preferably 20:400 to 200:400, and more preferably 30:400 to 150:400. [17B] The combination according to [1B], wherein the immunogen is contained in an amount of 10 to 400 μg, preferably 20 to 200 μg, more preferably 30 to 150 μg. [18B] The combination described in [1B], administered to a subject three times at four-week intervals.

[0009] [1C] 1. Use of an immunizing antigen and an adjuvant in the manufacture of a medicament for sublingual administration to confer immunogenicity to the immunizing antigen in a subject. [2C] The use described in [1C], wherein the medicament comprises a mucolytic agent. [3C] The use described in [1C], wherein the medicament is administered after administering a mucolytic agent sublingually to the subject. [4C] The use according to [2C] or [3C], wherein the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturing agents, deoxyribonucleases, reducing agents, and calcium chelating agents. [5C] The use according to [4C], wherein the reducing agent is cysteine ​​or a derivative thereof. [6C] The use according to [5C], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine. [7C] The use according to [4C], wherein the proteolytic enzyme is trypsin, papain or bromelain. [8C] The use according to [4C], wherein the protein denaturing agent is urea or guanidinium hydrochloride. [9C] The use according to [4C], wherein the deoxyribonuclease is human DNase I. [10C] The use according to [4C], wherein the calcium chelating agent is bicarbonate or ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). [11C] The use according to [1C], wherein the immunogen is derived from a virus selected from chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, and HIV; wherein preferably, the medicament is a medicament for preventing or treating infection with the virus. [12C] The use described in [1C], wherein the immunogen comprises the spike protein receptor-binding domain of SARS-CoV-2. [13C] The use according to [1C], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene, or an aluminum salt. [14C] The use according to [13C], wherein the TLR ligand is double-stranded RNA (dsRNA). [15C] The use according to [14C], wherein the dsRNA is Poly(I:C). [16C] The use according to [1C], wherein the weight ratio of the immunogen to the adjuvant contained in the medicament is 10:400 to 400:400, preferably 20:400 to 200:400, more preferably 30:400 to 150:400. [17C] The use according to [1C], wherein the medicament contains 10 to 400 μg, preferably 20 to 200 μg, more preferably 30 to 150 μg of the immunogen. [18C] The use described in [1C], wherein the medicament is administered to the subject three times at four-week intervals. [Effects of the Invention]

[0010] The vaccine composition or medicine for sublingual administration and the method of administering the same according to the present invention are effective in activating not only systemic immune responses but also mucosal immunity, thereby preventing or treating viral infections from the upper respiratory tract mucosa. [Brief explanation of the drawings]

[0011] [Figure 1] The results of a preclinical study in cynomolgus monkeys, a primate model, using a sublingual vaccine consisting of SARS-CoV-2 RBD antigen and poly(I:C) adjuvant are shown in Figure 1A: salivary levels of anti-RBD IgA; Figure 1B: plasma levels of anti-RBD IgG; Figure 1C: plasma levels of anti-RBD IgA; and Figure 1D: plasma levels of anti-RBD IgE. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, "comprising" encompasses "essentially comprising," "comprising an effective amount of," and "consisting of."

[0013] The upper respiratory tract refers to the respiratory tract from the nasal cavity to the lungs, including the nasal cavity, paranasal sinuses, pharynx, and larynx. An upper respiratory tract infection occurs when viruses or bacteria that have invaded from the outside world (mostly from the atmosphere) attach to the upper respiratory tract and multiply (infect) there, causing symptoms such as runny nose, nasal congestion, and sore throat. This includes acute upper respiratory tract inflammation (commonly known as the common cold syndrome), acute pharyngitis / tonsillitis, acute laryngitis, and acute epiglottitis. On the other hand, a lower respiratory tract infection occurs when a virus that has infects the upper respiratory tract migrates from the trachea to the respiratory bronchioles and multiplies in these areas, causing symptoms of bronchitis. In this specification, infection includes both upper and lower respiratory tract infections.

[0014] One embodiment of the present invention is a vaccine composition or medicament comprising an immunogen and an adjuvant. The vaccine composition or medicament is preferably administered sublingually. The subject to be administered is not particularly limited as long as it is a mammal capable of immune response, but is preferably a primate, and more preferably a human.

[0015] In one embodiment of the present invention, the immunogen (also simply referred to as antigen) is not particularly limited as long as it is capable of possessing antigenicity, and includes not only peptidic antigens (i.e., antigenic peptides) but also non-peptidic antigens such as phospholipids and complex carbohydrates (e.g., bacterial membrane components such as mycolic acid and lipoarabinoanthracene). The immunogen preferably comprises a component molecule derived from a pathogen such as a virus. Such pathogens include, but are not limited to, protozoa (e.g., Plasmodium, Leishmania species, Trypanosoma species), bacteria (e.g., gram-positive cocci, gram-positive bacilli, gram-negative bacteria, anaerobic bacteria), fungi (e.g., Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, Sporothrix), viruses (e.g., varicella virus, measles virus, mumps virus, poliovirus, rotavirus, adenovirus, herpes simplex virus, papillomavirus, respiratory syncytial virus, poxvirus, HIV, influenza virus, coronaviruses such as SARS-CoV and SARS-CoV2), intracellular parasites (e.g., Chlamydiaceae, Mycoplasmataceae, Acholeplasmataceae, Rickettsiaceae), and helminths (e.g., nematodes, trematodes, cestodes). Of these, viruses that infect the upper respiratory tract (e.g., chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, or HIV) are suitable for the present invention because they are expected to confer not only humoral immunity but also mucosal immunity. Therefore, the present invention can be applied to the prevention and treatment of infections by infectious pathogens such as protozoa, bacteria, fungi, viruses, intracellular parasites, and helminths (i.e., suppressing the proliferation of these infectious pathogens in the body).

[0016] As used herein, the term "antigenic peptide" is not particularly limited as long as it is a peptide composed of two or more amino acids (including those composed of more than 50 amino acids) that can serve as an antigen, and may be naturally derived, synthetically derived, or commercially available. Antigenic peptides may include the full-length amino acid sequence of a gene product or a partial amino acid sequence thereof. Examples include antigenic peptides (including full-length and partial sequences) derived from infectious pathogens such as the above-mentioned protozoa, bacteria, fungi, viruses, intracellular parasites, and helminths. For example, the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is an antigenic peptide. The antigen peptide may be optionally processed or modified (for example, phosphorylated or glycosylated).

[0017] In one embodiment of the present invention, an adjuvant refers to a substance used in combination with an immunizing antigen to enhance its effect (immunogenicity). Examples of adjuvants include, but are not limited to, those containing aluminum salts (e.g., AS04), those containing squalene (e.g., MF59, AS03, AddaS03), and those containing Toll-like receptor (TLR) ligands. Examples of TLR ligands include, but are not limited to, triacyl lipoproteins (TLR1 / 2 ligands); peptidoglycan, lipoarabinomannan, and porins (TLR2 ligands); double-stranded RNAs such as Poly(I:C) (TLR3 ligands); lipopolysaccharides (TLR4 ligands); flagellin (TLR5 ligands); diacyl lipoproteins and zymosan (TLR2 / 6 ligands); single-stranded RNAs (TLR7 / 8 ligands); and CpG-DNA (TLR9 ​​ligands). Binding of these ligands to TLRs on immune cells results in the formation of homodimers or heterodimers, transducing signals into the cells and activating immune cells (i.e., initiating an immune response), resulting in the induction of inflammatory cytokine production and type I interferon production.

[0018] In one embodiment of the present invention, the vaccine composition or medicament may contain 10 to 400 μg, preferably 20 to 200 μg, more preferably 30 to 150 μg of an immunogen per dose.

[0019] In one embodiment of the present invention, the weight ratio of the immunogen to the adjuvant may be 10:400 to 400:400, preferably 20:400 to 200:400, and more preferably 30:400 to 150:400.

[0020] In one embodiment of the invention, the vaccine composition or medicament may comprise a mucolytic agent and may be administered after or simultaneously with the administration of the mucolytic agent. Mucus covers the epithelial surfaces of the respiratory tract (including the nasal cavity and oral cavity), digestive organs (such as the stomach and intestines), vagina, joints, eyes, etc., forming a barrier against foreign substances and pathogens. Mucin, the main structural component of mucus, is a macromolecule formed by glycosylation of apomucin, which consists of a protein backbone with intermittent cysteine-rich regions involved in mucin-mucin interactions via disulfide bonds, and a central region containing tandem repeats rich in threonine, serine, and proline (hereinafter, the above barrier may be referred to as the mucin layer, mucus gel, or mucus layer). Mucolytic agents are drugs that reduce the viscosity of mucous gel. Examples of mucolytic agents include, but are not limited to, proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelators. Mucolytic agents increase the permeability of the mucous gel present under the tongue, allowing the immunogen and adjuvant to be absorbed into the body via the sublingual passage, thereby inducing a mucosal immune response and / or a systemic immune response.

[0021] Because mucus is a complex network of cross-linked proteins, proteolytic enzymes can be utilized to break peptide bonds and cleave non-glycosylated mucin domains, degrading the mucin protein backbone and / or proteins in the mucus gel and increasing the permeability of the mucus gel. Such proteolytic enzymes include trypsin, papain or bromelain. In the present invention, the protease may include a glycosidase that degrades glycosylation of mucin. By degrading glycosylation, the same effect as that of a protease can be obtained.

[0022] Mucus also contains DNA, a large molecule, so the permeability of the mucus gel can be increased by decomposing the DNA in the mucus. Such deoxyribonucleases (DNA degrading enzymes) include human DNase I (Enzyme No. 3.1.21.1) or active fragments thereof.

[0023] The permeability of mucus gel can be increased by reducing agents that reduce and break disulfide bonds between mucins in mucus. Such reducing agents include cysteine ​​and modified cysteine, such as N-acetylcysteine ​​(NAC) and S-carboxymethylcysteine ​​(carbocysteine).

[0024] The permeability of mucus gels can be increased by protein denaturants that disrupt the inter- and intramolecular hydrogen bonds of mucin molecules in mucus. Such protein denaturants include urea and guanidinium hydrochloride.

[0025] The mucin layer in mucus can be hydrated, swelled, and dispersed by calcium chelators, thereby increasing its permeability. Such calcium chelating agents include bicarbonate and ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0026] In one embodiment of the invention, the vaccine composition is preferably administered to a subject at least three times, with the administration interval being 3 to 5 weeks, preferably 4 weeks. Additionally, at least one additional dose may be administered to elicit an immune response.

[0027] In one embodiment of the invention, the vaccine composition or medicament may further comprise pharmaceutically acceptable carriers; antioxidants; preservatives; colorants; diluents; emulsifiers; suspending agents; solvents; fillers; bulking agents; buffers; delivery vehicles; diluents; excipients, etc.

[0028] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0029] Example 1 A sublingual vaccine consisting of SARS-CoV-2 RBD antigen and poly(I:C) adjuvant was tested in a preclinical study using the primate model, cynomolgus monkeys.

[0030] 1. Materials and Methods 1.1. Reagents and antibodies N-acetylcysteine ​​(NAC), bovine serum albumin (BSA), sodium caseinate, sodium azide (NaN3), and polyoxyethylene sorbitan monolaurate 20 (Tween 20) were purchased from Fujifilm-Wako Co., Ltd. Phosphate-buffered saline (PBS) was purchased from Nissui Co., Ltd. (Japan), polyester disinfectant cotton was purchased from Nippon Cotton Swabs Co., Ltd. (Japan), filter spin columns were purchased from Notgen Biotech, Inc. Nunc-immune module, F8 Maxisorp, was purchased from ThermoFisher Scientific, Inc. Streptavidin-HRP conjugate (SA-HRP) was purchased from Invitrogen, and tetramethylbenzidine (TMB) was purchased from Sigma-Aldrich. Poly(I:C) (HMW vaccine grade) was purchased from InvivoGen, recombinant SARSCoV-2 spike protein receptor-binding domain (RBD) from Creative Diagnostics, and ELAST ELISA enhancement system from PerkinElmer. Biotin-labeled (BT)-monkey IgA antibody was purchased from Mabtech; BT-monkey IgA (α chain) antibody was purchased from Merck; HRP-human IgG antibody was purchased from EY Laboratories; and BT IgE antibody was purchased from Bio-Rad Laboratories.

[0031] 1.2.Animals Nine cynomolgus monkeys (male and female; aged 12.1-20.6 years) were used. The nine cynomolgus monkeys were divided into three groups, each consisting of three monkeys: a control group (mP01-03), a low-dose group (mP04-06), and a high-dose group (mP07-09).

[0032] 1.3. Vaccination and Sampling Before vaccination, the monkeys were given an anesthetic injection of a mixture of meditomedin and ketamine to induce sleep. The monkeys were then treated with cotton soaked in 1% NAC for 5 minutes to break down the mucin layer on the sublingual surface, and then thoroughly washed with PBS to remove the NAC. The sublingual surface was then gently wiped with dry cotton to remove moisture. 0.7 ml of the test solution containing only 400 μg of poly(I:C) adjuvant was administered to the sublingual site of each monkey in the control group using a pipette, and the solution was allowed to stand for 1 minute. 0.7 ml of the test solution containing 30 μg of RBD antigen and 400 μg of poly(I:C) adjuvant was administered sublingually to monkeys in the low dose group using a pipette and allowed to stand for 1 minute. 0.7 ml of the test solution containing 150 μg of RBD antigen and 400 μg of poly(I:C) adjuvant was administered sublingually to monkeys in the high dose group using a pipette and allowed to stand for 1 minute. The monkeys in each group were then injected with atipamezole to induce awakening. The vaccine composition was administered three times at 4-week intervals, with a booster dose administered 15 weeks after the third vaccination to collect samples for ELISA. Blood and nasal secretion samples were collected under anesthesia as described above. Plasma samples obtained by centrifugation of blood were used to measure RBD-specific IgA, IgG, IgG, and IgE antibodies. Nasal secretion samples were absorbed onto polystyrene fiber swabs, collected by centrifugation using a spin column, and used for ELISA measurement of RBD-specific secretory IgA.

[0033] ELISA A Nunc Immunomodule plate was coated with 5 μg of RBD antigen (100 μl), incubated at 37°C for 1 hour, and then left overnight at 4°C. The plate was washed with PBS containing 0.05% Tween 20, and then a blocking agent consisting of PBS containing 1% sodium caseinate and 0.02% NaN3 was added. The plate was incubated at 37°C for 1 hour and then left overnight at 4°C. Nasal secretion and plasma samples were diluted 100-500 times and used as ELISA samples. After removing the blocking agent, an equal volume of 1 M sodium chloride solution (final concentration 0.5 M) was added to 50 μl of the ELISA sample to prevent nonspecific reactions. After incubation at 37°C for 1 hour, the sample was removed and the plate was washed with PBS containing 0.05% Tween 20. Appropriately diluted detection antibodies (BT monkey IgA antibody, BT monkey IgA (alpha chain) antibody, HRP human IgG antibody, or BT IgE antibody) were then added and incubated at 37°C for 1 hour. After washing, the plate was subjected to sensitization using the ELAST System containing SA-HRP and biotinyl tyramide. This sensitization increased the ELISA sensitivity by 10-30 times. The sensitized plate was washed with PBS containing 0.05% Tween 20, and then diluted SA-HRP was added and incubated at 37°C for 1 hour. Color development was performed using TMB (3,3',5,5'-tetramethylbenzidine), and the reaction was stopped with sulfuric acid. Absorbance at 450 nm and 600 nm was measured using an iMark microplate reader (Bio-Rad Laboratories).

[0034] 2. result The results are shown in Figure 1. RBD-specific IgA antibodies were detected in the nasal secretions of both the low-dose (30 μg / animal) and high-dose (150 μg / animal) vaccine administration groups, demonstrating that the sublingual vaccine of the present invention induces the production of RBD antigen-specific secretory IgA through mucosal immunity in the nasal and oral cavities. Furthermore, IgG and IgA were detected in plasma, but IgE was not. This indicates that the sublingual vaccine composition of the present invention conferred humoral immunity against RBD without causing any side effects associated with an allergic response. Furthermore, even when the vaccine composition for sublingual administration according to the present invention was administered, no redness, edema, weight loss, loss of appetite, etc. were observed around the administration site.

[0035] Example 2 A sublingual vaccine consisting of SARSCoV2 RBD antigen and AddaS03 or poly(I:C) adjuvant will be tested in preclinical trials using the primate model, cynomolgus monkeys. 1. Materials and Methods 1.1. Reagents and antibodies The same as in Example 1 is used. AddaS03 is purchased from InvivoGen.

[0036] 1.2.Animals Thirteen cynomolgus monkeys were used and divided into three groups: a control group (B1: mP01-03), an AddaS03 adjuvant-administered group (B2: mP04-08), and a poly(I:C) adjuvant-administered group (B3: mP09-13) (Table 1). [Table 1]

[0037] 1.3. Vaccination and Sampling Before vaccination, the monkeys were given an anesthetic injection of a mixture of meditomedin and ketamine to put them to sleep. The monkeys were then treated with cotton soaked in 1.5% NAC for 5 minutes to break down the mucin layer on the sublingual surface, and then thoroughly rinsed with saline to remove the NAC. The sublingual surface was then gently wiped with dry cotton to remove moisture. 500 μl of PBS alone is administered to the sublingual site of each monkey in group B1 using a pipette, and allowed to stand for 5 minutes or more. 500 μl of the test solution containing 150 μg of RBD antigen and 200 μl of AddaS03 adjuvant is administered sublingually to the monkeys in group B2 using a pipette and allowed to stand for 5 minutes or more. 500 μl of a test solution containing 150 μg of RBD antigen and 400 μg of poly(I:C) adjuvant is administered sublingually to monkeys in group B3 using a pipette and allowed to stand for 5 minutes or more. Each group of monkeys is then injected with atipamezole to induce wakefulness. The above vaccine will be administered three times at four-week intervals, with a booster dose for collecting samples for ELISA measurement administered 15 weeks after the first vaccination. Blood samples, saliva samples, and nasal wash samples will be collected under anesthesia before and every two weeks after vaccine administration. If the vaccine administration date and collection date are the same, the samples will be collected before vaccine administration. Saliva samples will be collected by leaving a swab in the oral cavity for at least five minutes. Nasal wash samples will be collected by spraying 200 μL of PBS into each of the left and right nostrils with a sprayer (100 μL / 1 push), holding the spray horizontally, and then collecting the wash sample from the nostrils after a few minutes.

[0038] ELISA As in Example 1, RBD-specific IgA, IgG, and IgE antibodies in each sample are detected and measured.

[0039] 1.5. Blood cytokines The antiviral effect of the vaccine and inflammatory response will be evaluated by detecting and measuring IL-12, type I interferon (IFN), IFN-γ (type II IFN), and IL-17 in the collected blood samples (serum and plasma).

[0040] 1.6. NK activation measurement and safety evaluation index gene expression study The expression level of the Granzyme B (GzmB) gene, which is an indicator of NK cell (natural killer cell) activation, will be detected and measured in peripheral blood mononuclear cells (PBMC) from the collected blood sample. The expression levels of other genes related to pro-inflammatory response and IFN response will also be detected and measured as safety evaluation indicators.

[0041] 1.7.DNA microarray analysis using white blood cells (WBC) RNA is isolated from white blood cells in the collected blood samples and used for DNA microarray analysis.

[0042] 1.8. Blood CRP measurement and hematological and blood biochemistry tests CRP is detected and measured in collected blood samples. Hematological tests are performed to detect and calculate the red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), reticulocyte count (RET), white blood cell count (WBC), and differential white blood cell percentages (lymphocytes (LYMPH), neutrophils (NEUT), monocytes (MONO), eosinophils (EO), basophils (BASO)] in collected blood samples. Blood biochemistry tests detect and measure the amounts of AST, ALT, ALP, γ-GTP, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, phospholipids, triglycerides, total protein, albumin, albumin (A) / globulin (G) ratio, LDH, inorganic phosphorus, calcium, magnesium, sodium, potassium, and chlorine in the collected blood sample. [Industrial Applicability]

[0043] The vaccine composition or medicine for sublingual administration according to the present invention activates not only systemic immune responses but also mucosal immunity, and is therefore effective in preventing or treating viral infections from the upper respiratory tract mucosa.

Claims

1. A vaccine composition for sublingual administration to a subject, comprising an immunizing antigen, Poly(I:C) and N-acetylcysteine ​​(NAC).

2. A vaccine composition for sublingual administration to a subject, comprising an immunizing antigen and Poly(I:C), wherein the vaccine composition is administered after administering N-acetylcysteine ​​(NAC) sublingually to the subject.

3. A vaccine composition for sublingual administration to a subject, comprising an immunizing antigen, wherein the vaccine composition is administered sublingually in combination with Poly(I:C) after N-acetylcysteine ​​(NAC) has been administered sublingually to the subject.

4. The vaccine composition according to any one of claims 1 to 3, wherein the immunogen is derived from chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, or HIV.

5. The vaccine composition of any one of claims 1 to 3, wherein the immunizing antigen comprises a spike protein receptor-binding domain of SARS-CoV-2.

6. 3. The vaccine composition according to claim 1, wherein the weight ratio of the immunogen to Poly(I:C) contained therein is 10:400 to 400:

400.

7. The vaccine composition according to any one of claims 1 to 3, wherein the immunogen is contained in an amount of 10 to 400 µg.

8. The vaccine composition according to any one of claims 1 to 3, which is administered to a subject three times at four-week intervals.

9. Use of an immunizing antigen, Poly(I:C) and N-acetylcysteine ​​(NAC) in the manufacture of a pharmaceutical for sublingual administration to confer immunogenicity to the immunizing antigen in a subject.

10. Use of an immunizing antigen and Poly(I:C) in the manufacture of a medicament for sublingual administration to confer immunogenicity to an immunizing antigen in a subject, wherein the medicament is administered sublingually after administering N-acetylcysteine ​​(NAC) sublingually to the subject.

11. Use of an immunizing antigen in the manufacture of a medicament for sublingual administration to confer immunogenicity to the immunizing antigen in a subject, wherein the medicament is administered sublingually in combination with Poly(I:C) after N-acetylcysteine ​​(NAC) has been administered sublingually to the subject.

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