Vaccine composition for sublingual administration

Abstract

Provided are a vaccine composition suitable for sublingual administration, a vaccine production method, and a vaccine administration method. A vaccine composition for administration to the sublingual of a subject comprising an immunizing antigen and an adjuvant is provided.

Description

Technical Field

[0001] The present invention relates to vaccine compositions for sublingual administration. Background Art

[0002] The effect of vaccine is sometimes also affected by route of administration except being affected by the selection of immune antigen and adjuvant.For influenza virus, coronavirus and other viruses that infect epithelial cells of bronchus and lung, compared with IgG antibody in blood, it is more important to induce secretory IgA antibody of viral antigen specificity in upper respiratory tract mucosa (non-patent literature 1).Recently developed vaccine administered by nasal or oral route, it is different from blood IgG antibody / systemic immune response, can induce secretory IgA antibody / mucosal immune response (non-patent literature 2).On the other hand, practical sublingual administration protein vaccine almost has no progress.In primates including humans and monkeys, there is a large space in sublingual part, which is easier to carry out vaccine administration than nasal cavity, but the effect of sublingual vaccine has not been confirmed (non-patent literature 3 and 4).

[0003] Prior art literature

[0004] Non-patent literature

[0005] Non-patent document 1: A. Ainai, et al. Human immune responses elicited by anintranasal inactivated H5 influenza vaccine, Microbiology and Immunology. 2020; 64: 313-325.

[0006] Non-patent literature 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.

[0007] Non-patent document 3: RSVeazey, 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.

[0008] Non-patent document 4: Alan D.Curtis II, et al, A simultaneous oral and intramuscular prime / sublingual boost with a DNA / Modified Vaccinia Ankaraviral vector-based vaccine induces simian immunodeficiency virus-specific systemic and mucosal immune responses in juvenile rhesus macaques, J MedPrimatol. 2018 October; 47(5):288-297. Summary of the invention

[0009] Problem that the invention aims to solve

[0010] Provided are a vaccine composition suitable for sublingual administration, a vaccine production method, and a vaccine administration method.

[0011] Solutions for solving problems

[0012] The inventors of the present application have conducted intensive studies and, as a result, have newly discovered a vaccine composition suitable for upper respiratory tract mucosal administration, particularly sublingual administration.

[0013] The invention of this application includes the following [1] to "18C".

[0014] [1] A vaccine composition for sublingual administration to a subject, comprising an immunizing antigen and an adjuvant.

[0015] [2] The vaccine composition according to [1], further comprising a mucolytic agent.

[0016] [3] The vaccine composition according to [1], which is administered after the mucolytic agent is administered sublingually to the subject.

[0017] [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.

[0018] [5] The vaccine composition according to [4], wherein the reducing agent is cysteine ​​or a derivative thereof.

[0019] [6] The vaccine composition according to [5], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine.

[0020] [7] The vaccine composition according to [4], wherein the proteolytic enzyme is trypsin, papain or bromelain.

[0021] [8] The vaccine composition according to [4], wherein the protein denaturing agent is urea or guanidine hydrochloride.

[0022] [9] The vaccine composition according to [4], wherein the deoxyribonuclease is human DNaseI.

[0023]

[10] The vaccine composition according to [4], wherein the calcium chelator is bicarbonate or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0024]

[11] The vaccine composition according to [1], wherein the immune antigen is derived from varicella virus, measles virus, mumps virus, polio virus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus or HIV.

[0025]

[12] The vaccine composition according to [1], wherein the immune antigen comprises the receptor binding domain of the spike protein of SARS-CoV-2.

[0026]

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

[0027]

[14] The composition according to

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

[0028]

[15] The composition according to

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

[0029]

[16] The vaccine composition according to [1], 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.

[0030]

[17] The vaccine composition according to [1], comprising 10 to 400 μg, preferably 20 to 200 μg, and more preferably 30 to 150 μg of the above-mentioned immunizing antigen.

[0031]

[18] The vaccine composition according to [1], wherein the vaccine composition is administered to the subject three times at intervals of 4 weeks.

[0032] [1A] A method of conferring immunogenicity to an immunizing antigen in a subject,

[0033] The method comprises the steps of 1) administering an immunizing antigen and an adjuvant under the tongue of a subject.

[0034] [2A] The method according to [1A], wherein the immunizing antigen and adjuvant are administered sublingually together with a mucolytic agent.

[0035] [3A] The method according to [1A], wherein the immunizing antigen and adjuvant are administered after the mucolytic agent is administered sublingually to the subject.

[0036] [4A] The method according to [2A] or [3A], wherein the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelators.

[0037] [5A] The method according to [4A], wherein the reducing agent is cysteine ​​or a derivative thereof.

[0038] [6A] The method according to [5A], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine.

[0039] [7A] The method according to [4A], wherein the proteolytic enzyme is trypsin, papain or bromelain.

[0040] [8A] The method according to [4A], wherein the protein denaturing agent is urea or guanidine hydrochloride.

[0041] [9A] The method according to [4A], wherein the deoxyribonuclease is human DNaseI.

[0042] [10A] The method according to [4A], wherein the calcium chelating agent is bicarbonate or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0043] [11A] The method according to [1A], wherein the immune antigen is derived from a virus selected from varicella virus, measles virus, mumps virus, polio virus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus and HIV; here, it is preferred to prevent or treat infection by the above virus in the subject by administering the drug.

[0044] [12A] The method according to [1A], wherein the immune antigen comprises the receptor binding domain of the spike protein of SARS-CoV-2.

[0045] [13A] The method according to [1A], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene or an aluminum salt.

[0046] [14A] The method according to [13A], wherein the TLR ligand is double-stranded RNA (dsRNA).

[0047] [15A] The method according to [14A], wherein the dsRNA is Poly(I:C).

[0048] [16A] The method according to [1A], 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.

[0049] [17A] The method according to [1A], comprising 10 to 400 μg, preferably 20 to 200 μg, and more preferably 30 to 150 μg of the above-mentioned immunizing antigen.

[0050] [18A] The method according to [1A], wherein the immunizing antigen and adjuvant are administered to the subject three times at intervals of four weeks.

[0051] [1B] A combination of an immunizing antigen and an adjuvant for administration under the tongue of a subject to confer immunogenicity against the immunizing antigen.

[0052] [2B] The combination according to [1B], wherein the drug is administered sublingually together with a mucolytic agent.

[0053] [3B] The combination according to [1B], which is administered sublingually after the mucolytic agent is administered sublingually to the subject.

[0054] [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.

[0055] [5B] The combination according to [4B], wherein the reducing agent is cysteine ​​or a derivative thereof.

[0056] [6B] The combination according to [5B], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine.

[0057] [7B] The combination according to [4B], wherein the proteolytic enzyme is trypsin, papain or bromelain.

[0058] [8B] The combination according to [4B], wherein the protein denaturing agent is urea or guanidine hydrochloride.

[0059] [9B] The combination according to [4B], wherein the deoxyribonuclease is human DNaseI.

[0060] [10B] The combination according to [4B], wherein the calcium chelator is bicarbonate or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0061] [11B] A combination according to [1B], wherein the immune antigen is derived from a virus selected from varicella virus, measles virus, mumps virus, polio virus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus and HIV; preferably, the combination is used to prevent or treat infection by the above viruses.

[0062] [12B] The combination according to [1B], wherein the immune antigen comprises the spike protein receptor binding domain of SARS-CoV-2.

[0063] [13B] The combination according to [1B], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene or an aluminum salt.

[0064] [14B] The combination according to [13B], wherein the TLR ligand is double-stranded RNA (dsRNA).

[0065] [15B] The combination according to [14B], wherein the dsRNA is Poly(I:C).

[0066] [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.

[0067] [17B] The combination according to [1B], comprising 10 to 400 μg, preferably 20 to 200 μg, and more preferably 30 to 150 μg of the above-mentioned immunizing antigen.

[0068] [18B] The combination according to [1B], wherein the drug is administered to the subject three times at intervals of 4 weeks.

[0069] [1C] Use of an immunizing antigen and an adjuvant in the manufacture of a medicament for sublingual administration for conferring immunogenicity against the immunizing antigen to a subject.

[0070] [2C] The use according to [1C], wherein the drug comprises a mucolytic agent.

[0071] [3C] The use according to [1C], wherein the drug is administered after the mucolytic agent is administered sublingually to the subject.

[0072] [4C] The use according to [2C] or [3C], wherein the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelators.

[0073] [5C] The use according to [4C], wherein the reducing agent is cysteine ​​or a derivative thereof.

[0074] [6C] The use according to [5C], wherein the cysteine ​​derivative is N-acetylcysteine ​​(NAC) or carbocysteine.

[0075] [7C] The use according to [4C], wherein the proteolytic enzyme is trypsin, papain or bromelain.

[0076] [8C] The use according to [4C], wherein the protein denaturing agent is urea or guanidine hydrochloride.

[0077] [9C] The use according to [4C], wherein the deoxyribonuclease is human DNaseI.

[0078] [10C] The use according to [4C], wherein the calcium chelating agent is bicarbonate or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0079] [11C] The use according to [1C], wherein the immune antigen is derived from a virus selected from varicella virus, measles virus, mumps virus, polio virus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus and HIV; here, preferably, the drug is a drug for preventing or treating infection by the above virus.

[0080] [12C] The use according to [1C], wherein the immune antigen comprises the spike protein receptor binding domain of SARS-CoV-2.

[0081] [13C] The use according to [1C], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene or an aluminum salt.

[0082] [14C] The use according to [13C], wherein the TLR ligand is double-stranded RNA (dsRNA).

[0083] [15C] The use according to [14C], wherein the dsRNA is Poly(I:C).

[0084] [16C] The use according to [1C], wherein the weight ratio of the immune antigen to the adjuvant contained in the above-mentioned drug is 10:400 to 400:400, preferably 20:400 to 200:400, and more preferably 30:400 to 150:400.

[0085] [17C] The use according to [1C], wherein the drug contains 10 to 400 μg, preferably 20 to 200 μg, and more preferably 30 to 150 μg of the immune antigen.

[0086] [18C] The use according to [1C], wherein the drug is administered to the subject three times at intervals of 4 weeks.

[0087] Effects of the Invention

[0088] The vaccine composition or drug for sublingual administration of the present invention, and the method for administration thereof, activate not only systemic immune response but also mucosal immunity, and are also effective for the prevention or treatment of viral infection via the upper respiratory tract mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The results of preclinical trials in cynomolgus monkeys, a primate model, using a sublingual vaccine composed of SARS-CoV-2 RBD antigen and poly(I:C) adjuvant are shown. Figure 1 A: the amount of anti-RBDIgA in saliva; Figure 1 B: the amount of anti-RBD IgG in plasma; Figure 1 C: amount of anti-RBDIgA in plasma; Figure 1 D: The amount of anti-RBDIgE in plasma. DETAILED DESCRIPTION

[0090] In the present specification, “comprising” includes “essentially comprising”, “comprising an effective amount of” and “consisting of”.

[0091] Upper respiratory tract refers to the nasal cavity, paranasal sinuses, pharynx, and larynx in the respiratory tract from the nasal cavity to the lungs. Viruses and bacteria invaded from the outside world (mostly in the atmosphere) are attached to the upper respiratory tract and multiplied (infected), so that symptoms such as runny nose, nasal congestion, pharyngitis, and sore throat are called upper respiratory tract infection. It includes acute upper respiratory tract inflammation (i.e. cold syndrome), acute pharyngitis / tonsillitis, acute laryngitis, and acute epiglottitis. On the other hand, the virus infecting the upper respiratory tract is transferred from the trachea to the respiratory bronchioles and multiplied at these positions and the symptoms of bronchitis are called lower respiratory tract infection. In this manual, infection includes upper respiratory tract infection and lower respiratory tract infection.

[0092] One embodiment of the present invention is a vaccine composition or a drug comprising an immune antigen and an adjuvant. The vaccine composition or the drug is preferably administered sublingually. The subject of administration is not particularly limited, as long as it is a mammal with an immune response, preferably a primate, and more preferably a human.

[0093] In one embodiment of the present invention, the immune antigen (also referred to as antigen) is not particularly limited as long as it is antigenic, and includes not only peptide antigens (i.e., antigenic peptides), but also non-peptide antigens such as phospholipids and complex carbohydrates (e.g., bacterial membrane constituents, such as mycolic acid, lipoarabinomannan, etc.).

[0094] The immunizing antigen preferably includes a constituent molecule derived from a pathogen such as a virus.

[0095] The pathogens are not particularly limited, and examples thereof include protozoa (e.g., Plasmodium, Leishmania, and Trypanosoma), bacteria (e.g., Gram-positive cocci, Gram-positive bacilli, Gram-negative bacteria, and anaerobic bacteria), fungi (e.g., Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, and Sporothrix), viruses (e.g., varicella virus, measles virus, mumps virus, polio virus, rotavirus, adenovirus, herpes simplex virus, papillomavirus, respiratory syncytial virus, poxvirus, HIV, influenza virus, and coronaviruses such as SARS-CoV and SARS-CoV2), intracellular parasites (e.g., Chlamydiaceae, Mycoplasmaceae, Achoestrogensaceae, and Rickettsiaceae), helminths (e.g., nematodes, trematodes, and tapeworms), and the like. Among them, for viruses that infect the upper respiratory tract (e.g., varicella virus, measles virus, mumps virus, polio virus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, or HIV), not only humoral immunity but also mucosal immunity can be expected, and thus they are preferred in the present invention. Therefore, the present invention can be used to prevent and treat infections by infectious pathogens such as these protozoa, bacteria, fungi, viruses, intracellular parasites, worms, etc. (i.e., inhibit the proliferation of these infectious pathogens in the body).

[0096] As used in this specification, "antigenic peptide" is not particularly limited as long as it is a peptide consisting of more than 2 amino acids (including those consisting of more than 50 amino acids) that can be used as an antigen, and can be derived from nature, synthesis, or a commercially available product. The antigenic peptide can include the full-length amino sequence of a gene product or a partial amino acid sequence thereof. For example, antigenic peptides (including full-length sequences and partial sequences thereof) derived from infectious pathogens such as the above-mentioned protozoa, bacteria, fungi, viruses, intracellular parasites, worms, etc. can be cited. For example, the SARSCoV-2 spike protein receptor binding domain (RBD) belongs to the antigenic peptide.

[0097] The antigen peptide may be arbitrarily processed or modified (eg, phosphorylation or sugar chain modification).

[0098] In one embodiment of the present invention, an adjuvant refers to a substance used to enhance the effect (immunogenicity) of an immune antigen by administration together with the immune antigen. The adjuvant may include, without particular limitation, an adjuvant containing an aluminum salt (e.g., AS04), an adjuvant containing squalene (e.g., MF59, AS03, AddaS03), or an adjuvant containing a Toll-like receptor (TLR) ligand.

[0099] TLR ligands are not particularly limited, and examples thereof include triacyl lipoproteins (as TLR1 / 2 ligands); peptidoglycan, lipoarabinomannan, and porin (as TLR2 ligands); double-stranded RNA such as Poly (I:C) (as TLR3 ligands); lipopolysaccharide (as TLR4 ligands); flagellin (as TLR5 ligands); diacyl lipoproteins and zymosan (as TLR2 / 6 ligands); single-stranded RNA (as TLR7 / 8 ligands); CpG-DNA (as TLR9 ligands), etc. By binding these ligands to TLRs of immune cells, TLRs form homodimers or heterodimers to transmit signals into cells, activate immune cells (i.e., trigger immune responses), and thereby induce the production of inflammatory cytokines and type I interferons.

[0100] In one embodiment of the present invention, the vaccine composition or drug may contain 10 to 400 μg, preferably 20 to 200 μg, and more preferably 30 to 150 μg of the immunizing antigen per administration.

[0101] In one embodiment of the present invention, the weight ratio of the immunizing antigen to the adjuvant may be 10:400 to 400:400, preferably 20:400 to 200:400, and more preferably 30:400 to 150:400.

[0102] In one embodiment of the present invention, the vaccine composition or medicament may contain a mucolytic agent, and may be administered after or at the same time as the mucolytic agent.

[0103] Mucus covers the epithelial surfaces of the airways including the nose and mouth, digestive organs such as the stomach and intestines, the vagina, joints, eyes, etc., and forms a barrier to foreign matter and pathogens. Mucin, the main structural component present in mucus, is a macromolecule formed by sugar chain modification of apomucin composed of a protein backbone and a central region, wherein the protein backbone has a cysteine-rich region that is indispensable for participating in mucin-mucin interactions via disulfide bonds, and the central region includes longitudinal repeats rich in threonine, serine, and proline (hereinafter, the above barrier may be referred to as a mucin layer, mucus gel, or mucus layer).

[0104] Mucolytic agents refer to agents that reduce the viscosity of mucus gel. Although not particularly limited, mucolytic agents may include proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelators. Mucolytic agents can be used to increase the permeability of sublingual mucus gel, making it easier for immune antigens and adjuvants to be absorbed into the body from the sublingual region, thereby inducing a mucosal immune response and / or a systemic immune response.

[0105] Mucus is a complex network of cross-linked proteins, so the permeability of mucus gels can be improved by using proteolytic enzymes to break peptide bonds, cleave non-glycosylated mucin domains, and decompose the mucin protein backbone and / or proteins in mucus gels.

[0106] Examples of the proteolytic enzyme include trypsin, papain, and bromelain.

[0107] In the present invention, the proteolytic enzyme may include a glucosidase that can degrade the sugar chain modification of mucin. By degrading the sugar chain modification, the same effect as that of the proteolytic enzyme can be obtained.

[0108] Mucus also contains DNA, which is a giant molecule. Therefore, by decomposing the DNA in the mucus, the permeability of the mucus gel can be improved.

[0109] The deoxyribonuclease (DNA degrading enzyme) includes human DNaseI (enzyme number 3.1.21.1) or an active fragment thereof.

[0110] The permeability of mucus gel can be improved by using a reducing agent that reduces and cleaves the disulfide bonds between mucins in mucus.

[0111] Examples of the reducing agent include cysteine ​​and its modified form. Examples of the modified form of cysteine ​​include N-acetylcysteine ​​(NAC) and S-carboxymethylcysteine ​​(carbocysteine).

[0112] The permeability of mucus gel can be improved by using protein denaturants that disrupt the hydrogen bonds between and within mucin molecules in the mucus.

[0113] Examples of the protein denaturing agent include urea and guanidine hydrochloride.

[0114] By using calcium chelators to hydrate, swell and disperse the mucin layer in the mucus, its permeability can be improved.

[0115] Examples of the calcium chelating agent include bicarbonate and ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0116] In one embodiment of the present invention, the vaccine composition is preferably administered to a subject at least three times. The administration interval is preferably 3 to 5 weeks, preferably 4 weeks.

[0117] Furthermore, the drug may be administered at least once more in order to induce an immune response.

[0118] In one embodiment of the invention, the vaccine composition or drug may further include a pharmaceutically acceptable carrier; an antioxidant; a preservative; a colorant; a diluent; an emulsifier; a suspending agent; a solvent; a filler; an extender; a buffer; a delivery vehicle; a diluent; an excipient, etc.

[0119] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[0120] Example

[0121] Example 1

[0122] Preclinical trials and verification were carried out on a sublingual vaccine consisting of SARSCoV2 RBD antigen and poly(I:C) adjuvant using the primate model, cynomolgus monkey.

[0123] 1. Materials and Methods

[0124] 1.1. Reagents and antibodies

[0125] N-acetylcysteine ​​(NAC), bovine serum albumin (BSA), caseinate sodium, sodium azide (NaN 3 ) and polyoxyethylene sorbitan monolaurate 20 (Tween 20) were purchased and used from Fujifilm Wako Co., Ltd. Phosphate buffered saline (PBS) was purchased and used from NISSUI Co., Ltd. (Japan); polyester sterile cotton was purchased and used from Nippon Cotton Swab Co., Ltd. (Japan); filtration spin column was purchased and used from Notgen Biotech Co., Ltd.; Nunc-immune module, F8 Maxisorp was purchased and used from ThermoFisherScientific Co., Ltd.; streptavidin-HRP conjugate (SA-HRP) was purchased and used from Invitrogen Co., Ltd.; Tetramethylbenzidine (TMB) was purchased and used from Sigma-Aldrich Co., Ltd.

[0126] Poly(I:C) (HMW vaccine grade) was purchased and used from InvivoGen, and the recombinant SARSCoV-2 spike protein receptor binding domain (RBD) was purchased and used from Creative Diagnostics; in addition, the ELAST ELISA sensitization system was purchased and used from PerkinElmer.

[0127] Biotin-labeled (BT)-monkey IgA antibody was purchased from Mabtech and used; BT-monkey IgA (α chain) antibody was purchased from Merck and used; HRP-human IgG antibody was purchased from EY Laboratories and used; in addition, BTIgE antibody was purchased from Bio-Rad Laboratories and used.

[0128] 1.2. Animals

[0129] Nine cynomolgus monkeys (male and female; 12.1-20.6 years old) were used and divided into three groups: a control group (mP01-03), a low-dose group (mP04-06), and a high-dose group (mP07-09), each consisting of three monkeys.

[0130] 1.3. Vaccine administration and sampling

[0131] Before vaccine administration, the monkeys were injected with a mixture of medetomidine and ketamine as an anesthetic to induce sleep, and then treated with a cotton ball soaked in 1% NAC for 5 minutes to destroy the mucin layer on the lower surface of the tongue, followed by thorough washing with PBS to remove NAC. To remove moisture from the lower surface of the tongue, the monkeys were gently wiped with a dry cotton ball.

[0132] 0.7 ml of the test solution containing 400 μg of poly(I:C) adjuvant alone was administered to the sublingual area of ​​monkeys in the control group (Control) by pipette, and the mixture was allowed to stand for 1 minute.

[0133] 0.7 ml of a test solution containing 30 μg of RBD antigen and 400 μg of poly(I:C) adjuvant was administered to the sublingual area of ​​monkeys in the low-dose group using a pipette and allowed to stand for 1 minute.

[0134] 0.7 ml of a test solution containing 150 μg of RBD antigen and 400 μg of poly(I:C) adjuvant was administered to the sublingual area of ​​monkeys in the high-dose group (High Dose) by pipette, and the mixture was allowed to stand for 1 minute.

[0135] Afterwards, each group of monkeys was injected with atipamezole to promote wakefulness.

[0136] The above-mentioned vaccine composition was administered three times at intervals of 4 weeks, and the booster administration for collecting samples for ELISA determination was performed 15 weeks after the third vaccine. Blood collection and nasal mucus collection were performed under the above-mentioned anesthesia. The plasma sample obtained by centrifugation of the blood was used for RBD-specific IgA, IgG, IgG and IgE antibody determination. In addition, the nasal mucus sample was absorbed into a polystyrene fiber swab and collected, and recovered by centrifugation using a spin column for ELISA determination of RBD-specific secretory IgA.

[0137] 1.4.ELISA

[0138] 5 μg of RBD antigen (100 μl) was coated on the NUNC immune module plate, incubated at 37°C for 1 hour and then left to stand at 4°C overnight. The plate was washed with PBS containing 0.05% Tween 20 and then added with 1% sodium caseinate, 0.02% NaN 3 The cells were blocked with PBS and incubated at 37°C for 1 hour and then incubated at 4°C overnight.

[0139] Nasal mucus and plasma samples were diluted 100 to 500 times as ELISA samples. After removing the blocking agent, an equal amount of 1M sodium chloride aqueous solution (final concentration 0.5M) was added to 50μl of ELISA sample to prevent nonspecific reactions. After incubation at 37°C for 1 hour, the above samples were removed and the plate was washed with PBS containing 0.05% Tween20. Then, appropriately diluted detection antibodies (BT monkey IgA antibody, BT monkey IgA (alpha chain) antibody, HRP human IgG antibody or BT IgE antibody) were added and incubated at 37°C for 1 hour. After washing the plate, sensitization was performed using the ELAST System containing SA-HRP and biotinyl tyrosinamide. Through this sensitization, the sensitivity of ELISA is increased by 10 to 30 times.

[0140] After washing the sensitized plate with PBS containing 0.05% Tween20, diluted SA-HRP was added and incubated at 37°C for 1 hour. TMB (3,3',5,5'-tetramethylbenzidine) was used for color development, and sulfuric acid was used to stop the reaction. The absorption at 450nm and 600nm was measured using an iMark microplate reader (Bio-rad laboratories).

[0141] 2. result

[0142] The results are shown in Figure 1 . RBD-specific IgA antibodies were confirmed in the nasal mucus of the two vaccine administration groups, the low-dose (30μg / 1) and the high-dose (150μg / 1), indicating that the sublingual vaccine of the present invention induced the production of RBD antigen-specific secretory IgA through nasal and oral mucosal immunization. In addition, IgG and IgA were detected in the plasma, but IgE was not detected. This shows that humoral immunity against RBD was obtained using the sublingual vaccine composition of the present invention, but there was no side effect associated with an allergic response.

[0143] Furthermore, even after administration of the vaccine composition for sublingual administration of the present invention, no redness, edema, weight loss, loss of appetite, etc. were observed around the administration site.

[0144] Example 2

[0145] For the sublingual vaccine composed of SARSCoV2RBD antigen and AddaS03 adjuvant or poly(I:C) adjuvant, preclinical trials and verification were carried out using the primate model - crab-eating macaque.

[0146] 1. Materials and Methods

[0147] 1.1. Reagents and antibodies

[0148] The same reagents and antibodies as in Example 1 were used.

[0149] AddaS03 was purchased from InvivoGen.

[0150] 1.2. Animals

[0151] 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).

[0152] [Table 1]

[0153]

[0154] 1.3. Vaccine administration and sampling

[0155] Before the vaccine administration, the monkeys were injected with a mixture of medetomidine and ketamine as an anesthetic to induce sleep, and then treated with a cotton ball soaked in 1.5% NAC for 5 minutes to destroy the mucin layer on the lower surface of the tongue. The monkeys were then washed thoroughly with saline to remove the NAC. To remove moisture from the lower surface of the tongue, they were gently wiped with a dry cotton ball.

[0156] 500 μl of PBS was administered to each monkey in the sublingual area of ​​the monkeys in group B1 using a pipette and allowed to stand for more than 5 minutes.

[0157] 500 μl of a test solution containing 150 μg of RBD antigen and 200 μl of AddaS03 adjuvant was administered to the sublingual area of ​​the monkeys in group B2 using a pipette and allowed to stand for more than 5 minutes.

[0158] To the sublingual site of the monkeys in group B3, 500 μl of a test solution containing 150 μg of RBD antigen and 400 μg of poly(I:C) adjuvant was administered by pipette and allowed to stand for more than 5 minutes.

[0159] Afterwards, each group of monkeys was injected with atipamezole to promote wakefulness.

[0160] The above-mentioned vaccine administration was carried out three times at intervals of 4 weeks, and booster administration for collecting samples for ELISA measurement was carried out 15 weeks after the first vaccination.

[0161] Blood and saliva / nasal wash fluid were collected under the above anesthesia before and after vaccine administration at intervals of 2 weeks. If the vaccine administration day and the collection day were the same day, the collection was performed before vaccine administration. Saliva was collected by leaving the swab in the mouth for more than 5 minutes. For nasal wash fluid, 200 μL of PBS (100 μL / 1 push) was sprayed into the left and right nasal cavities respectively, kept horizontal, and the wash fluid was recovered from the nostrils after a few minutes.

[0162] 1.4.ELISA

[0163] In the same manner as in Example 1, the RBD-specific IgA, IgG, and IgE antibodies in each sample were detected and measured.

[0164] 1.5. Cytokines in blood

[0165] IL-12, type I interferon (IFN), IFN-γ (type II IFN), and IL-17 in the collected blood samples (serum and plasma) were detected and measured to evaluate the antiviral effect of the vaccine and the inflammatory response.

[0166] 1.6. NK activation assay and safety evaluation index gene expression study

[0167] The expression level of GranzymeB (GzmB), an activation index of NK cells (natural killer cells), in peripheral blood mononuclear cells (PBMC) in the collected blood samples was detected and measured. As other safety evaluation index genes, the expression levels of genes related to inflammation-promoting response and IFN response were detected and measured.

[0168] 1.7. DNA microarray analysis using white blood cells (WBC)

[0169] RNA was isolated from leukocytes of the collected blood samples and used for DNA microarray analysis.

[0170] 1.8. Blood CRP measurement, hematological examination, and blood biochemistry examination

[0171] CRP in the collected blood sample is detected and measured. As a hematological test, the red blood cell count (RBC), hemoglobin content (HGB), hematocrit value (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), reticulocyte rate (RET), white blood cell count (WBC), and white blood cell type percentage (DIFFERENTIAL WHITE BLOOD CELL PERCENTAGES) [lymphocyte (LYMPH), neutrophil (NEUT), monocyte (MONO), eosinophil (EO), basophil (BASO)] ratio in the collected blood sample are detected and calculated.

[0172] As a blood biochemistry test, the amount of AST, ALT, ALP, γ-GTP, total bilirubin, urea nitrogen, creatinine, glucose, total cholesterol, phospholipids, neutral fat, total protein, albumin, albumin (A) / globulin (G) ratio, LDH, inorganic phosphorus, calcium, magnesium, sodium, potassium and chloride in the collected blood sample are detected and measured.

[0173] Industrial Applicability

[0174] The vaccine composition or drug for sublingual administration of the present invention can activate not only systemic immune response but also mucosal immunity, and is also effective for preventing or treating viral infection via the upper respiratory tract mucosa.

Claims

1. A vaccine composition for sublingual administration to a subject, comprising an immunizing antigen and an adjuvant.

2. The vaccine composition according to claim 1, in, Also contains a mucolytic agent.

3. The vaccine composition according to claim 1, which is administered after administering a mucolytic agent sublingually to a subject.

4. The vaccine composition according to claim 2 or 3, in, 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 claim 4, in, The reducing agent is cysteine ​​or a cysteine ​​derivative.

6. The vaccine composition according to claim 5, in, The derivatives of cysteine ​​are N-acetylcysteine ​​(NAC) or carbocysteine.

7. The vaccine composition according to claim 4, in, The proteolytic enzyme is trypsin, papain or bromelain.

8. The vaccine composition according to claim 4, in, The protein denaturant is urea or guanidine hydrochloride.

9. The vaccine composition according to claim 4, in, The deoxyribonuclease is human DNaseI.

10. The vaccine composition according to claim 4, in, The calcium chelator is bicarbonate or ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

11. The vaccine composition according to claim 1, in, The immune antigen is derived from varicella virus, measles virus, mumps virus, polio virus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus or HIV.

12. The vaccine composition according to claim 1, in, The immune antigen comprises the spike protein receptor binding domain of SARS-CoV-2.

13. The vaccine composition according to claim 1, in, Adjuvants include Toll-like receptor (TLR) ligands, squalene or aluminum salts.

14. The composition according to claim 13, in, TLR ligands are double-stranded RNA (dsRNA).

15. The composition according to claim 14, in, The dsRNA is Poly(I:C).

16. The vaccine composition according to claim 1, in, The weight ratio of the immune antigen to the adjuvant is 10:400 to 400:

400.

17. The vaccine composition according to claim 1, in, Contains 10 to 400 μg of the immune antigen.

18. The vaccine composition according to claim 1, in, The subjects were administered 3 times at 4-week intervals.

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