Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
  • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20071—Demonstrated in vivo effect
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a vaccine composition for sublingual administration.
• Non-patent research In addition to the selection of the immunizing antigen and adjuvant, the effectiveness of a vaccine may be affected by the route of administration. For viruses such as influenza viruses and coronaviruses that infect epithelial cells of the bronchi and lungs, it is more important to induce secreted IgA antibodies specific for virus antigens in the upper respiratory mucosa than IgG antibodies in the blood (non-patent research). Reference 1). Recently, vaccine administration through the nasal or oral route has been developed, which induces a secreted IgA antibody/mucosal immune response, unlike a blood IgG antibody/systemic immune response (Non-Patent Document 2). On the other hand, little progress has been made regarding practical protein vaccines for sublingual administration. In primates, including humans and monkeys, the sublingual region has a large space and is easier to administer vaccines than the nasal cavity, but the effectiveness of vaccines administered sublingually has not been confirmed (Non-Patent Documents 3 and 4).
• a vaccine composition suitable for sublingual administration, a vaccine production method, and a vaccine administration method are provided.
• the present invention includes the following [1] to "18C".
• a vaccine composition for sublingual administration to a subject comprising an immunizing antigen and an adjuvant.
• the vaccine composition according to [1] further comprising a mucolytic agent.
• EGTA ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid
• the immunizing antigen is derived from varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, or HIV.
• the vaccine composition according to [1], wherein the immunizing antigen comprises the spike protein receptor binding domain of SARS-CoV-2.
• the vaccine composition according to [1], wherein the adjuvant comprises a Toll-like receptor (TLR) ligand, squalene or an aluminum salt.
• TLR Toll-like receptor
• dsRNA double-stranded RNA
• the composition according to [14], wherein the dsRNA is Poly(I:C).
• the vaccine composition according to [1], wherein the weight ratio of the immunizing antigen and adjuvant contained is 10:400 to 400:400, preferably 20:400 to 200:400, more preferably 30:400 to 150:400. thing.
• [1A] A method for imparting immunogenicity to an immunizing antigen in a subject, the method comprising: 1) sublingually administering the immunizing antigen and an adjuvant to the 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 immunizing antigen and adjuvant are administered after administering the mucolytic agent sublingually to the subject.
• the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelating agents.
• [5A] The method according to [4A], wherein the reducing agent is cysteine or a derivative thereof.
• the cysteine derivative is N-acetylcysteine (NAC) or carbocysteine.
• NAC N-acetylcysteine
• the proteolytic enzyme is trypsin, papain or bromelain.
• the protein denaturant 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).
• the immunizing antigen is derived from a virus selected from varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, and HIV.
• the administration prevents or treats infection of the virus in the subject.
• the immunizing antigen comprises the spike protein receptor binding domain of SARS-CoV-2.
• the adjuvant comprises a Toll like receptor (TLR) ligand, squalene or an aluminum salt.
• TLR Toll like receptor
• dsRNA double-stranded RNA
• the dsRNA is Poly(I:C).
• [16A] The method according to [1A], wherein the weight ratio of the immunizing antigen and 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 immunizing antigen is contained in an amount of 10 to 400 ⁇ g, preferably 20 to 200 ⁇ g, more preferably 30 to 150 ⁇ g.
• [18A] The method according to [1A], wherein the immunizing antigen and adjuvant are administered to the subject three times at four-week intervals.
• [1B] A combination of an immunizing antigen and an adjuvant for use in a subject for sublingual administration to confer immunogenicity to the immunizing antigen.
• [2B] The combination according to [1B], which is administered sublingually together with a mucolytic agent.
• [3B] The combination according to [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 chelating agents.
• [5B] The combination according to [4B], wherein the reducing agent is cysteine or a derivative thereof.
• the cysteine derivative is N-acetylcysteine (NAC) or carbocysteine.
• NAC N-acetylcysteine
• the protease is trypsin, papain or bromelain.
• the protein denaturant 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).
• the immunizing antigen is derived from a virus selected from varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, and HIV. combination; here preferably a combination for use in the prevention or treatment of infection with said virus.
• the immunizing antigen comprises the spike protein receptor binding domain of SARS-CoV-2.
• the adjuvant comprises a Toll like receptor (TLR) ligand, squalene or an aluminum salt.
• TLR Toll like receptor
• dsRNA double-stranded RNA
• 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, more preferably 30:400 to 150:400.
• the combination according to [1B] which is administered to a subject three times at four-week intervals.
• [1C] Use of an immunizing antigen and an adjuvant in the manufacture of a medicament for sublingual administration to confer immunogenicity to an immunizing antigen in a subject.
• the medicament includes a mucolytic agent.
• the mucolytic agent is selected from the group consisting of proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents and calcium chelators.
• 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.
• NAC N-acetylcysteine
• the proteolytic enzyme is trypsin, papain or bromelain.
• the protein denaturing agent is urea or guanidinium hydrochloride.
• 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).
• the immunizing antigen is derived from a virus selected from varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, and HIV.
• the medicament is a medicament for preventing or treating infection with the virus.
• the adjuvant comprises a Toll like receptor (TLR) ligand, squalene or an aluminum salt.
• TLR Toll like receptor
• dsRNA double-stranded RNA
• the dsRNA is Poly(I:C).
• the weight ratio of the immunizing antigen and adjuvant contained in the pharmaceutical is 10:400 to 400:400, preferably 20:400 to 200:400, more preferably 30:400 to 150:400.
• the medicament contains 10 to 400 ⁇ g, preferably 20 to 200 ⁇ g, more preferably 30 to 150 ⁇ g of the immunogenic antigen.
• the use according to [1C] wherein the medicament is administered to the subject three times at four-week intervals.
• the vaccine composition or medicament for sublingual administration according to the present invention and its administration method activate not only systemic immune responses but also mucosal immunity, and are effective in preventing or treating viral infections from the upper respiratory tract mucosa. be.
• FIG. 1A Amount of anti-RBDIgA in saliva
• FIG. 1B Amount of anti-RBDIgG in plasma
• FIG. 1C Amount of anti-RBDIgA in plasma
• FIG. 1D Amount of anti-RBDIgE in plasma.
• composition includes “essentially comprising”, “comprising an effective amount of”, and “consisting of”.
• the upper respiratory tract refers to the airway that extends from the nasal cavity to the lungs, including the nasal cavity, sinuses, pharynx, and larynx.
• An upper respiratory tract infection is when viruses and bacteria that have invaded from the outside world (mostly from the atmosphere) attach to the upper respiratory tract and multiply (infect), resulting in symptoms such as nasal discharge, nasal congestion, and sore throat.
• Infection includes upper respiratory tract infections and lower respiratory tract infections.
• One embodiment of the present invention is a vaccine composition or medicament comprising an immunizing antigen and an adjuvant.
• the vaccine composition or medicament is administered sublingually.
• the subject to be administered is not particularly limited as long as it is a mammal that has an immune response, but primates are preferred, and humans are more preferred.
• the immunizing antigen (also simply referred to as antigen) is not particularly limited as long as it can have antigenicity, and includes not only peptidic antigens (i.e., antigenic peptides), Non-peptidic antigens such as phospholipids and complex carbohydrates (eg, bacterial membrane components such as mycolic acids and lipoarabinoannan) are also included.
• the immunizing antigen includes constituent molecules derived from a pathogen such as a virus.
• pathogens include protozoa (e.g., Plasmodium spp., Leishmania spp., Trypanosoma spp.), bacteria (e.g., gram-positive cocci, gram-positive rods, gram-negative bacteria, anaerobes), 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 syncytiaviruses, poxviruses, HIV, influenza viruses, coronaviruses such as SARS-CoV and SARS-CoV2), intracellular parasites (e.g. Chlamydiaceae, Mycoplasmadaceae, Acholeplasmaceae, Rickettsiaceae), helminths (e.g. , nematodes, trematodes, tapeworms), etc.
• viruses that infect the upper respiratory tract e.g.
• varicella virus varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, rubella virus, SARS virus, or HIV
• infectious agents such as protozoa, bacteria, fungi, viruses, intracellular parasites, and helminths (i.e., to suppress the proliferation of these infectious agents in the body). can.
• the "antigen peptide” used herein is not particularly limited as long as it is a peptide composed of two or more amino acids (including one composed of more than 50 amino acids) that can serve as an antigen, and is a naturally occurring peptide. It may be derived from natural sources, synthetically derived, or commercially available.
• the antigenic peptide may include the full-length amino acid sequence of the gene product or a partial amino acid sequence thereof. Examples include antigenic peptides (including full-length sequences and partial sequences thereof) derived from infectious agents such as the protozoa, bacteria, fungi, viruses, intracellular parasites, and helminths.
• infectious agents such as the protozoa, bacteria, fungi, viruses, intracellular parasites, and helminths.
• the SARS CoV-2 spike protein receptor binding domain (RBD) is an antigenic peptide.
• the antigenic peptide may be subjected to any processing or modification (for example, phosphorylation or sugar chain modification).
• an adjuvant refers to a substance used to be administered together with an immunizing antigen to increase its efficacy (immunogenicity).
• Adjuvants include, but are not limited to, those containing aluminum salts (eg, AS04), squalene (eg, MF59, AS03, AddaS03), or Toll-like receptor (TLR) ligands.
• TLR ligands include, but are not limited to, triacyl lipoproteins (as TLR1/2 ligands); peptidoglycan, lipoarabinomannan, porin (as TLR2 ligands); double-stranded RNAs such as Poly(I:C) ( lipopolysaccharide (as TLR4 ligand); flagellin (as TLR5 ligand); diacyllipoprotein, zymosan (as TLR2/6 ligand); single-stranded RNA (as TLR7/8 ligand); CpG-DNA (TLR9 (as a ligand), etc.
• TLR1/2 ligands triacyl lipoproteins
• peptidoglycan peptidoglycan
• lipoarabinomannan porin
• porin as TLR2 ligands
• double-stranded RNAs such as Poly(I:C) ( lipopolysaccharide (as TLR4 lig
• TLRs When these ligands bind to the TLRs of immune cells, the TLRs become homodimers or heterodimers and transmit signals into the cells, activating the immune cells (i.e., eliciting an immune response) and, as a result, producing inflammatory cytokines. production and production of type I interferon is induced.
• the vaccine composition or medicament may contain 10 to 400 ⁇ g, preferably 20 to 200 ⁇ g, and more preferably 30 to 150 ⁇ g of the immunizing antigen per dose.
• 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.
• the vaccine composition or medicament may include 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, the digestive organs such as the stomach and intestines, the vagina, joints, and eyes, and forms a barrier against foreign substances and pathogens.
• Mucin the main structural component present in mucus, contains a protein backbone with intermittent cysteine-rich regions that participate in mucin-mucin interactions via disulfide bonds and tandem repeats rich in threonine, serine, and proline.
• Mucolytic agents are agents that reduce the viscosity of mucus gels. Mucolytic agents include, but are not limited to, proteolytic enzymes, protein denaturants, deoxyribonucleases, reducing agents, and calcium chelating agents.
• a mucolytic agent increases the permeability of the mucus gel present under the tongue, making it easier for immune antigens and adjuvants to be absorbed into the body from under the tongue, and can induce a mucosal immune response and/or a systemic immune response.
• proteolytic enzymes are utilized to break peptide bonds and cleave non-glycosylated mucin domains to degrade the mucin protein backbone and/or proteins in the mucus gel. , can increase the permeability of mucus gel.
• proteolytic enzymes include trypsin, papain or bromelain.
• the protease may include a glycosisase that decomposes sugar chain modification of mucin. By decomposing sugar chain modifications, the same effects as proteolytic enzymes can be obtained.
• mucus also contains DNA, which is a macromolecule
• the permeability of mucus gel can be increased by decomposing the DNA in mucus.
• deoxyribonucleases include human DNase I (enzyme number 3.1.21.1) or an active fragment thereof.
• the permeability of mucus gels can be increased by reducing agents that reduce and cleave disulfide bonds between mucins in mucus.
• reducing agents include cysteine or modified forms thereof.
• Modified forms of cysteine include N-acetylcysteine (NAC), S-carboxymethylcysteine (carbocysteine), and the like.
• the permeability of mucus gels can be increased by protein denaturants that disrupt inter- and intramolecular hydrogen bonds in mucin molecules.
• protein denaturants include urea and guanidinium hydrochloride.
• Such calcium chelators include bicarbonate and ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).
• EGTA ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid
• the vaccine composition is administered to a subject at least three times.
• the administration interval is preferably 3 to 5 weeks, preferably 4 weeks.
• at least one additional dose may be administered to elicit an immune response.
• the vaccine composition or medicament further comprises a pharmaceutically acceptable carrier; an antioxidant; a preservative; a coloring agent; a diluent; an emulsifier; a suspending agent; a solvent; a filler; ; buffering agents; delivery vehicles; diluents; excipients, and the like.
• Example 1 A preclinical test was conducted and verified in a primate model, cynomolgus monkey, for a sublingual vaccine composed of the SARS CoV2 RBD antigen and a poly(I:C) adjuvant.
• N-acetylcysteine (NAC), bovine serum albumin (BSA), sodium caseinate, sodium azide (NaN 3 ), and polyoxyethylene sorbitan monolaurate 20 (Tween 20) were products of Fuji Film-Wako Co., Ltd. Purchased and used.
• Phosphate buffered saline (PBS) was from Nissui Co., Ltd. (Japan); polyester sterile cotton was from Nippon Cotton Swab Co., Ltd.
• Biotin-labeled (BT)-monkey IgA antibody was from Mabtech; BT-monkey IgA ( ⁇ chain) antibody was from Merck; HRP-human IgG antibody was from EY Laboratories; and BT IgE antibody was from Bio-Rad Laboratories. Each was purchased and used.
• Vaccine administration and sampling Before vaccine administration, a mixture of meditomedine and ketamine was injected as an anesthetic to put the monkey to sleep, and then the monkey was injected with cotton soaked in 1% NAC for 5 minutes to break down the mucin layer on the sublingual surface. and then washed extensively with PBS to remove NAC. The sublingual surface was gently wiped with dry cotton to remove moisture. 0.7 ml of the test solution containing only 400 ⁇ g poly(I:C) adjuvant was administered to the sublingual site of each monkey in the control group (Control) using a pipette, and the test solution was allowed to stand for 1 minute.
• 0.7 ml of the test solution containing 30 ⁇ g RBD antigen and 400 ⁇ g poly(I:C) adjuvant was administered to the sublingual site of the monkeys in the low dose group (Low Dose) using a pipette, and the test solution was allowed to stand for 1 minute.
• 0.7 ml of the test solution containing 150 ⁇ g RBD antigen and 400 ⁇ g poly(I:C) adjuvant was administered to the sublingual site of the monkeys in the high dose group (High Dose) using a pipette, and the test solution was allowed to stand for 1 minute.
• the monkeys in each group were then injected with atipamezole to induce awakening.
• the above vaccine composition was administered three times at four-week intervals, and a booster administration for sample collection for ELISA measurement was performed 15 weeks after the third vaccination.
• Blood and nasal secretion were collected under anesthesia as described above. Plasma samples obtained by centrifuging the blood were subjected to RBD-specific IgA, IgG, IgG, and IgE antibody measurements.
• the nasal secretion sample was absorbed into a polystyrene fiber swab, collected by centrifugation using a spin column, and used for ELISA measurement of RBD-specific secreted IgA.
• ELISA A Nunc immunomodule plate was coated with 5 ⁇ g RBD antigen (100 ⁇ l), incubated at 37°C for 1 hour, and then left at 4°C overnight. After washing the plate with PBS containing 0.05% Tween20, a blocking agent of PBS containing 1% sodium caseinate and 0.02% NaN3 was added, and after incubating at 37°C for 1 hour, the plate was left standing at 4°C overnight. I placed it. Nasal secretion and plasma samples were diluted 100 to 500 times and used as ELISA samples. After removing the blocking agent, the same amount of 1 M sodium chloride aqueous solution was added to 50 ⁇ l of the ELISA sample (final concentration 0.5 M) to prevent non-specific reactions.
• Example 2 A sublingual vaccine composed of SARS CoV2 RBD antigen and AddaS03 adjuvant or poly(I:C) adjuvant will be tested in a preclinical primate model cynomolgus monkey. 1. Materials and Methods 1.1. Reagents and antibodies The same ones as in Example 1 are used. AddaS03 is purchased from InvivoGen.
• Vaccine administration and sampling Before vaccine administration, a mixture of meditomedine and ketamine was administered as an anesthetic to put the monkeys to sleep, and then a cotton ball soaked in 1.5% NAC was used to break down the mucin layer on the sublingual surface of the monkeys. The cells were treated for 5 minutes and then thoroughly washed with saline to remove NAC. Gently wipe the sublingual surface with dry cotton to remove moisture. Administer 500 ⁇ l of PBS per monkey to the sublingual site of Group B1 monkeys using a pipette, and leave it for at least 5 minutes.
• 500 ⁇ l of the test solution containing 150 ⁇ g RBD antigen and 200 ⁇ l AddaS03 adjuvant is administered to the sublingual site of the monkeys in group B2 using a pipette, and the test solution is allowed to stand for 5 minutes or more.
• 500 ⁇ l of the test solution containing 150 ⁇ g RBD antigen and 400 ⁇ g poly(I:C) adjuvant is administered to the sublingual site of group B3 monkeys using a pipette, and the test solution is allowed to stand for 5 minutes or more.
• Each group of monkeys was then injected with atipamezole to induce awakening.
• the above vaccine administration is carried out three times at 4-week intervals, and a booster administration for sample collection for ELISA measurement is carried out 15 weeks after the first vaccination.
• Blood, saliva, and nasal washes will be collected under anesthesia before and every two weeks after vaccine administration. If the vaccine administration date and sampling date are on the same day, samples should be collected before vaccine administration.
• Saliva is collected by leaving a swab in the oral cavity for at least 5 minutes.
• the antiviral effects of vaccines can be determined by detecting and measuring IL-12, type I interferon (IFN), IFN- ⁇ (type II IFN), and IL-17 in collected blood samples (serum/plasma). and evaluate the inflammatory response.
• IFN type I interferon
• IFN- ⁇ type II IFN
• IL-17 in collected blood samples (serum/plasma).
• GzmB GranzymeB
• DNA microarray analysis using white blood cells (WBC) RNA is isolated from leukocytes in the collected blood sample and used for DNA microarray analysis.
• WBC white blood cells
• 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 percentage by white blood cell type (DIFFERENTIAL WHITE BLOOD CELL PERCENTAGES) [lymphocytes (LYMPH), neutrophils (NEUT), monocytes (MONO), eosinophil (EO), basophil (BASO)] ratio is detected and calculated.
• RBC Red blood cell count
• HGB hemoglobin content
• HCT hematocrit value
• MCV mean corpuscular volume
• MH mean corpuscular hemoglobin content
• MHC mean corpuscular hemoglobin concentration
• PHT platelet count
• RET reticulocyte rate
• WBC white blood cell count
• Blood biochemical tests include AST amount, ALT amount, ALP amount, ⁇ -GTP amount, total bilirubin amount, urea nitrogen amount, creatinine amount, glucose amount, total cholesterol amount, phospholipid amount, The amount of neutral fat, total protein, albumin, albumin (A)/globulin (G) ratio, LDH amount, inorganic phosphorus amount, calcium amount, magnesium amount, sodium amount, potassium amount, and chlorine amount are detected and measured.
• the vaccine composition or medicament for sublingual administration according to the present invention activates not only systemic immune response but also mucosal immunity, and is effective in preventing or treating viral infections from the upper respiratory tract mucosa.

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