WO2025058011A1 - Composition for nasal administration - Google Patents

Abstract

The present invention addresses the problem of providing a technique for improving the antibody-inducing ability of a nasal vaccine, and a composition for nasal administration having improved antibody-inducing ability. The problem is solved by a composition for nasal administration containing: at least one substance selected from the group consisting of antigen polypeptides and polynucleotides including a coding sequence of an antigen polypeptide; and a sugar component that is a sugar and/or a sugar alcohol, wherein the content of the sugar component is more than 4.5% by mass/volume.

Description

Composition for nasal administration

The present invention relates to a composition for nasal administration, etc.

　Unlike the injectable type, the nasal vaccine induces IgA in the upper respiratory tract in addition to IgG in the blood, making it possible to protect against initial infection and, in the unlikely event that the virus migrates to the lower respiratory tract, prevent the disease from becoming severe. Furthermore, since it can also prevent person-to-person transmission of infection that accompanies upper respiratory tract infection, a nasal influenza vaccine (Flumist (registered trademark)) that uses an attenuated live virus and contains gelatin and other additives has been approved for manufacture and sale.

International Publication No. 2019/021305

In the course of their research, the inventors have focused on the problem that nasal vaccines have insufficient antibody induction capacity (particularly in the upper respiratory tract). This problem is more pronounced when a relatively small amount is administered intranasally to target only the upper respiratory tract (in general, nasal administration to humans is primarily intended for the upper respiratory tract) than when a relatively large amount is administered intranasally to target the entire respiratory tract, including the lungs (corresponding to nasal administration in a typical mouse model).

Recently, a global pandemic of SARS-coronavirus 2 (SARS-CoV-2) has occurred, and with no established treatment technology, it continues to cause severe damage in many areas, including healthcare and the economy.

The objective of the present invention is to provide a technique for improving the antibody-inducing ability of nasal vaccines and a composition for nasal administration with improved antibody-inducing ability.

As a result of intensive research, the inventors have discovered that sugar and/or sugar alcohol improves the antibody-inducing ability of nasal vaccines.

Conventionally, glycerol, a type of sugar and/or sugar alcohol, was intended to be used as a stabilizer, etc., and its content was relatively low in accordance with its action. Glycerol is only known to be incorporated into vaccines as a stabilizer, etc. (Patent Document 1), and it was completely unexpected that sugars and/or sugar alcohols containing glycerol would improve antibody induction ability. Based on the knowledge that sugars and/or sugar alcohols improve antibody induction ability, the present inventors have succeeded in effectively exerting antibody induction ability by setting the sugar and/or sugar alcohol content to a certain amount or more. Based on this knowledge, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

Item 1. A composition for nasal administration comprising at least one selected from the group consisting of a polynucleotide comprising a coding sequence for an antigenic polypeptide and an antigenic polypeptide, and a sugar component which is a sugar and/or a sugar alcohol, wherein the content of the sugar component is more than 4.5% by volume by mass.
Item 1A. A method for administering a composition comprising at least one selected from the group consisting of a polynucleotide comprising a coding sequence for an antigen polypeptide and an antigen polypeptide, and a sugar component which is a sugar and/or sugar alcohol, and the content of the sugar component is more than 4.5% by mass by volume, to a subject organism via the nasal administration.
Item 1B. A composition for use as a composition for nasal administration (preferably a vaccine composition for nasal administration, a composition for nasal administration for use in preventing the onset and/or aggravation of an infectious disease caused by a microorganism), comprising at least one selected from the group consisting of a polynucleotide comprising a coding sequence for an antigenic polypeptide and an antigenic polypeptide, and a sugar component which is a sugar and/or a sugar alcohol, wherein the content of the sugar component is more than 4.5% by mass/volume.
Item 1C: Use of a composition comprising at least one selected from the group consisting of a polynucleotide comprising a coding sequence for an antigenic polypeptide and an antigenic polypeptide, and a sugar component which is a sugar and/or a sugar alcohol, the content of which is more than 4.5% by mass, for the manufacture of a composition for nasal administration (preferably a vaccine composition for nasal administration, or a composition for nasal administration used for preventing the onset and/or aggravation of an infectious disease caused by a microorganism).
Item 1D. Use of a composition comprising at least one selected from the group consisting of a polynucleotide comprising a coding sequence for an antigen polypeptide and an antigen polypeptide, and a sugar component which is a sugar and/or a sugar alcohol, wherein the content of the sugar component is more than 4.5% by mass, as a composition for nasal administration (preferably a vaccine composition for nasal administration, or a composition for nasal administration used for preventing the onset and/or aggravation of an infectious disease caused by a microorganism).

Item 2. The composition for nasal administration according to Item 1, wherein the sugar component content is 7% by mass or more by volume.

Item 3. The composition for nasal administration according to Item 1, wherein the sugar component is a sugar and/or sugar alcohol having three or more hydroxyl groups.

Item 4. The composition for nasal administration according to Item 3, wherein the sugar component has 3 to 6 hydroxyl groups.

Item 5. The composition for nasal administration according to Item 1, which contains a virus particle containing the polynucleotide and/or the antigen polypeptide and an adjuvant.

Item 6. The composition for nasal administration according to Item 5, which contains the virus particles.

Item 7. The composition for nasal administration according to Item 6, wherein the virus particles are adenovirus particles.

Item 8. A composition for nasal administration according to item 1 for use in administration to the upper respiratory tract.

Item 9. The composition for nasal administration according to any one of items 1 to 8, which is a vaccine composition.

Item 10. The composition for nasal administration according to any one of Items 1 to 8, wherein the antigen polypeptide is an antigen protein derived from a microorganism or a partial polypeptide thereof.

Item 11. The composition for nasal administration described in Item 10 for use in preventing the onset and/or aggravation of an infectious disease caused by the microorganism.

Item 12. The composition for nasal administration according to Item 10, wherein the microorganism is at least one selected from the group consisting of viruses, bacteria, fungi, and parasites.

Item 13. The composition for nasal administration according to Item 1, wherein the sugar component is glycerol and the content of the sugar component is more than 5% by volume.

Item 14. An additive for use in the manufacture of a composition for nasal administration according to any one of Items 1 to 8, which contains a sugar component that is a sugar and/or a sugar alcohol.

Item 15. The additive according to Item 14, wherein the sugar component is glycerol and the content of the sugar component is more than 5% by volume.

The present invention provides a technique for improving the antibody-inducing ability of nasal vaccines and a composition for nasal administration with improved antibody-inducing ability.

The results of an analysis of the effect of glycerol on nasal administration of AdV-OVA are shown (Test Example 1). The objects to be measured are shown above the graph. The administered substance is shown on the horizontal axis (Naive indicates the value in control mice that received no administration treatment). Each group had n = 5, and the bars in the graph indicate the median value. *P < 0.05, ***P < 0.001, ****P < 0.0001. The dotted line indicates the limit of quantitation. The results of a comparison of the effects of intranasal administration of AdV-OVA (glycerol and CVP) are shown (Test Example 2). The objects to be measured are shown above the graph. The administered substances are shown on the horizontal axis. n = 3 for each group, and the bars in the graph indicate the median. *P < 0.05, **P < 0.01. The dotted line indicates the limit of quantitation. The results of a comparison of the effects of nasal administration of AdV-OVA (glycerol and various thickening agents) are shown (Test Example 3). % indicates (w/v) %. The objects measured are shown in the graph. The administered substance and its concentration are shown on the horizontal axis. n = 3 for each group, and the bars in the graph indicate the median. *P < 0.05, ***P < 0.001, ****P < 0.0001. The dotted line indicates the limit of quantitation. The results of an analysis of the effect of glycerol on intranasal administration of AdV-HA/AdV-S are shown (Test Example 4). The objects to be measured are shown above the graph. The administered substance is shown on the horizontal axis. n = 5 for each group, and the bars in the graph indicate the median. **P < 0.01, ****P < 0.0001. The dotted line indicates the limit of quantitation. The results of an analysis of the effect of glycerol on nasal administration of OVA are shown (Test Example 5). The objects to be measured are shown above the graph. The administered substance is shown on the horizontal axis. n = 5 for each group, and the bars in the graph indicate the median. **P < 0.01. The dotted line indicates the limit of quantitation. The results of an analysis of the mechanism of enhanced efficacy of AdV nasal vaccine are shown (Test Example 6). The objects to be measured are shown above the graph. The administered substance is shown on the horizontal axis. n = 3 for each group, and the bars in the graph indicate the median. *P < 0.05, **P < 0.01. The dotted line indicates the limit of quantitation. The results of an analysis of the effect of glycerol on nasal retention are shown (Test Example 7). The vertical axis indicates the amount of luciferase in the nasal wash. The horizontal axis indicates the time elapsed since administration. The administered substance is shown in the legend (Blank indicates a control without administration treatment = limit of quantitation). For the PBS-administered group, n = 8, and for the glycerol or CVP-administered groups, n = 4, and the plots show the average values. *P < 0.05, ****P < 0.0001 vs PBS. This shows the results of an analysis of the effect of glycerol on intramuscular injection of AdV-OVA (Test Example 8). The objects to be measured are shown above the graph. The administered substance is shown on the horizontal axis (Naive indicates the value in control mice that received no administration treatment). For each group, n=5, and the bars in the graph indicate the median value. The dotted line indicates the limit of quantitation. The results of the analysis of the effect of glycerol concentration are shown (Test Example 9). The objects to be measured are shown above the graph. The administered substances are shown on the horizontal axis. For each group, n = 5, and the columns show the median values. **P < 0.01, ***P < 0.001. The dotted lines indicate the limit of quantitation. The results of an analysis of the antibody-inducing ability of components other than glycerol are shown (Test Example 10). The objects to be measured are shown above the graph. The administered substance is shown on the horizontal axis (Naive indicates the value in control mice that received no administration treatment). For each group, n = 6 or 7, and columns indicate the median value. ***P < 0.001, ****P < 0.0001. The dotted line indicates the limit of quantitation. The results of the analysis of the mechanism of action are shown (Test Example 11). The subjects to be measured are shown on the left side of the graph. The administered substance is shown on the horizontal axis (Naive indicates the value in control mice that received no administration treatment). n = 4 for each group, and columns indicate the median value. **P < 0.01, ***P < 0.001.

In this specification, the expressions "contain" and "include" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

　When the upper and lower limits of a numerical range are separately described in this specification, any combination of any upper limit and any lower limit is also considered to be disclosed in this specification.

Unless otherwise noted in this specification, "mass volume %" refers to w/v % (mass to volume percentage).

In one aspect, the present invention relates to a composition for nasal administration (sometimes referred to as the "composition of the present invention" in this specification) that contains at least one selected from the group consisting of a polynucleotide including a coding sequence for an antigenic polypeptide and an antigenic polypeptide, and a sugar component that is a sugar and/or a sugar alcohol, and the content of the sugar component is more than 4.5% by mass/volume.

The composition of the present invention contains a sugar component which is a sugar and/or a sugar alcohol. This can improve the antibody induction ability when the composition of the present invention is administered intranasally.

The sugars are not particularly limited, but examples include monosaccharides, oligosaccharides, etc.

The monosaccharide is not particularly limited, and known monosaccharides can be used. Examples include heptose, hexose, pentose, tetraose, and triose. Examples of monosaccharides include aldohexoses, ketohexoses, and hexose derivatives such as glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, mannose, fructose, allose, talose, gulose, altrose, idose, psicose, sorbose, and tagatose, among which aldohexoses are preferred.

Oligosaccharides are sugars in which two or more monosaccharide molecules are linked together into one molecule by glycosidic bonds. The number of monosaccharide molecules constituting an oligosaccharide can be, for example, 2 to 10, preferably 2 to 5, more preferably 2 to 3, and even more preferably 2.

Sugar alcohols are components obtained by reducing the carbonyl group of monosaccharides or disaccharides that are aldoses or ketoses, and are not particularly limited in this respect. Examples of sugar alcohols include glycerol, erythritol, threitol, arabinitol, xylitol, ribitol, mannitol, iditol, galactitol, sorbitol, volemitol, maltitol, etc., and among these, sugar alcohols derived from monosaccharides are preferred.

The above sugar components can be one type alone or a combination of two or more types.

The sugar component is preferably a sugar and/or sugar alcohol having three or more hydroxyl groups. In this case, the number of hydroxyl groups is more preferably 3 to 6, and even more preferably 3 to 5, 3 to 4, or 3. In these preferred embodiments, the number of carbon atoms in the sugar component is preferably 3 to 10, more preferably 3 to 8, even more preferably 3 to 6, even more preferably 3 to 5, particularly preferably 3 to 4, and especially preferably 3.

Specific preferred examples of the sugar components include glycerol, glucose, mannitol, etc., with glycerol being particularly preferred.

The content of the sugar component in the composition of the present invention is more than 4.5% by volume. The content is preferably 4.6% by volume or more, 4.7% by volume or more, 4.8% by volume or more, 4.9% by volume or more, 5.0% by volume or more, more than 5.0% by volume, 5.1% by volume or more, 5.2% by volume or more, 5.3% by volume or more, 5.4% by volume or more, 5.5% by volume or more, 5.6% by volume or more, 5.7% by volume or more, 5.8% by volume or more, or 5.9% by volume or more. The content is more preferably 5% by volume or more, even more preferably 6% by volume or more, even more preferably 7% by volume or more, particularly preferably 8% by volume or more, and particularly preferably 9% by volume or more (alternatively, 9.1% by volume or more, 9.2% by volume or more, 9.3% by volume or more, 9.4% by volume or more, 9.5% by volume or more, 9.6% by volume or more, 9.7% by volume or more, 9.8% by volume or more, or 9.9% by volume or more). By making the content closer to 10% by volume (for example, by making it 7% by volume or more, 8% by volume or more, or 9% by volume or more), the antibody-inducing ability can be more effectively exerted. The upper limit of the content is not particularly limited and is, for example, 100% by volume or less, but from the viewpoint of antibody induction ability (particularly antibody induction ability in the upper respiratory tract), it is preferably 95% by volume or less, 90% by volume or less, 80% by volume or less, 70% by volume or less, 60% by volume or less, 50% by volume or less, 40% by volume or less, 30% by volume or less, 25% by volume or less, 20% by volume or less, or 15% by volume or less. In one aspect of the present invention, the lower limit of the content can be set even higher than the above-mentioned value, and can be, for example, more than 10% by volume, 11% by volume or more, 12% by volume or more, 13% by volume or more, 14% by volume or more, 15% by volume or more, 20% by volume or more, or 25% by volume or more.

An antigenic polypeptide is a polypeptide that is an antigen that can be recognized by the immune system of a mammal and induce an immune response (antibody induction) when it is introduced (directly or, for example, when expressed in a DNA vaccine or mRNA vaccine) into a mammal having an immune system, and is not particularly limited in that respect.

Examples of antigenic polypeptides include microbial antigenic proteins or partial polypeptides thereof, and cancer antigenic proteins or partial polypeptides thereof. In the present invention, the antigenic polypeptide is particularly preferably a microbial antigenic protein or partial polypeptide thereof. Note that "derived from X" (where "X" is any microorganism) refers to something that "X" has, and is preferably exposed on the surface of "X".

Microorganisms include, for example, viruses, bacteria, fungi, parasites, etc. These are infectious microorganisms in one embodiment of the present invention, and can also be pathogenic microorganisms in one embodiment of the present invention. Particularly preferred examples of the microorganism include viruses, and among these, viruses that cause respiratory tract (upper and lower respiratory tract) infections are preferred.

The virus is not particularly limited, but examples thereof include influenza virus (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, chickenpox/shingles virus, herpes simplex virus, mumps virus, arbovirus, respiratory syncytial virus, SARS coronavirus, MERS coronavirus, SARS coronavirus 2, hepatitis virus (e.g., hepatitis B virus, hepatitis C virus, etc.), yellow fever virus, human immunodeficiency virus, rabies virus, hantavirus, dengue virus, Nipah virus, lyssavirus, and other enveloped viruses (viruses with an envelope); adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, rhinovirus, and other non-enveloped viruses (viruses without an envelope). Among these, preferred are enveloped viruses, and more preferred are influenza virus, SARS coronavirus, SARS coronavirus 2, and other coronaviruses.

SARS-coronavirus 2 includes, but is not limited to, known strains such as the Wuhan strain, alpha strain, delta strain, lambda strain, and Omicron strain, as well as various unknown strains that may be discovered in the future. It also includes sublineages of the above strains.

Bacteria or fungi include, but are not limited to, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Clostridium faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella, Klebsiella pneumoniae, Koinebacterium, Streptococcus hemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma, Candida, Aspergillus, etc.

Parasites include, but are not limited to, Entamoeba histolytica, Plasmodium, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, etc.

Cancer antigen proteins include, but are not limited to, ERK1, ERK2, MART-1/Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal-associated antigen (CRC)-C017-1A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/C D3-zeta chain, CD20, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A 12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, GAGE-1, GAGE-2 , GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, colorectal adenoma These include APC protein, fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7, CD20, c-erbB-2, etc.

Target cancers are not particularly limited, and examples include leukemia (including chronic lymphocytic leukemia and acute lymphocytic leukemia), lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, malignant lymphoma, diffuse lymphoma, and follicular lymphoma), myeloma (including multiple myeloma), breast cancer, colon cancer, kidney cancer, stomach cancer, ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer, and esophageal cancer. , liver cancer, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, urinary tract cancer, prostate cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer, thecoma, male germinoma, endometrial hyperplasia, endometriosis, embryonal tumor, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous hemangioma, hemangioblastoma, retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma, neuroblastoma, glioma, rhabdomyosarcoma, osteoblastoma, leiomyosarcoma, thyroid sarcoma, and Wilms' tumor.

The length of the antigen polypeptide is not particularly limited as long as the composition of the present invention can induce an immune response by nasal administration. The number of amino acid residues in the antigen polypeptide is, for example, about 2 or more, preferably about 35,000 or less, more preferably about 13 to about 3,000, even more preferably about 80 to about 2,000, and most preferably about 150 to about 1,500. When using an antigen polypeptide with low immunogenicity (e.g., a polypeptide with fewer than about 80 amino acid residues), it is preferable to use the antigen polypeptide by binding or conjugating it to a carrier protein such as a protein derived from an organism other than the target organism or a denatured protein derived from the target organism.

An antigen polypeptide may be naturally derived (e.g., disrupted and/or extracted microorganisms/cells, or purified products thereof), may be a genetically recombinant product, or may be a chemically synthesized product. The amino acid sequence of the antigen polypeptide may be wild-type or mutant. The amino acid sequence of the mutant antigen polypeptide has, for example, 70% or more identity, preferably 80% or more identity, more preferably 90% or more identity, and even more preferably 95% or more identity to the amino acid sequence of the corresponding wild-type antigen polypeptide.

As used herein, "identity" of amino acid sequences refers to the degree of agreement between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the identity between two amino acid sequences, the greater the identity or similarity of those sequences. The level of identity of amino acid sequences is determined, for example, using the sequence analysis tool FASTA, using default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, which is based on the BLAST algorithm, has been developed. The specific techniques for these analysis methods are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/).

An antigen polypeptide (antigen protein) may contain other amino acid sequences (e.g., tag sequences such as histidine tag, HA tag, FLAG tag, etc.) as long as it is capable of inducing antibodies or eliciting an immune response.

The antigen polypeptide (antigen protein) may be chemically modified as long as it is capable of inducing an immune response (antibody induction).

The C-terminus of the antigenic polypeptide (antigen protein) may be any of a carboxyl group (--COOH), a carboxylate (--COO ^- ), an amide (--CONH ₂ ) or an ester (--COOR).

Here, examples of R in the ester include _C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl; _C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; _C6-12 aryl groups such as phenyl and α-naphthyl; C1-2 phenyl- _C1-2 alkyl groups such as benzyl and phenethyl; _C7-14 aralkyl groups such as α-naphthyl- _C1-2 alkyl groups such as α-naphthylmethyl; and pivaloyloxymethyl groups.

The antigen polypeptide (antigen protein) may have a carboxyl group (or carboxylate) other than that at the C-terminus amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

Furthermore, antigen polypeptides (antigen proteins) also include those in which the amino group of the N-terminal amino acid residue is protected with a protecting group (e.g., a _C1-6 acyl group, such as a formyl group, a _C1-6 alkanoyl group such as an acetyl group), those in which the N-terminal glutamine residue that may be generated by cleavage in the body is pyroglutamylated, and those in which substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) are protected with an appropriate protecting group (e.g., a _C1-6 acyl group, such as a formyl group, a _C1-6 alkanoyl group such as an acetyl group).

An antigenic polypeptide (antigenic protein) may be a glycoprotein in which a glycan is bound to some of the amino acids that make up the antigenic polypeptide (antigenic protein). Examples of sugars that form glycans include glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and xylose.

An antigenic polypeptide (antigenic protein) may be a monomer or a multimer such as a dimer, trimer, tetramer, or pentamer. In the case of a multimer, it may be a homomultimer formed from the same subunits, or a heteromultimer formed from different subunits.

The antigen polypeptide (antigen protein) may be in the form of a pharma- ceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharma- ceutically acceptable salt, and either an acid salt or a basic salt can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; amino acid salts such as aspartate and glutamate; and the like. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

The antigen polypeptide (antigen protein) may be in the form of a solvate. The solvent is not particularly limited as long as it is pharma- ceutically acceptable, and examples of the solvent include water, ethanol, glycerol, and acetic acid.

The antigen polypeptide may be one type alone or two or more types in combination. In addition, for example, to analyze the antibody inducing ability of the composition of the present invention, polypeptides such as albumin, e.g., ovalbumin, fluorescent proteins, e.g., luciferase, green fluorescent protein, eGFP, and antibodies, e.g., anti-Ep-CAM (CD326) antibody, may be used as the antigen polypeptide.

The coding sequence of an antigen polypeptide is a base sequence that can express the antigen polypeptide by a replication mechanism, a transcription mechanism, or a translation mechanism, and is not particularly limited in that respect. When the polynucleotide containing the coding sequence is single-stranded, the coding sequence can be either the sense strand or the antisense strand.

The polynucleotide containing the coding sequence of the antigen polypeptide is not particularly limited as long as it is capable of expressing the antigen polypeptide in the target organism, and is usually a polynucleotide containing an expression cassette containing the coding sequence of the antigen polypeptide, or an mRNA containing the coding sequence of the antigen polypeptide, and preferably contains an expression cassette. A typical example of an expression cassette is a polynucleotide containing a promoter and a coding sequence of the antigen polypeptide placed under the control of the promoter.

As the promoter, for example, various pol II promoters can be used. Pol II promoters are not particularly limited, but examples include the CMV (cytomegalovirus) promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, etc.

Polynucleotides can be single-stranded or double-stranded. Polynucleotides can be linear or circular.

From the viewpoint of antibody induction ability, it is particularly preferable that the composition of the present invention contains a virus particle containing a polynucleotide including a coding sequence of an antigen polypeptide. In this embodiment, the polynucleotide is usually a virus vector. The virus vector is not particularly limited, but examples thereof include adenovirus vectors, Sindbis virus/Semliki Forest virus vectors, retrovirus vectors, lentivirus vectors, rabies virus vectors, Sendai virus vectors, adeno-associated virus vectors, and herpes simplex virus vectors. Among these, adenovirus vectors are preferable. The virus particles are usually those corresponding to the virus vector used. For example, when an adenovirus vector is used, the virus particles are usually adenovirus.

The polynucleotide may typically be DNA or RNA (preferably mRNA). The polynucleotide may also be chemically modified as shown below. To prevent degradation by hydrolases such as nucleases, the phosphoric acid residue (phosphate) of each nucleotide may be replaced with a chemically modified phosphoric acid residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may also be replaced with -OR (R represents, for example, CH3(2'-O-Me), _CH2CH2OCH3 (2'-O-MOE), _CH2CH2NHC (NH) _NH2 , _CH2CONHCH3 , or _{CH2CH2CN )} . _The base portion (pyrimidine, purine) may also be chemically modified, for example by introducing a methyl group or a _cationic functional group into _the 5-position _of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. In addition, BNA (LNA), in which the conformation of the sugar moiety is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide, can also be preferably used.

The polynucleotide may be of one type alone or of two or more types in combination. In addition, for example, in order to analyze the antibody inducing ability of the composition of the present invention, a polynucleotide containing a coding sequence for a polypeptide such as albumin, e.g., ovalbumin, luciferase, fluorescent protein, e.g., green fluorescent protein, eGFP, or antibody, e.g., anti-Ep-CAM (CD326) antibody, may be used.

The composition of the present invention may contain an adjuvant. When the composition of the present invention contains an antigen polypeptide, it is particularly preferable that the composition contains an adjuvant in order to enhance its antibody-inducing ability.

Adjuvants include, but are not limited to, CpG oligoDNA, poly IC RNA, imidazoquinolines (e.g., R848 and imiquimod), STING agonists (e.g., cyclic di-GMP), lipopolysaccharides, lipid A, monophosphoryl lipid A, lipopeptides, nanoparticles (e.g., nanoparticles containing saponin, cholesterol and phospholipids), o/w emulsions (e.g., o/w emulsions consisting of squalene, o/w emulsions containing nonionic surfactants), liposomes, Alum (aluminum compounds such as aluminum hydroxide gel), mineral oil, vegetable oil, alum, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, interferon, polysaccharides (e.g., chitosan), sphingoglycolipids, GM-CSF, toxins (e.g., cholera toxin, E. coli heat-labile toxin), TLR5 ligands (e.g., flagellin), or mixtures thereof. Among these, nucleic acid adjuvants are preferred.

The adjuvant may be a single type or a combination of two or more types.

The amount of adjuvant contained in the composition of the present invention varies depending on the type of adjuvant, the type and amount of antigen, the subject of administration, the dosage form, etc., and is not limited. However, in the case of a nucleic acid adjuvant such as cyclic di-GMP, the amount can be, for example, 0.001 to 100 parts by weight, preferably 0.01 to 10 parts by weight, per part by weight of antigen (antigen polypeptide).

In addition to the above components, the composition of the present invention may contain bases, carriers, diluents, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

The composition of the present invention can be prepared by mixing the components constituting the composition by a method known per se. For example, it may be prepared by mixing all the components at once, or by mixing some of the components and then mixing them with the remaining components. Furthermore, after mixing, the composition may be diluted with a diluent or concentrated, if necessary.

The properties of the composition of the present invention are not particularly limited as long as they are suitable for nasal administration. The composition of the present invention can be, for example, a liquid (e.g., an aqueous solution, an emulsion), a gel, or a cream, and is particularly preferably a liquid. In a preferred embodiment of the present invention, the total content of the sugar component and water in the composition of the present invention is, for example, 30% by weight (w/w%) or more, preferably 50% by weight (w/w%) or more, more preferably 60% by weight (w/w%) or more, even more preferably 70% by weight (w/w%) or more, even more preferably 80% by weight (w/w%) or more, and particularly preferably 90% by weight (w/w%) or more.

The composition of the present invention has low toxicity and side effects and can be administered intranasally to target organisms for prophylactic or therapeutic purposes.

The method of nasal administration is not particularly limited, and known methods can be used. Such methods can be, for example, application, spraying, dropping, placement, etc. The target site for nasal administration (the site with which the composition of the present invention comes into contact during administration) is, for example, the upper respiratory tract, preferably the nasal cavity.

The target organisms of the composition of the present invention are not particularly limited. Examples of such organisms include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits. Of these, humans are preferred.

The content of an antigen in the composition of the present invention depends on the type of antigen, the subject of administration, the dosage form, the condition of the patient, the doctor's judgment, etc., and is not limited thereto, but in the case of an antigen polypeptide, the content can be, for example, 0.0001 to 95% by volume (w/v%), preferably 0.001 to 50% by volume (w/v%). In the case of a virus particle containing a polynucleotide comprising a coding sequence for an antigen polypeptide, the content can be, for example, 0.000025 (2.5 x ^10-5 ) to 95% by volume (w/v%), preferably 0.00025 (2.5 x ^10-4 ) to 50% by volume (w/v%).

The amount of the composition of the present invention to be used depends on the type of antigen, the subject to be administered, the dosage form, the condition of the patient, the doctor's judgment, etc., and is not limited thereto. For example, in the case of an antigen polypeptide, the amount of the antigen polypeptide administered in one administration is 1 μg to 10 mg/kg (body weight), and preferably 10 to 1000 μg/kg (body weight). In the case of a virus particle containing a polynucleotide including a coding sequence for an antigen polypeptide, the amount of the virus particle administered in one administration is 0.0025 μg to 10 mg/kg (body weight), and preferably 0.025 to 1000 μg/kg (body weight). The interval and number of administrations are not particularly limited, but may be, for example, a single administration, or 2 to 5 administrations at intervals of about 1 to 8 weeks.

The composition of the present invention is suitable for use as a vaccine composition. Furthermore, when the composition of the present invention contains a microorganism-derived antigen protein or a partial polypeptide thereof, the composition of the present invention can be used, for example, to prevent the onset and/or aggravation of an infectious disease caused by a microorganism, preferably the microorganism. Furthermore, when the composition of the present invention contains a cancer antigen protein or a partial polypeptide thereof, the composition of the present invention can be used, for example, to prevent or treat cancer, preferably the cancer.

The composition of the present invention can increase the amount of antibodies in the body (particularly, antibodies specific to an antigen polypeptide encoded by a polynucleotide contained in the composition of the present invention, or an antigen polypeptide contained in the composition of the present invention (antigen-specific antibodies)).

Sites in the body where the amount of antibodies can be improved include, for example, the upper respiratory tract and body fluids (for example, blood). In addition, types of antibodies include, for example, IgA, IgG, IgM, IgD, IgE, etc., and among these, IgA and/or IgG are preferred. According to the composition of the present invention, it is possible to improve the amount of IgA on the surface of the upper respiratory tract and/or IgG in the blood, and preferably the amount of IgA on the surface of the upper respiratory tract and IgG in the blood.

In a preferred embodiment of the present invention, the composition of the present invention can increase the number of immune cells in a living body (particularly immune cells specific to an antigen polypeptide encoded by a polynucleotide contained in the composition of the present invention, or specific to an antigen polypeptide contained in the composition of the present invention).

The site in the body where the amount of antibody can be increased is, for example, the upper respiratory tract and body fluids (for example, blood). The type of immune cells is, for example, T cells, and preferably CD8 ⁺ T cells. The composition of the present invention can increase the number of immune cells, particularly in the tissue of the upper respiratory tract.

The present invention will be described in detail below based on test examples, but the present invention is not limited to these test examples.

In Test Examples 1 to 4, 6, and 8 to 10, a polynucleotide (DNA) containing a coding sequence for an antigen polypeptide placed under the control of a CMV promoter was used.

Test Example 1. Analysis of the effect of glycerol on nasal administration of AdV-OVA The effect of glycerol on nasal administration of chicken ovalbumin (OVA) gene-loaded human serum type 5 adenovirus vector (AdV-OVA) to the upper respiratory tract was analyzed.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered substance: chicken ovalbumin (OVA) gene-carrying human serum type 5 adenovirus vector (AdV-OVA).
-Additive: glycerol.

<Method>
5 × 10 ⁸ IFU of AdV-OVA (3.2 × 10 ⁹ VP) was suspended in PBS or PBS containing 10 (w/v) % (final concentration) glycerol, and a total of 6 μL was administered intranasally to mice (upper respiratory tract administration: 3 μL/nostril). 28 days after administration, blood was collected and OVA-specific IgG in plasma was evaluated by ELISA. In addition, 34 days after administration, nasal washes and nasal tissues were collected. OVA-specific IgA in nasal washes was evaluated by ELISA, and OVA-specific CD8 ⁺ T cells in nasal mucosa were evaluated by flow cytometry.

The results are shown in Figure 1. When AdV-OVA was administered intranasally to the upper respiratory tract, the induction of OVA-specific IgG in the blood was low, and IgA production on the mucosal surface was hardly observed. On the other hand, in the group to which glycerol was added, the OVA-specific IgG in the blood increased by about 100-fold, and the OVA-specific IgA and CD8 ⁺ T cells in the nasal wash increased by about 7-fold (compared to the median values), indicating that the intranasal vaccine effect of AdV was greatly enhanced.

Test Example 2. Comparison of the effects of nasal administration of AdV-OVA (glycerol and CVP) The effects of glycerol and carboxyvinyl polymer (CVP) on the nasal administration of chicken ovalbumin (OVA) gene-loaded human serum type 5 adenovirus vector (AdV-OVA) to the upper respiratory tract were compared. Specifically, the procedure was as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered substance: chicken ovalbumin (OVA) gene-carrying human serum type 5 adenovirus vector (AdV-OVA).
Additives: glycerol or carboxyvinyl polymer (CVP).

<Method>
5 × 10 ⁸ IFU of AdV-OVA (3.2 × 10 ⁹ VP) was suspended in PBS, 10 (w/v) % (final concentration) glycerol, or 0.55 (w/v) % (final concentration) CVP, which is listed in the Pharmaceutical Excipients Standards and is expected to be used as a thickening agent for nasal vaccines, and 6 μL in total was administered intranasally to mice (upper respiratory tract administration: 3 μL/nostril). 28 days after administration, blood, nasal washes, and nasal tissues were collected, and OVA-specific IgG in plasma and OVA-specific IgA in nasal washes were evaluated by ELISA, and OVA-specific CD8 ⁺ T cells in the nasal mucosa were evaluated by flow cytometry.

The results are shown in Figure 2. When CVP was used as an additive, unlike the addition of glycerol, it was shown that there was almost no increase in the induction of OVA-specific IgG by nasal administration of AdV-OVA to the upper respiratory tract or in IgA production on the mucosal surface. On the other hand, the addition of CVP significantly promoted the induction of CD8 ⁺ T cells in the nasal tissue. Overall, it was suggested that glycerol has a stronger effect of enhancing the efficacy of nasal AdV vaccines than the addition of general thickening agents.

Test Example 3. Comparison of the effects of nasal administration of AdV-OVA (glycerol and various thickeners) The effects of glycerol and various thickeners on nasal administration of chicken ovalbumin (OVA) gene-loaded human serum type 5 adenovirus vector (AdV-OVA) to the upper respiratory tract were compared. Specifically, the procedure was as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered substance: chicken ovalbumin (OVA) gene-carrying human serum type 5 adenovirus vector (AdV-OVA).
Additives: Glycerol, polyacrylic acid, pork-derived gelatin hydrolysate, methylcellulose, sodium alginate.

<Method>
5 × ¹⁰⁸ IFU of AdV-OVA (3.2 × ¹⁰⁹ VP) was suspended in PBS or in PBS containing various thickeners at the final concentrations (w/v%) shown in Figure 3, and 6 μL of the suspension was administered intranasally to mice (upper respiratory tract administration: 3 μL/nostril). 28 days after administration, blood, nasal washes, and nasal tissues were collected, and OVA-specific IgG in plasma and OVA-specific IgA in nasal washes were evaluated by ELISA.

The results are shown in Figure 3. Unlike the addition of glycerol, the newly used thickening agent was found to not result in the induction of OVA-specific IgG or improved IgA production on the mucosal surface. This suggests that the enhancement of the nasal vaccine effect of AdV-OVA by glycerol is due to properties other than its thickening effect.

Test Example 4. Analysis of the effect of glycerol on nasal administration of AdV-HA/AdV-S The effect of glycerol on nasal administration of human serotype 5 adenovirus vectors (AdV-HA and AdV-S, respectively) carrying the hemagglutinin (HA) gene derived from the H1N1 influenza virus (PR8 strain) or the spike (S) gene of SARS-CoV-2 (Wuhan-Hu-1 strain) (modified Furin Cleavage Site 682-685 RRAR to GSAS, including the D614G mutation) to the upper respiratory tract was analyzed. Specifically, the procedure was as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice (AdV-HA), BALB/c mice (AdV-S) Administered substance: Human serotype 5 adenovirus vector carrying the hemagglutinin (HA) gene derived from the H1N1 influenza virus (PR8 strain) or the spike (S) gene of SARS-CoV-2 (Wuhan-Hu-1 strain) (Furin Cleavage Site 682-685 RRAR modified to GSAS, including the D614G mutation) (AdV-HA, AdV-S, respectively)
-Additive: glycerol.

<Method>
5 × 10 ⁸ IFU of AdV-HA (2.9 × 10 ⁹ VP) or AdV-S (3.7 × 10 ⁹ VP) was suspended in PBS or PBS containing 10 (w/v) % (final concentration) glycerol, and a total of 6 μL was administered intranasally to mice (upper respiratory tract administration: 3 μL/nostril). 28 days after administration, blood was collected and antigen-specific IgG in plasma was evaluated by ELISA.

The results are shown in Figure 4. As with the AdV-OVA study, it was found that in the group to which glycerol was added, HA-specific IgG in the blood was approximately 150 times higher, and S-specific IgG was approximately 15 times higher (median comparison) compared to when AdV-HA or AdV-S was simply suspended in PBS and administered. In addition, S-specific IgA in the nasal lavage fluid also increased significantly. Therefore, the fact that glycerol is effective regardless of the antigen gene carried suggests that glycerol is highly versatile.

Test Example 5. Analysis of the effect of glycerol on nasal administration of OVA To investigate whether glycerol is effective for substances other than AdV, the effect of glycerol on nasal administration of chicken ovalbumin (OVA) to the upper respiratory tract was analyzed.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered substance (antigen): chicken ovalbumin (OVA).
・Adjuvant: c-di-GMP
-Additive: glycerol.

<Method>
Mice were intranasally administered 6 μL of 5 μg OVA and 3 μg c-di-GMP in PBS or PBS containing 10 (w/v) % (final concentration) glycerol twice at 21-day intervals (upper respiratory tract administration: 3 μL/nostril). 14 days after the final administration, blood was collected and OVA-specific IgG in plasma was evaluated by ELISA.

The results are shown in Figure 5. Nasal administration of OVA and c-di-GMP induced a certain level of OVA-specific IgG, but the addition of 10 (w/v)% glycerol promoted the induction by approximately nine-fold. Thus, it was demonstrated that glycerol enhances the efficacy of not only AdV but also nasal vaccines using protein antigens.

Test Example 6. Analysis of the mechanism of enhancing the efficacy of AdV nasal vaccine We verified whether glycerol promotes the infection efficiency of AdV in nasal tissue and analyzed the mechanism of enhancing the efficacy of AdV nasal vaccine by glycerol. Specifically, the procedure was as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered substance: human serotype 5 adenovirus vector carrying the eGFP gene (AdV-GFP). Additive: glycerol.

<Method>
5 × 10 ⁸ IFU of AdV-GFP (4.0 × 10 ⁹ VP) was suspended in PBS or 10 (w/v) % (final concentration) glycerol-containing PBS, and a total of 6 μL was administered intranasally to mice (upper respiratory tract administration: 3 μL/nostril). Three days after administration, nasal tissues were collected and the number of GFP-positive cells in the nasal tissues was analyzed by flow cytometry.

The results are shown in Figure 6. In the group where glycerol was added at a concentration of 10 (w/v) %, the number of GFP ⁺ cells increased in both the CD45 ⁺ immune cells and the CD45 ^- tissue-constituting cells in the nasal tissue, suggesting that AdV infection of the nasal tissue was increased. Therefore, it was suggested that glycerol enhances the nasal vaccine effect of AdV by promoting AdV infection of the nasal tissue.

Test Example 7. Analysis of the effect of glycerol on nasal retention Because glycerol has a thickening effect, it is possible that glycerol improves the retention of AdV in the nasal cavity, which may result in enhanced infection. Therefore, we investigated the possibility that the addition of glycerol improves the retention of the administered reagent in the nasal cavity, using the amount of recombinant luciferase protein recovered in the nasal wash as an indicator.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administration reagent: recombinant luciferase protein. Additives: glycerol or carboxyvinyl polymer (CVP).

<Method>
1 × 10 ⁶ units of recombinant luciferase protein was suspended in PBS, 10 (w/v) % (final concentration) glycerol, or 0.55 (w/v) % (final concentration) CVP, which is listed in the Pharmaceutical Excipients Standards and is expected to be used as a thickening agent for nasal vaccines, and 6 μL in total was administered intranasally to mice (upper respiratory tract administration: 3 μL/nostril). Nasal washes were collected 1, 4, and 8 hours after administration, and the amount of luciferase protein in the nasal washes was evaluated by luciferase assay.

The results are shown in Figure 7. In the CVP-added group, higher luciferase activity was detected in the nasal wash 4 hours after administration compared to the group administered recombinant luciferase protein in PBS. This suggests that the thickening agent CVP improved the retention of luciferase in the nasal cavity. On the other hand, in the glycerol-added group, luciferase activity in the nasal wash was actually lower 4 hours after administration, suggesting that at least there is no effect of improving the retention of luciferase in the nasal cavity. This suggests that the enhancement of the effectiveness of nasal vaccines by the addition of glycerol is not due to improved retention of the vaccine in the nasal cavity.

Test Example 8. Analysis of the effect of glycerol on intramuscular administration of AdV-OVA The effect of glycerol on intramuscular administration of chicken ovalbumin (OVA) gene-loaded human serum type 5 adenovirus vector (AdV-OVA) was analyzed. Specifically, the procedure was as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered substance: chicken ovalbumin (OVA) gene-carrying human serum type 5 adenovirus vector (AdV-OVA).
-Additive: glycerol.

<Method>
Mice were administered 5 × ¹⁰⁶ or 5 × ¹⁰⁸ IFU of AdV-OVA (3.2 × ¹⁰⁷ VP and 3.2 × ¹⁰⁹ VP, respectively) by intramuscular injection in 30 μL in PBS or PBS containing 10 (w/v)% (final concentration) glycerol. Twenty-eight days after administration, blood was collected and OVA-specific IgG in plasma was assessed by ELISA.

The results are shown in Figure 8. When administered by intramuscular injection, no significant difference was observed in OVA-specific IgG production by AdV-OVA when glycerol was used as an additive, compared to when glycerol was not used as an additive. This suggests that the enhancement of vaccine efficacy by the addition of glycerol is limited to intranasal administration, and that even when glycerol is administered into the body by injection, it does not promote immunity due to so-called inflammatory properties.

Test Example 9. Analysis of the effect of glycerol concentration The effect of glycerol concentration was analyzed. Specifically, the analysis was performed as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice Immunization: Chicken ovalbumin (OVA) gene-carrying human serum type 5 adenovirus vector (AdV-OVA)
-Additive: glycerol.

<Method>
Glycerol was diluted with purified water to the concentrations (w/v%, final concentration) shown in the graph, and mice were intranasally immunized with 1 × 10 ⁸ IFU of AdV-OVA (6.4 × 10 ⁸ VP) in a total of 6 μL (upper respiratory tract immunization: 3 μL/nostril). As a positive control, 1 × 10 ⁸ IFU of AdV-OVA (6.4 × 10 ⁸ VP) was suspended in PBS containing 10 (w/v)% glycerol at a final concentration, and mice were intranasally immunized with a total of 6 μL (upper respiratory tract immunization: 3 μL/nostril). 28 days after immunization, blood, nasal washes, and nasal tissues were collected, and OVA-specific IgG in plasma and OVA-specific IgA in nasal washes were evaluated by ELISA, and OVA-specific CD8 ⁺ T cells in the nasal mucosa were evaluated by flow cytometry.

The results are shown in Figure 9. When the glycerol concentration was 2.5% (w/v), no improvement in antibody induction was observed, but when the concentration was 5% (w/v) or higher, an improvement in antibody induction was observed.

Test Example 10. Analysis of antibody-inducing ability of components other than glycerol The antibody-inducing ability of components other than glycerol was analyzed. Specifically, the analysis was performed as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice Immunization: Chicken ovalbumin (OVA) gene-carrying human serum type 5 adenovirus vector (AdV-OVA)
Additives: glycerol, glucose, mannitol.

<Method>
AdV-OVA (6.4 × ¹⁰⁸ VP) (1 × ¹⁰⁸ IFU) was suspended in PBS or PBS containing various additives at the concentrations shown in the graph (w/v%, final concentration), and mice were immunized intranasally with a total of 6 μL (upper respiratory tract immunization: 3 μL/nostril). 28 days after immunization, blood, nasal washes, and nasal tissues were collected, and OVA-specific IgG in plasma and OVA-specific IgA in nasal washes were evaluated by ELISA, and OVA-specific CD8 ⁺ T cells in the nasal mucosa were evaluated by flow cytometry.

The results are shown in Figure 10. In addition to glycerol, glucose and mannitol were also found to have an effect of improving antibody induction. Sugars or sugar alcohols were also found to have an effect of improving antibody induction.

Test Example 11. Analysis of mechanism of action The mechanism of action of the improving effect of antibody induction was analyzed. Specifically, the analysis was carried out as follows.

<Experimental animals, reagents, etc.>
Mice: 7-week-old female C57BL/6 mice. Administered reagent: anti-Ep-CAM antibody. Additive: glycerol.

<Method>
Anti-Ep-CAM antibody (0.1 μg) was diluted in PBS or 10 (w/v)% (final concentration) glycerol-containing PBS and administered intranasally to mice at a total volume of 6 μL (upper respiratory tract administration: 3 μL/nostril). Two hours after administration, nasal tissues were collected and the number of antibody-bound epithelial cells was evaluated by flow cytometry.

The results are shown in Figure 11. Figure 11 shows that the addition of glycerol enabled anti-Ep-CAM antibodies administered intranasally to bind well to epithelial cells.

Claims (15)

A composition for nasal administration, comprising at least one selected from the group consisting of a polynucleotide containing a coding sequence for an antigenic polypeptide and an antigenic polypeptide, and a sugar component which is a sugar and/or a sugar alcohol, the sugar component having a content of more than 4.5% by mass/volume. The composition for nasal administration according to claim 1, wherein the sugar component content is 7% by mass or more by volume. The composition for nasal administration according to claim 1, wherein the sugar component is a sugar and/or sugar alcohol having three or more hydroxyl groups. The composition for nasal administration according to claim 3, wherein the number of hydroxyl groups in the sugar component is 3 to 6. The composition for nasal administration according to claim 1, comprising a virus particle containing the polynucleotide and/or the antigen polypeptide and an adjuvant. The composition for nasal administration according to claim 5, which contains the virus particles. The composition for nasal administration according to claim 6, wherein the virus particles are adenovirus particles. The composition for nasal administration according to claim 1, for use in administration to the upper respiratory tract. The composition for nasal administration according to any one of claims 1 to 8, which is a vaccine composition. The composition for nasal administration according to any one of claims 1 to 8, wherein the antigen polypeptide is an antigen protein derived from a microorganism or a partial polypeptide thereof. The composition for nasal administration according to claim 10, for use in preventing the onset and/or aggravation of an infectious disease caused by the microorganism. The composition for nasal administration according to claim 10, wherein the microorganism is at least one selected from the group consisting of viruses, bacteria, fungi, and parasites. The composition for nasal administration according to claim 1, wherein the sugar and/or sugar alcohol is glycerol and the content of the sugar component is more than 5% by volume. An additive for use in the manufacture of a composition for nasal administration according to any one of claims 1 to 8, which contains a sugar component that is a sugar and/or a sugar alcohol. The additive of claim 14, wherein the sugar component is glycerol and the content of the sugar component is greater than 5% by volume.

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