Composition for nasal administration

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

Nasal administration composition

[0001] The present invention relates to a composition for nasal administration, etc.

[0002] Unlike injectable vaccines, nasal vaccines induce IgA in the upper respiratory tract in addition to IgG in the blood, making it possible to protect against initial infection and also prevent the disease from becoming severe even if the virus migrates to the lower respiratory tract. Furthermore, because it can also prevent person-to-person transmission of infection associated with 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.

[0003] International Publication No. 2019 / 021305

[0004] In the course of their research, the present 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 of vaccine is administered intranasally to target only the upper respiratory tract (in general, nasal administration to humans primarily targets only 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 general mouse models).

[0005] Recently, a global pandemic of 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.

[0006] An objective of the present invention is to provide a technique for improving the antibody-inducing ability of a nasal vaccine and a composition for nasal administration with improved antibody-inducing ability.

[0007] As a result of extensive research, the present inventors have found that sugars and / or sugar alcohols improve the antibody-inducing ability of nasal vaccines.

[0008] Conventionally, glycerol, a type of sugar and / or sugar alcohol, has been intended for use as a stabilizer or the like, and its content has been relatively low in accordance with its function. Glycerol is only known to be incorporated into vaccines as a stabilizer or the like (Patent Document 1), and it was completely unexpected that sugars and / or sugar alcohols, including glycerol, would improve antibody induction ability. Based on the finding 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 finding, the present inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following aspects.

[0009] 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 sugar alcohol, wherein the sugar component content is greater than 4.5% by volume by mass. Item 1A. A method of administration, comprising nasally administering to a subject organism 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 sugar alcohol, wherein the sugar component content is greater than 4.5% by volume by mass. Item 1B. A composition for use as a nasal administration composition (preferably a nasal vaccine composition, or a nasal composition for use in preventing the onset and / or aggravation of infectious diseases caused by microorganisms), 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 sugar alcohol, wherein the sugar component content is greater than 4.5% by volume by mass. Item 1C. Item 1D. 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 sugar alcohol, wherein the sugar component content is greater than 4.5% by volume by mass, for the manufacture of a composition for nasal administration (preferably a vaccine composition for nasal administration, a composition for nasal administration used to prevent the onset and / or aggravation of infectious diseases caused by microorganisms). Item 1D. 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 sugar alcohol, wherein the sugar component content is greater than 4.5% by volume by mass, for the manufacture of a composition for nasal administration (preferably a vaccine composition for nasal administration, a composition for nasal administration used to prevent the onset and / or aggravation of infectious diseases caused by microorganisms).

[0010] Item 2. The composition for nasal administration according to Item 1, wherein the sugar component content is 7% by volume or more.

[0011] 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.

[0012] Item 4. The composition for nasal administration according to Item 3, wherein the sugar moiety has 3 to 6 hydroxyl groups.

[0013] Item 5. The composition for nasal administration according to Item 1, which comprises a virus particle containing the polynucleotide and / or the antigen polypeptide and an adjuvant.

[0014] Item 6. The composition for nasal administration according to Item 5, which contains the virus particles.

[0015] Item 7. The composition for nasal administration according to Item 6, wherein the virus particles are adenovirus particles.

[0016] Item 8. The composition for nasal administration according to Item 1, which is for administration to the upper respiratory tract.

[0017] Item 9. The composition for nasal administration according to any one of Items 1 to 8, which is a vaccine composition.

[0018] Item 10. The composition for nasal administration according to any one of Items 1 to 8, wherein the antigenic polypeptide is an antigenic protein derived from a microorganism or a partial polypeptide thereof.

[0019] Item 11. The composition for nasal administration according to Item 10, which is used to prevent the onset and / or aggravation of an infectious disease caused by the microorganism.

[0020] 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.

[0021] 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 by mass.

[0022] Item 14. An additive for use in producing the 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.

[0023] 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.

[0024] According to the present invention, it is possible to provide a technique for improving the antibody-inducing ability of a nasal vaccine and a composition for nasal administration with improved antibody-inducing ability.

[0025] Figure 1 shows the results of an analysis of the effect of glycerol on nasal administration of AdV-OVA (Test Example 1). The measurement target is indicated above the graph. The administered substance is indicated on the horizontal axis (Naive indicates the value in control mice that received no administration treatment). n = 5 for each group, and the bar in the graph indicates the median. *P < 0.05, ***P < 0.001, ****P < 0.0001. The dotted line indicates the limit of quantitation. Figure 2 shows the results of a comparison of the effects of nasal administration of AdV-OVA (glycerol and CVP) (Test Example 2). The measurement target is indicated above the graph. The administered substance is indicated on the horizontal axis. n = 3 for each group, and the bar in the graph indicates the median. *P < 0.05, **P < 0.01. The dotted line indicates the limit of quantitation. Figure 3 shows the results of a comparison of the effects of nasal administration of AdV-OVA (glycerol and various thickeners) (Test Example 3). % indicates (w / v) %. The measurement targets are shown in the graph. The horizontal axis indicates the administered substance and its concentration. n = 3 for each group, and the bar in the graph indicates the median. *P < 0.05, ***P < 0.001, ****P < 0.0001. The dotted line indicates the limit of quantitation. Analysis results of the effect of glycerol on nasal administration of AdV-HA / AdV-S are shown (Test Example 4). The measurement targets are shown above the graph. The administered substance is shown on the horizontal axis. n = 5 for each group, and the bar in the graph indicates the median. **P < 0.01, ****P < 0.0001. The dotted line indicates the limit of quantitation. Analysis results of the effect of glycerol on nasal administration of OVA are shown (Test Example 5). The measurement targets are shown above the graph. The administered substance is shown on the horizontal axis. n = 5 for each group, and the bar in the graph indicates the median. **P < 0.01. The dotted line indicates the limit of quantitation. This shows the results of an analysis of the mechanism by which AdV nasal vaccine efficacy is enhanced (Test Example 6). The measurement target is shown above the graph. The administered substance is shown on the horizontal axis. n = 3 for each group, and the bar in the graph indicates the median. *P < 0.05, **P < 0.01. The dotted line indicates the limit of quantitation. This shows the results of an analysis of the effect of glycerol on nasal retention (Test Example 7).The vertical axis shows the amount of luciferase in the nasal wash fluid. The horizontal axis shows the time elapsed since administration. The administration substance is shown in the legend (Blank indicates a control without administration = limit of quantitation). n = 8 for the PBS administration group, and n = 4 for the glycerol or CVP administration groups. The plots show the mean values. *P < 0.05, ****P < 0.0001 vs PBS. This figure shows the results of an analysis of the effect of glycerol on intramuscular administration of AdV-OVA (Test Example 8). The measurement target is shown above the graph. The horizontal axis shows the administration target (Naive indicates the value in control mice without administration). n = 5 for each group, and the bar in the graph indicates the median. The dotted line indicates the limit of quantitation. This figure shows the results of an analysis of the effect of glycerol concentration (Test Example 9). The measurement target is shown above the graph. The horizontal axis shows the administration target. n = 5 for each group, and the column indicates the median. **P < 0.01, ***P < 0.001. The dotted line indicates the limit of quantitation. This figure shows the results of an analysis of the antibody-inducing ability of components other than glycerol (Test Example 10). The measurement targets are shown above the graph. The horizontal axis indicates the administered substance (Naive indicates the value in control mice that received no administration treatment). For each group, n = 6 or 7, and the columns indicate the median. ***P < 0.001, ****P < 0.0001. The dotted line indicates the limit of quantitation. This figure shows the results of an analysis of the mechanism of action (Test Example 11). The measurement targets are shown on the left side of the graph. The horizontal axis indicates the administered substance (Naive indicates the value in control mice that received no administration treatment). For each group, n = 4, and the columns indicate the median. **P < 0.01, ***P < 0.001.

[0026] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "include," "consist essentially of," and "consist only of."

[0027] When the upper and lower limits of a numerical range are separately stated herein, any combination of any upper and lower limits is also intended to be disclosed herein.

[0028] Unless otherwise specified herein, "mass volume %" refers to w / v % (mass to volume percentage).

[0029] In one aspect, the present invention relates to a composition for nasal administration (sometimes referred to herein as the "composition of the present invention"), which comprises 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, and the content of the sugar component is more than 4.5% by mass / volume.

[0030] The composition of the present invention contains a sugar component that is a sugar and / or a sugar alcohol, which can improve the antibody-inducing ability of the composition of the present invention when administered intranasally.

[0031] The sugar is not particularly limited, and examples thereof include monosaccharides and oligosaccharides.

[0032] 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, with aldohexoses being preferred.

[0033] Oligosaccharides are sugars in which two or more monosaccharide molecules are linked together via 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.

[0034] Sugar alcohols are components obtained by reducing the carbonyl group of monosaccharides or disaccharides that are aldoses or ketoses, and are not particularly limited thereto. Examples of sugar alcohols include glycerol, erythritol, threitol, arabinitol, xylitol, ribitol, mannitol, iditol, galactitol, sorbitol, volemitol, and maltitol, with monosaccharide-derived sugar alcohols being preferred.

[0035] The sugar component may be one type alone or a combination of two or more types.

[0036] 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, 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, still more preferably 3 to 5, particularly preferably 3 to 4, and particularly preferably 3.

[0037] More specific preferred examples of the sugar component include glycerol, glucose, mannitol, etc., with glycerol being particularly preferred.

[0038] The content of the sugar component in the composition of the present invention is more than 4.5% by volume, 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 by mass or more, even more preferably 6% by volume by mass or more, even more preferably 7% by volume by mass or more, particularly preferably 8% by volume by mass or more, and especially preferably 9% by volume by mass or more (alternatively, 9.1% by volume by mass or more, 9.2% by volume by mass or more, 9.3% by volume by mass or more, 9.4% by volume by mass or more, 9.5% by volume by mass or more, 9.6% by volume by mass or more, 9.7% by volume by mass or more, 9.8% by volume by mass or more, or 9.9% by volume by mass or more). By making the content closer to 10% by volume by mass (for example, by making it 7% by volume by mass or more, 8% by volume by mass or more, or 9% by volume by mass 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, 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.

[0039] 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 introduced into the mammal (directly or upon expression, for example, in a DNA vaccine or mRNA vaccine) into the mammal having an immune system, and is not particularly limited insofar as such polypeptide is so introduced.

[0040] Examples of antigenic polypeptides include microbial-derived 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-derived antigenic protein or partial polypeptides thereof. Note that "derived from X" (where "X" is any microorganism) refers to something that "X" possesses, preferably something that is exposed on the surface of "X."

[0041] Examples of microorganisms include viruses, bacteria, fungi, and parasites. 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 microorganisms include viruses, and particularly preferred are viruses that cause respiratory tract (upper and lower respiratory tract) infections.

[0042] Examples of viruses include, but are not limited to, enveloped viruses (viruses with an envelope) such as influenza virus (e.g., type A, type B, etc.), rubella virus, Ebola virus, coronavirus, measles virus, varicella-zoster 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, and lyssavirus; and non-enveloped viruses (viruses without an envelope) such as adenovirus, norovirus, rotavirus, human papillomavirus, poliovirus, enterovirus, coxsackievirus, human parvovirus, encephalomyocarditis virus, and rhinovirus. Among these, preferred are enveloped viruses, and more preferred are influenza virus and coronaviruses such as SARS coronavirus and SARS coronavirus 2.

[0043] SARS-CoV-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. Sublineages of the above strains are also included.

[0044] Examples of bacteria or fungi include, but are not limited to, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella enterica, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus staphylococcus, Bacillus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Clostridium faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella pneumoniae, Klebsiella pneumoniae, Koinebacterium hemolyticum, Streptococcus hemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma, Candida, and Aspergillus.

[0045] Examples of parasites include, but are not limited to, Entamoeba histolytica, Plasmodium, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, and Trichomonas.

[0046] Examples of 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 / CD3-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-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp2 (MAGE-B2), and MAGE-Xp2 (MAGE-Xp2). E-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, CDK 4, 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, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75 GM2 ganglioside, GD2 ganglioside, human papillomavirus proteins, the 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, and c-erbB-2.

[0047] Target cancers are not particularly limited, and examples thereof 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.

[0048] The length of the antigen polypeptide is not particularly limited, as long as the composition of the present invention can elicit an immune response when administered intranasally. The number of amino acid residues in the antigen polypeptide is, for example, at least about 2, preferably not more than about 35,000, 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 bound or conjugated 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.

[0049] The antigen polypeptide may be naturally derived (e.g., disrupted and / or extracted microorganisms / cells, or purified products thereof), recombinant, or chemically synthesized. 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.

[0050] As used herein, "identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with 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, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0051] The antigen polypeptide (antigen protein) may contain other amino acid sequences (e.g., tag sequences such as a histidine tag, HA tag, or FLAG tag) as long as it is capable of inducing antibodies or eliciting an immune response.

[0052] The antigen polypeptide (antigen protein) may be chemically modified as long as it is capable of inducing an immune response (antibody induction).

[0053] The antigenic polypeptide (antigenic protein) has a carboxyl group (-COOH) at the C-terminus, a carboxylate group (-COO － ), amide (-CONH2) or ester (-COOR).

[0054] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1－2 C such as alkyl group 7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0055] The antigenic polypeptide (antigenic protein) may be amidated or esterified at a carboxyl group (or carboxylate) other than the C-terminus. In this case, the ester used may be, for example, the C-terminal ester described above.

[0056] Furthermore, in the antigen polypeptide (antigen protein), the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1－6 C such as alkanoyl 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1－6 C such as alkanoyl group 1－6 Also included are those protected with an alkyl group such as an acyl group.

[0057] The antigenic polypeptide (antigenic protein) may be a glycoprotein in which a sugar chain is bound to a portion of the amino acids constituting the antigenic polypeptide (antigenic protein). Examples of sugars that form the sugar chain include glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and xylose.

[0058] The antigen polypeptide (antigen protein) may be a monomer or a multimer such as a dimer, trimer, tetramer, pentamer, etc. In the case of a multimer, it may be a homomultimer formed from the same subunits or a heteromultimer formed from different subunits.

[0059] The antigen polypeptide (antigen protein) may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and both acidic and basic salts can be used. Examples of acidic 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; and amino acid salts such as aspartate and glutamate. 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.

[0060] The antigen polypeptide (antigen protein) may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0061] The antigen polypeptide may be a single type or a combination of two or more types. For example, to analyze the antibody-inducing ability of the composition of the present invention, polypeptides such as albumin (e.g., ovalbumin), luciferase, fluorescent proteins (e.g., green fluorescent protein and eGFP), and antibodies (e.g., anti-Ep-CAM (CD326) antibody) may be used as the antigen polypeptide.

[0062] The coding sequence of an antigen polypeptide is not particularly limited, as long as it is a nucleotide sequence that can express the antigen polypeptide using replication, transcription, and translation mechanisms. When the polynucleotide containing the coding sequence is single-stranded, the coding sequence can be either the sense strand or the antisense strand.

[0063] The polynucleotide comprising the coding sequence for an antigenic polypeptide is not particularly limited as long as it is capable of expressing the antigenic polypeptide in a target organism, and is usually a polynucleotide comprising an expression cassette comprising the coding sequence for the antigenic polypeptide, or an mRNA comprising the coding sequence for the antigenic polypeptide, and preferably comprises an expression cassette. Typical examples of expression cassettes include a promoter and a polynucleotide comprising the coding sequence for the antigenic polypeptide placed under the control of the promoter.

[0064] The promoter may be, for example, any of a variety of Pol II promoters, including, but not limited to, the CMV (cytomegalovirus) promoter, the EF1 promoter, the SV40 promoter, the MSCV promoter, the hTERT promoter, the β-actin promoter, and the CAG promoter.

[0065] A polynucleotide can be single-stranded or double-stranded. A polynucleotide can be linear or circular.

[0066] From the viewpoint of antibody induction ability, it is particularly preferable that the composition of the present invention contains viral particles containing a polynucleotide comprising a coding sequence for an antigen polypeptide. In this embodiment, the polynucleotide is usually a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, Sindbis virus / Semliki Forest virus vectors, retroviral vectors, lentiviral vectors, rabies virus vectors, Sendai virus vectors, adeno-associated virus vectors, and herpes simplex virus vectors. Among these, adenoviral vectors are preferred. The viral particles are usually those corresponding to the viral vector used. For example, when an adenoviral vector is used, the viral particles are usually adenovirus.

[0067] Polynucleotides can typically be DNA or RNA (preferably mRNA). Polynucleotides may also be chemically modified as described below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide can be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may also be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or 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 substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, 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. Also preferably used are 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.

[0068] The polynucleotide may be a single type or a combination of two or more types. Furthermore, for example, to analyze the antibody-inducing ability of the composition of the present invention, the polynucleotide may include 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).

[0069] 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 to contain an adjuvant in order to enhance its antibody-inducing ability.

[0070] Examples of adjuvants include, but are not limited to, CpG oligoDNA, polyIC 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, interleukins, interferons, polysaccharides (e.g., chitosan), sphingoglycolipids, GM-CSF, toxins (e.g., cholera toxin, Escherichia coli heat-labile toxin), TLR5 ligands (e.g., flagellin), and mixtures thereof. Among these, nucleic acid adjuvants are preferred.

[0071] The adjuvant may be one kind alone or a combination of two or more kinds.

[0072] The content of the adjuvant in the composition of the present invention varies depending on the type of adjuvant, the type and content of the 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 content can be, for example, 0.001 to 100 parts by weight, preferably 0.01 to 10 parts by weight, per part by weight of the antigen (antigenic polypeptide).

[0073] In addition to the above components, the composition of the present invention may contain a base, carrier, diluent, solvent, dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrant, lubricant, thickener, moisturizer, colorant, fragrance, chelating agent, etc.

[0074] The composition of the present invention can be prepared by mixing the components constituting the composition using a method known per se. For example, it may be prepared by mixing all of the components at once, or by mixing some of the components and then mixing them with the remaining components. Furthermore, after mixing, the mixture may be diluted with a diluent or concentrated, if necessary.

[0075] The form of the composition of the present invention is not particularly limited as long as it is 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.

[0076] The composition of the present invention has low toxicity and side effects and can be administered intranasally to target organisms for prophylaxis or treatment.

[0077] The method of nasal administration is not particularly limited, and known methods can be used. Such methods can include, 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 upon administration) is, for example, the upper respiratory tract, preferably the nasal cavity.

[0078] The target organisms for 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.

[0079] 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, and the like, and is not limited thereto. 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 weight / volume (w / v%), preferably 0.00025 (2.5 x 10 -4 ) to 50% by mass volume (w / v %).

[0080] The amount of the composition of the present invention used depends on factors such as the type of antigen, the recipient, the dosage form, the patient's condition, and the physician's judgment, and is not limited thereto. For example, in the case of an antigen polypeptide, the amount of antigen polypeptide administered per administration is 1 μg to 10 mg / kg (body weight), preferably 10 to 1,000 μg / kg (body weight). In the case of virus particles containing a polynucleotide comprising a coding sequence for an antigen polypeptide, the amount of virus particles administered per administration is 0.0025 μg to 10 mg / kg (body weight), preferably 0.025 to 1,000 μg / kg (body weight). The interval and number of administrations are not particularly limited, and may be, for example, a single administration, or 2 to 5 administrations at intervals of approximately 1 to 8 weeks.

[0081] The compositions of the present invention are suitable for use as vaccine compositions. Furthermore, when the compositions of the present invention contain a microorganism-derived antigen protein or a partial polypeptide thereof, the compositions of the present invention can be used, for example, to prevent the onset and / or aggravation of infectious diseases caused by a microorganism, preferably the microorganism. Furthermore, when the compositions of the present invention contain a cancer antigen protein or a partial polypeptide thereof, the compositions of the present invention can be used, for example, to prevent or treat cancer, preferably the cancer.

[0082] The composition of the present invention can increase the amount of antibodies in vivo (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)).

[0083] The sites in the body where the amount of antibodies can be increased include, for example, the upper respiratory tract and body fluids (e.g., blood). Examples of types of antibodies include IgA, IgG, IgM, IgD, and IgE, with IgA and / or IgG being preferred. The composition of the present invention can particularly increase the amount of IgA on the surface of the upper respiratory tract and / or IgG in the blood, preferably the amount of IgA on the surface of the upper respiratory tract and IgG in the blood.

[0084] Furthermore, 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 to an antigen polypeptide contained in the composition of the present invention).

[0085] The sites in the body where the amount of antibodies can be increased include the upper respiratory tract and body fluids (e.g., blood). The types of immune cells include T cells, preferably CD8 + The composition of the present invention can increase the number of immune cells, particularly in the upper respiratory tract tissue.

[0086] The present invention will be described in detail below based on test examples, but the present invention is not limited to these test examples.

[0087] 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.

[0088] Test Example 1. Analysis of the effect of glycerol on nasal administration of AdV-OVA The effect of glycerol on nasal administration of a human serotype 5 adenovirus vector (AdV-OVA) carrying the chicken ovalbumin (OVA) gene to the upper respiratory tract was analyzed. Specifically, the procedure was as follows.

[0089] <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.

[0090] <Method> 5 × 10 8 IFU AdV-OVA (3.2 x 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 the plasma was evaluated by ELISA. 34 days after administration, nasal washes and nasal tissues were collected. OVA-specific IgA in the nasal washes was assayed by ELISA, and OVA-specific CD8 in the nasal mucosa was assayed by ELISA. + T cells were assessed by flow cytometry.

[0091] The results are shown in Figure 1. Even 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 glycerol-added group, the OVA-specific IgG in the blood was approximately 100-fold higher, and the OVA-specific IgA and CD8 in the nasal lavage fluid were significantly higher. + It was shown that the effect of nasal vaccination by AdV was greatly enhanced, with T cells increasing by approximately 7-fold (compared to median values).

[0092] Test Example 2. Comparison of the effects on nasal administration of AdV-OVA (glycerol and CVP) The effects of glycerol and carboxyvinyl polymer (CVP) on nasal administration of chicken ovalbumin (OVA) gene-carrying human serotype 5 adenovirus vector (AdV-OVA) to the upper respiratory tract were compared. Specifically, the procedure was as follows.

[0093] <Experimental animals, reagents, etc.> Mice: 7-week-old female C57BL / 6 mice. Administered substance: chicken ovalbumin (OVA) gene-carrying human serotype 5 adenovirus vector (AdV-OVA). Additives: glycerol or carboxyvinyl polymer (CVP).

[0094] <Method> 5 × 10 8 IFU AdV-OVA (3.2 x 10 9 OVA 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 thickener in nasal vaccines. A total of 6 μL 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 measured by ELISA, and OVA-specific CD8 in nasal mucosa was measured. + T cells were assessed by flow cytometry.

[0095] The results are shown in Figure 2. When CVP was used as an additive, unlike when glycerol was added, it was shown that the induction of OVA-specific IgG by nasal administration of AdV-OVA to the upper respiratory tract and the production of IgA on the mucosal surface hardly increased. On the other hand, the CD8 + The addition of CVP significantly enhanced the induction of T cells. Overall, it was suggested that glycerol has a stronger effect on enhancing the efficacy of nasal AdV vaccination than the addition of general viscosity enhancers.

[0096] Test Example 3. Comparison of the effects on nasal administration of AdV-OVA (glycerol and various thickeners) The effects of glycerol and various thickeners on nasal administration of a chicken ovalbumin (OVA) gene-carrying human serotype 5 adenovirus vector (AdV-OVA) to the upper respiratory tract were compared. Specifically, the procedure was as follows.

[0097] <Laboratory animals, reagents, etc.> ・Mice: 7-week-old female C57BL / 6 mice ・Administered substance: chicken ovalbumin (OVA) gene-loaded human serum type 5 adenovirus vector (AdV-OVA) ・Additives: glycerol, polyacrylic acid, porcine-derived gelatin hydrolysate, methylcellulose, sodium alginate.

[0098] <Method> 5 × 10 8 IFU AdV-OVA (3.2 x 10 9 VP) was suspended in PBS or PBS containing various thickeners at the final concentrations (w / v%) shown in Figure 3, and a total of 6 μL 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.

[0099] The results are shown in Figure 3. Unlike the addition of glycerol, the newly used thickening agent did not result in the induction of OVA-specific IgG or an improvement in IgA production on the mucosal surface. This suggests that the enhancement of the nasal vaccine efficacy of AdV-OVA by glycerol is due to properties other than its thickening effect.

[0100] 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) (Furin cleavage site 682-685 RRAR modified to GSAS, containing the D614G mutation) was analyzed. Specifically, the procedure was as follows.

[0101] <Laboratory 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 (AdV-HA, AdV-S respectively) carrying the hemagglutinin (HA) gene derived from H1N1 influenza virus (PR8 strain) or SARS-CoV-2 (Wuhan-Hu-1 strain) spike (S) gene (Furin Cleavage Site 682-685 RRAR modified to GSAS, containing the D614G mutation) ・Additive: Glycerol.

[0102] <Method> 5 × 10 8 IFU AdV-HA (2.9 × 10 9 VP) or AdV-S (3.7 × 10 9 VP) was suspended in PBS or 10% (w / v) glycerol (final concentration) in PBS and administered intranasally to mice at a total volume of 6 μL (upper respiratory tract administration: 3 μL / nostril). 28 days after administration, blood was collected and antigen-specific IgG in the plasma was assessed by ELISA.

[0103] The results are shown in Figure 4. As with the AdV-OVA study, the glycerol-added group showed approximately 150-fold higher HA-specific IgG and approximately 15-fold higher S-specific IgG in the blood (median comparison) compared to when AdV-HA or AdV-S was administered simply suspended in PBS. Furthermore, S-specific IgA in nasal lavage fluid also increased significantly. Therefore, the effectiveness of glycerol is demonstrated regardless of the antigen gene carried, suggesting its versatile utility.

[0104] Test Example 5. Analysis of the effect of glycerol on nasal administration of OVA To investigate whether glycerol is effective against substances other than AdV, the effect of glycerol on nasal administration of chicken ovalbumin (OVA) to the upper respiratory tract was analyzed. Specifically, the procedure was as follows.

[0105] <Experimental animals, reagents, etc.> ・Mice: 7-week-old female C57BL / 6 mice ・Administered substance (antigen): chicken ovalbumin (OVA) ・Adjuvant: c-di-GMP ・Additive: glycerol.

[0106] <Method> 5 μg of OVA and 3 μg of c-di-GMP were administered intranasally to mice in PBS or PBS containing 10 (w / v)% (final concentration) glycerol (6 μL each time, 21 days apart). 14 days after the final administration, blood was collected and OVA-specific IgG in the plasma was assessed by ELISA.

[0107] 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 enhanced the induction by approximately ninefold. Therefore, glycerol was shown to enhance the efficacy of not only AdV but also nasal vaccines using protein antigens.

[0108] Test Example 6. Analysis of the mechanism by which glycerol enhances the efficacy of AdV nasal vaccines We investigated whether glycerol promotes the infection efficiency of AdV in nasal tissues and analyzed the mechanism by which glycerol enhances the efficacy of AdV nasal vaccines. Specifically, we performed the following procedure.

[0109] <Experimental animals, reagents, etc.> Mice: 7-week-old female C57BL / 6 mice. Administered substance: human serum type 5 adenovirus vector carrying the eGFP gene (AdV-GFP). Additive: glycerol.

[0110] <Method> 5 × 10 8 IFU of AdV-GFP (4.0 x 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 tissue was collected and the number of GFP-positive cells in the nasal tissue was analyzed by flow cytometry.

[0111] The results are shown in Figure 6. In the group to which glycerol was added at a concentration of 10 (w / v) %, CD45 in the nasal tissue was significantly increased. + Immune cells and CD45 - GFP was detected in all tissue constituent cell fractions. + The cell count increased, suggesting that AdV infection of the nasal tissues increased. Therefore, it was suggested that glycerol enhances the efficacy of nasal AdV vaccination by promoting AdV infection of the nasal tissues.

[0112] Test Example 7: Analysis of the effect of glycerol on nasal retention Because glycerol has a viscosity-increasing effect, it is possible that glycerol improves the retention of AdV in the nasal cavity, thereby promoting infection. Therefore, we investigated the possibility that the addition of glycerol could improve the retention of the administered reagent in the nasal cavity, using the recovery amount of nasally administered recombinant luciferase protein in nasal wash as an indicator.

[0113] <Experimental animals, reagents, etc.> Mice: 7-week-old female C57BL / 6 mice. Administered reagent: recombinant luciferase protein. Additive: glycerol or carboxyvinyl polymer (CVP).

[0114] <Method> 1 × 10 6Recombinant luciferase protein (1000 units) was suspended in PBS, 10 (w / v)% (final concentration) glycerol, or 0.55 (w / v)% (final concentration) CVP, a thickening agent listed in pharmaceutical excipient standards and expected to be used in nasal vaccines. A total of 6 μL of the suspension 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.

[0115] The results are shown in Figure 7. Compared with the group administered recombinant luciferase protein in PBS, the CVP-treated group showed higher luciferase activity in nasal washes 4 hours after administration. This suggests that the thickening agent CVP improved the retention of luciferase in the nasal cavity. In contrast, the glycerol-treated group showed lower luciferase activity in nasal washes 4 hours after administration, suggesting that glycerol does not improve the retention of luciferase in the nasal cavity. This suggests that the enhanced efficacy of nasal vaccines due to the addition of glycerol is not due to improved retention of the vaccine in the nasal cavity.

[0116] Test Example 8. Analysis of the effect of glycerol on intramuscular administration of AdV-OVA The effect of glycerol on intramuscular administration of a human serotype 5 adenovirus vector carrying the chicken ovalbumin (OVA) gene (AdV-OVA) was analyzed. Specifically, the procedure was as follows.

[0117] <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.

[0118] <Method> 5 × 10 6 , or 5 × 10 8 IFU of AdV-OVA (3.2 x 10 each) 7 VP, 3.2 × 10 9VP) was suspended in PBS or PBS containing 10% (w / v) glycerol (final concentration) and administered intramuscularly to mice at a volume of 30 μL. 28 days after administration, blood was collected and plasma OVA-specific IgG was assessed by ELISA.

[0119] The results are shown in Figure 8. When administered intramuscularly, there was no significant difference in OVA-specific IgG production by AdV-OVA with or without glycerol as an additive. This suggests that the enhancement of vaccine efficacy by the addition of glycerol is limited to intranasal administration and that glycerol administered intravenously does not promote immunity through its inflammatory properties.

[0120] Test Example 9: Analysis of the effect of glycerol concentration The effect of glycerol concentration was analyzed. Specifically, the analysis was carried out as follows.

[0121] <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.

[0122] <Method> Glycerol was diluted with purified water to the concentrations (w / v%, final concentration) shown in the graph, and 1 × 10 8 IFU of AdV-OVA (6.4 x 10 8 Mice were nasally immunized with a total of 6 μL of VP (upper respiratory tract immunization: 3 μL / nostril). 8 IFU AdV-OVA (6.4 x 10 8 OVA-specific IgG in plasma and OVA-specific IgA in nasal washes were assessed by ELISA. OVA-specific CD8+ / -10% nasal mucosa was also assessed. + T cells were assessed by flow cytometry.

[0123] The results are shown in Figure 9. When the glycerol concentration was 2.5 (w / v)%, no effect of improving antibody induction ability was observed, but when the concentration was 5 (w / v)% or higher, an effect of improving antibody induction ability was observed.

[0124] 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 carried out as follows.

[0125] <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.

[0126] <Method> 1 × 10 8 IFU AdV-OVA (6.4 x 10 8 VP) was suspended in PBS or PBS containing various additives at the concentrations (w / v%, final concentration) shown in the graph, 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. OVA-specific CD8 in the nasal mucosa was also evaluated. + T cells were assessed by flow cytometry.

[0127] The results are shown in Figure 10. In addition to glycerol, glucose and mannitol were also found to have the effect of improving antibody induction ability. Any sugar or sugar alcohol was found to have the effect of improving antibody induction ability.

[0128] Test Example 11. Analysis of mechanism of action The mechanism of action of the effect of improving antibody induction ability was analyzed. Specifically, this was carried out as follows.

[0129] <Experimental animals, reagents, etc.> Mice: 7-week-old female C57BL / 6 mice. Administered reagent: anti-Ep-CAM antibody. Additive: glycerol.

[0130] <Method> 0.1 μg of anti-Ep-CAM antibody was diluted in PBS or 10 (w / v)% (final concentration) glycerol-added PBS and administered intranasally to mice in 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 assessed by flow cytometry.

[0131] The results are shown in Figure 11. Figure 11 demonstrates that the addition of glycerol allows the anti-Ep-CAM antibody administered intranasally to bind well to epithelial cells.

Claims

1. A composition for nasal administration comprising at least one member 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 sugar alcohol, wherein the content of the sugar component is greater than 4.5% by mass / volume.

2. A composition for nasal administration as described in claim 1, wherein the content of the sugar component is 7% by mass or more by volume.

3. A composition for nasal administration as described in claim 1, wherein the sugar component is a sugar and / or sugar alcohol having three or more hydroxyl groups.

4. The composition for nasal administration described in claim 3, wherein the number of hydroxyl groups in the sugar moiety is 3 to 6.

5. A composition for nasal administration as described in claim 1, comprising a virus particle containing the polynucleotide and / or the antigenic polypeptide and an adjuvant.

6. A composition for nasal administration as described in claim 5, containing the virus particles.

7. The composition for nasal administration described in claim 6, wherein the viral particles are adenovirus particles.

8. A composition for nasal administration as described in claim 1 for use in administration to the upper respiratory tract.

9. A composition for nasal administration described in any one of claims 1 to 8, which is a vaccine composition.

10. A composition for nasal administration according to any one of claims 1 to 8, wherein the antigenic polypeptide is an antigenic protein derived from a microorganism or a partial polypeptide thereof.

11. A composition for nasal administration as described in claim 10 for use in preventing the onset and / or aggravation of infectious diseases caused by the microorganism.

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

13. A composition for nasal administration as described in claim 1, wherein the sugar and / or sugar alcohol is glycerol and the content of the sugar component is greater than 5% by volume.

14. 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 which is a sugar and / or a sugar alcohol.

15. The additive according to 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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