WO2022230485A1 - Vaccine composition for transpulmonary or transnasal administration - Google Patents

Abstract

A vaccine composition for transpulmonary or transnasal administration contains a nucleic-acid-containing carrier having such a structure that a complex comprising a nucleic acid encoding an antigen protein and a cationic molecule is coated with γ-polyglutamic acid or a salt thereof. In the vaccine composition for transpulmonary or transnasal administration, the charge ratio among the nucleic acid, the cationic molecule, and the γ-polyglutamic acid or the salt thereof may be 1:(2 to 8 inclusive):(4 to 16 inclusive). The cationic molecule may be 1,2-dioleoyl-3-trimethylammoniumpropane.

Description

Vaccine composition for pulmonary or nasal administration

The present invention relates to vaccine compositions for pulmonary or nasal administration.
This application claims priority based on Japanese Patent Application No. 2021-074100 filed in Japan on April 26, 2021, the content of which is incorporated herein.

Novel coronavirus infection (COVID-19; Coronavirus disease 2019) is caused by severe acute respiratory syndrome (Severe acute respiratory syndrome) coronavirus 2 (SARS-CoV-2) (hereinafter referred to as "SARS coronavirus-2") It is an infection caused by Several types of mRNA vaccines have been developed so far against the spread of novel coronavirus infections. An advantage of mRNA vaccines is that antigens are expressed immediately after being taken up into cells. Moreover, since it is not inserted into host DNA, it is also excellent in safety.

On the other hand, the inventors have so far proposed a drug-delivery complex containing a complex of a drug and a cationic molecule and an anionic molecule encapsulating it, and having a substantially uncharged or negative surface charge. wherein the anionic molecule is γ-polyglutamic acid or a salt thereof, a drug delivery complex (see, for example, Patent Document 1, etc.), or a complex consisting of a nucleic acid and dendrigraft poly-L-lysine is γ- We have developed a drug carrier for pulmonary delivery that is coated with polyglutamic acid, has a negative surface charge, and has a diameter of 50 nm to 250 nm (see, for example, Patent Document 2, etc.). These drug carriers have been shown to be less toxic to the body and capable of selectively delivering drugs to cells at the target site.

The currently developed mRNA vaccine is a formulation optimized for intramuscular administration, and although it can induce IgG antibodies in the blood and systemic cell-mediated immunity, it can induce infection and viral proliferation in local lungs. Inducing immunity is difficult. For this reason, the mRNA vaccines currently in use have been shown to be effective in preventing the onset and severity of COVID-19, but post-vaccination infections have also been confirmed. still have doubts.

Japanese Patent No. 5382682 Japanese Patent Application Laid-Open No. 2020-193160

The present invention has been made in view of the above circumstances, and provides a novel vaccine composition for pulmonary or nasal administration that can effectively induce immunity in the lungs or the whole body.

That is, the present invention includes the following aspects.
(1) A vaccine composition for pulmonary or nasal administration, comprising a nucleic acid-containing carrier in which a complex comprising a nucleic acid encoding an antigen protein and a cationic molecule is coated with γ-polyglutamic acid or a salt thereof.
(2) The transpulmonary or transnasal route according to (1), wherein the nucleic acid, the cationic molecule, and the γ-polyglutamic acid or its salt have a charge ratio of 1:2 or more and 8 or less:4 or more and 16 or less. Vaccine composition for administration.
(3) The vaccine composition for pulmonary or nasal administration according to (1) or (2), wherein the cationic molecule is 1,2-dioleoyl-3-trimethylammonium propane.
(4) The vaccine for pulmonary or nasal administration according to any one of (1) to (3), wherein the weight average molecular weight of the γ-polyglutamic acid or its salt is 2,000 or more and 3,000 or less. Composition.
(5) The vaccine composition for pulmonary or nasal administration according to any one of (1) to (4), wherein the nucleic acid is mRNA.
(6) The vaccine composition for pulmonary or nasal administration according to any one of (1) to (5), wherein the nucleic acid is mRNA encoding a viral antigen protein.
(7) The vaccine composition for pulmonary or nasal administration according to any one of (1) to (6), wherein the nucleic acid is mRNA encoding the spike protein of SARS coronavirus-2.
(8) any one of (1) to (7), wherein the nucleic acid comprises an mRNA encoding the receptor-binding domain of the SARS coronavirus-2 spike protein consisting of the amino acid sequence represented by SEQ ID NO: 1; A vaccine composition for pulmonary or nasal administration as described.
(9) The vaccine for pulmonary or nasal administration according to any one of (1) to (8), wherein the nucleic acid is operably linked to the 5' end or 3' end of a secretory signal sequence. Composition.
(10) The vaccine composition for pulmonary or nasal administration according to (9), wherein the secretory signal sequence is a sequence encoding a signal peptide derived from secretory luciferase.
(11) The vaccine composition for pulmonary or nasal administration according to any one of (1) to (10), further comprising an adjuvant.
(12) The vaccine composition for pulmonary or nasal administration according to (11), wherein the adjuvant is a CpG oligodeoxynucleotide.

According to the vaccine composition for pulmonary or nasal administration of the above aspect, it is possible to provide a novel vaccine composition for pulmonary or nasal administration that can effectively induce immunity in the lungs or the whole body.

1 is a schematic diagram showing an example of an antigen carrier contained in a vaccine composition for pulmonary or nasal administration of the present embodiment. FIG. 2 is a graph showing luciferase activity in lung tissue of mice to which each complex in Reference Example 1 was pulmonally administered. 1 is a graph showing luciferase activity in lung tissue of mice to which each nucleic acid-containing carrier in Reference Example 1 was pulmonally administered. 2 is an image showing the fluorescence of rhodamine and the luminescence of luciferin oxidized by luciferase in each organ of mice to which a nucleic acid-containing carrier and rhodamine-labeled phospholipid in Reference Example 1 were pulmonally administered. 1 is a graph showing luciferase activity in each organ of mice to which a nucleic acid-containing carrier in Reference Example 1 was pulmonally administered. 2 is a graph showing luciferase activity in the lung tissue of mice to which the nucleic acid-containing carrier in Reference Example 1 was pulmonally administered at each dosage. 2 is a graph showing changes over time in luciferase activity in lung tissue of mice to which a nucleic acid-containing carrier in Reference Example 1 was pulmonally administered. Fig. 2 shows the results of measuring anti-OVA IgG antibody and anti-OVA IgA antibody in serum and bronchoalveolar lavage fluid of mice pulmonally administered with a carrier containing mRNA encoding ovalbumin (OVA) in Reference Example 2 using ELISA. graph. 1 is a diagram showing the structure of each mRNA in Example 1. FIG. 1 is a diagram showing the results of measuring the expression levels of antigen proteins in the lysate and culture supernatant of HepG2 cells transfected with mRNA encoding each antigen protein in Example 1 by Western blotting. FIG. 1 is a graph showing the results of evaluating the inducibility of cell-mediated immunity in the lung and spleen of mice to which each nucleic acid-containing carrier in Example 1 was pulmonally administered. 1 is a graph showing the results of evaluating the induction of humoral immunity with serum and bronchoalveolar lavage fluid of mice to which each nucleic acid-containing carrier in Example 1 was pulmonally administered. 1 is a graph showing the results of evaluating the inducibility of cell-mediated immunity in the lung and spleen of mice to which each nucleic acid-containing carrier in Example 1 was pulmonally administered. 1 is a graph showing the results of evaluating the induction of humoral immunity with serum and bronchoalveolar lavage fluid of mice to which each nucleic acid-containing carrier in Example 1 was pulmonally administered. 2 is a graph showing the results of evaluating the induction of humoral immunity with serum and bronchoalveolar lavage fluid of mice to which a nucleic acid-containing carrier and various adjuvants in Example 2 were pulmonally administered. 2 is a graph showing the results of evaluating the induction of humoral immunity with serum and bronchoalveolar lavage fluid of mice to which each nucleic acid-containing carrier in Example 4 was pulmonally administered. FIG. 10 is a graph showing the results of evaluation of serum-induced humoral immunity induction and splenic cell-mediated immunity induction of mice to which each nucleic acid-containing carrier was pulmonally administered in Example 6. FIG. Fig. 10 is a graph showing the results of evaluating the inducibility of cell-mediated immunity in the lungs and spleens of mice to which each nucleic acid-containing carrier in Example 7 was administered through the lungs or through the nose.

<<Vaccine composition for pulmonary or nasal administration>>
A vaccine composition for pulmonary or nasal administration according to one embodiment of the present invention (hereinafter referred to as "vaccine composition for pulmonary or nasal administration of this embodiment") comprises a nucleic acid encoding an antigen protein and a cation. A complex consisting of a sexual molecule contains a nucleic acid-containing carrier coated with γ-polyglutamic acid or a salt thereof.

The vaccine composition for transpulmonary or nasal administration of the present embodiment can effectively induce immunity in the lungs or the whole body by having the above configuration. Immunity here includes cell-mediated immunity and humoral immunity. Both cell-mediated immunity and humoral immunity can be effectively induced.

In addition, in the vaccine composition for pulmonary or nasal administration of the present embodiment, the nucleic acid is encapsulated in a nucleic acid-containing carrier and can maintain a stable structure. Therefore, conventional RNA vaccines are basically stored frozen at −80° C., and it is recommended that they be used within a short period of time, such as within 6 hours after thawing. Vaccine compositions can be stored more stably even when the nucleic acid is easily degraded by RNA or the like.

Cell-mediated immunity refers to an immune mechanism that uses cells as the main effectors in eliminating foreign substances such as pathogens themselves, virus-infected cells, and cancer cells. It is an elimination mechanism by immunocompetent cells such as macrophages, cytotoxic T cells (CTL, killer T cells), and natural killer cells (NK cells). On the other hand, humoral immunity refers to an immune system centered on B cells and antibodies. Cytokines produced by helper T cells (Th2 cells) stimulate B cells to differentiate into plasma cells and produce large amounts of antibodies, which circulate in body fluids and spread throughout the body. . In addition, some of the stimulated B cells become memory B cells that store antigen information, and when reinfected, they react more quickly than the initial response and have higher affinity for the antigen. Antibodies can be produced in large amounts.

Next, each component contained in the vaccine composition for pulmonary or nasal administration of the present embodiment will be described in detail below.

<Nucleic acid-containing carrier>
FIG. 1 is a schematic diagram showing an example of a nucleic acid-containing carrier 10. FIG.
A nucleic acid-containing carrier 10 is obtained by coating a complex 3 composed of a nucleic acid 1 encoding an antigen protein and a cationic molecule 2 with γ-polyglutamic acid or a salt 4 thereof.

The size of the nucleic acid-containing carrier 10 is nanoscale. Specifically, the average particle diameter of the nucleic acid-containing carrier 10 can be 1 nm or more and 1000 nm or less, preferably 10 nm or more and 500 nm or less, and 30 nm. It is more preferably 300 nm or less, and further preferably 50 nm or more and 150 nm or less.
When the average particle diameter is at least the above lower limit, a more sufficient amount of nucleic acid can be contained, and when it is at most the above upper limit, more sufficient retention can be achieved, and , more efficiently deliver nucleic acids to cells around the lungs and nose.
The average particle size can be calculated from the scattering intensity distribution obtained using, for example, a dynamic light scattering measurement device.

The charge ratio of the nucleic acid 1, the cationic molecule 2, and γ-polyglutamic acid or its salt 4 (the phosphate group of the nucleic acid, the cationic group (eg, amino group) of the cationic molecule, and the carboxyl group of γ-polyglutamic acid molar ratio) is preferably 1:2 or more and 8 or less: 4 or more and 16 or less, more preferably 1:2 or more and 6 or less: 6 or more and 10 or less, and further preferably 1:4:8. preferable. When the charge ratio is within the above numerical range, it is possible to further suppress the formation of fine particles composed of surplus lipids and macromolecules that do not contain nucleic acids.

The nucleic acid-containing carrier 10 has a neutral to negative surface charge.

[Nucleic acid]
A nucleic acid encodes an antigen protein.

The type of nucleic acid is not particularly limited, and includes, for example, DNA, RNA, chimeric nucleic acids of DNA and RNA, DNA/RNA hybrids, and the like. In addition, the nucleic acid may be single-stranded or more and three-stranded or less, preferably single-stranded or double-stranded. Nucleic acids may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers with non-nucleotide backbones (such as commercially available peptide nucleic acids (PNA)) or other oligomers containing special linkages. (However, the oligomer contains nucleotides with a configuration that permits base pairing and base attachment as found in DNA and RNA). Furthermore, those with known modifications, such as those with labels known in the art, those with caps, those that are methylated, those in which one or more natural nucleotides are replaced with analogues, Intramolecular nucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages or sulfur-containing linkages (e.g., phosphorothioates, phosphorothioates, dithioates, etc.), such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.), sugars (e.g., monosaccharides, etc.), intercurrent compounds ( acridine, psoralen, etc.), those containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those containing modified linkages. (eg, α-anomeric nucleic acid, etc.).

For example, the type of DNA can be appropriately selected depending on the purpose of use, and is not particularly limited, but examples include cDNA, chromosomal DNA, and the like. A form in which these cDNAs and chromosomal DNAs are introduced into plasmid DNAs may also be used. Circular DNA such as plasmid DNA can be appropriately digested with a restriction enzyme or the like and used as linear DNA.

In addition, the type of RNA can be appropriately selected depending on the purpose of use, and is not particularly limited. For example, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, Examples include ribosomal RNA and the like. Among them, mRNA is preferable.

The size of the nucleic acid is not particularly limited, and ranges from huge nucleic acid molecules such as chromosomes (such as artificial chromosomes) (for example, about 100 kb (p) in size) to low-molecular nucleic acids (for example, in about 5 b (p) in size). However, considering the efficiency of nucleic acid introduction into cells, it is preferably 15 kbp or less. For example, the size of macromolecular nucleic acids such as chromosomal DNA, plasmid DNA, and mRNA can be 2 kb (p) or more and 15 kb (p) or less, and can be 2 kb (p) or more and 10 kb (p) or less. It is preferably 4 kb(p) or more and 10 kb(p) or less. In addition, the size of relatively low-molecular-weight nucleic acids can be 5b(p) or more and 2000b(p) or less, preferably 10b(p) or more and 1000b(p) or less, and 15b(p) or more. It is more preferably 800b(p) or less. The unit for a single-stranded nucleic acid is b (base), while the unit for a double-stranded nucleic acid is bp (base pair).

Nucleic acids may be either naturally occurring or synthesized nucleic acids, but if they have a size of about 100b(p) or less, they can be synthesized by a commonly used automatic nucleic acid synthesizer by the phosphotriethyl method, the phosphodiester method, or the like. can be synthesized using

The type of antigen protein encoded by the nucleic acid is not particularly limited. origin, etc.). Among them, it is preferably a virus antigen protein. That is, the nucleic acid is preferably mRNA encoding a viral antigen protein.

The type of virus is not particularly limited, but includes, for example, SARS coronavirus-2, MERS coronavirus, influenza virus, cytomegalovirus, and the like. Among them, SARS coronavirus-2 is preferred.
More preferably, the SARS coronavirus-2 spike protein consists of the amino acid sequence represented by SEQ ID NO: 3, and the nucleic acid is mRNA encoding the SARS coronavirus-2 spike protein. The mRNA encoding the SARS coronavirus-2 spike protein consists of the base sequence represented by SEQ ID NO:4.
The SARS coronavirus-2 spike protein may be a full-length protein or a partial protein, but preferably comprises at least the receptor-binding domain of the SARS coronavirus-2 spike protein. The receptor binding domain of the SARS coronavirus-2 spike protein consists of the amino acid sequence represented by SEQ ID NO:1. More preferably, the nucleic acid comprises an mRNA encoding the receptor-binding domain of the SARS coronavirus-2 spike protein consisting of the amino acid sequence represented by SEQ ID NO:1. The mRNA encoding the receptor-binding domain of the spike protein of SARS coronavirus-2 consists of the base sequence represented by SEQ ID NO:2.

Preferably, the nucleic acid is operably linked to the 5' end of a secretory signal sequence. This makes it possible to more effectively induce immunity (particularly cell-mediated immunity) in the lungs or the whole body, as shown in the examples below. As used herein, the term “operably linked” means a nucleic acid expression control sequence (e.g., promoter, a series of transcription factor binding sites, a specific modification structure, etc.) and a nucleic acid to be expressed (in this embodiment, an antigen nucleic acid encoding a protein).
A secretory signal sequence means an amino acid sequence that encodes a secretory signal peptide, and is not particularly limited as long as it can function in a subject animal in which the nucleic acid is expressed. Secretory signals include, for example, a signal peptide (amino acid sequence: SEQ ID NO: 17, mRNA base sequence: SEQ ID NO: 18) derived from secretory luciferase (Lucia (registered trademark)), a signal peptide derived from IL-6 (amino acid Sequence: SEQ ID NO: 21, mRNA base sequence: SEQ ID NO: 22), and the like. Among them, the secretory signal sequence is preferably a sequence encoding a signal peptide derived from a secretory luciferase (Lucia (registered trademark)).

In addition, when the nucleic acid is DNA, a polyadenylation signal required for polyadenylation of the 3' end of mRNA may be operably linked downstream (3' side) thereof. Examples of polyadenylation signals include polyadenylation signals contained in the above-mentioned virus-derived, various human or non-human animal-derived genes, e.g., SV40 late gene or early gene, rabbit β-globin gene, bovine growth hormone gene, human A3. Examples include polyadenylation signals of adenosine receptor genes and the like.
In addition, when the nucleic acid is DNA, the splicing signal, the enhancer region, a part of the intron, etc. are placed upstream (5′ side) of the promoter region, the promoter region and the translation region, in order to highly express the antigen protein in vivo. It may be ligated between or downstream (3' side) of the translation region.
Alternatively, when the nucleic acid is RNA, a cap structure may be operably linked upstream (5' side) or a polyadenine (poly A) chain downstream (3' side).
In addition to the translational region, the nucleic acid can further contain an untranslated region within a range that does not impede the effects of the vaccine composition for pulmonary or nasal administration of the present embodiment.

[Cationic molecule]
The cationic molecule may be one that can form a complex by electrostatic interaction with nucleic acids, and examples thereof include cationic polymers and cationic lipids.

Examples of cationic polymers include polyethyleneimine (hereinafter sometimes abbreviated as "PEI"); polycationic polysaccharides such as chitin and chitosan; polycationic polypeptides such as polylysine, polyarginine, polyornithine, and protamine. etc., but not limited to these.

Examples of cationic lipids include phosphatidylcholines such as soybean phosphatidylcholine, egg yolk phosphatidylcholine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine; phosphatidylethanolamines such as distearoylphosphatidylethanolamine; , sphingomyelin, egg yolk lecithin, soybean lecithin, hydrogenated phospholipids; A quaternary ammonium group such as an amino group, an alkylamino group, a dialkylamino group, a trialkylammonium group, a monoacyloxyalkyl-dialkylammonium group, or a diacyloxyalkyl-monoalkylammonium group is introduced into a glycosphingolipid such as a ganglioside. lipids; N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium (DOTMA), didodecyldimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium Propane (DOTAP), 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), dioleoyl-3-dimethylammonium propane (DODAP), dioctadecyl-dimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl -3-dimethylhydroxyethylammonium (DMRIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 3β -N-(N',N'-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol, O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine chloride, etc. , but not limited to.

Among them, cationic lipids are preferred, and 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP) is more preferred.

These cationic molecules may be prepared by known methods or may be commercially available products.

[γ-polyglutamic acid or its salt]
Examples of salts of γ-polyglutamic acid include γ-polyglutamic acid and alkali metal atoms such as sodium, potassium and lithium; , tertiary amines such as ethanolamine; and salts with quaternary amines such as tetramethylamine and tetraethylamine.

The weight average molecular weight of γ-polyglutamic acid or a salt thereof can be 500 or more and 100,000 or less, preferably 1,000 or more and 50,000 or less, and 1,500 or more and 8,000 or less. is more preferable, and more preferably 2,000 or more and 3,000 or less. When the weight-average molecular weight is at least the above lower limit, more stable fine particles can be formed. It can be suppressed and the antigen protein can be expressed more effectively.
A weight average molecular weight can be measured by a gel permeation chromatography (GPC), for example.

γ-polyglutamic acid or a salt thereof may be prepared by a known method, or a commercially available product may be used.

<Adjuvant>
The vaccine composition for pulmonary or nasal administration of the present embodiment preferably further contains an adjuvant in addition to the nucleic acid-containing carrier. As a result, immunity (particularly humoral immunity) can be more effectively induced in the lungs or the whole body, as shown in the examples below.

The adjuvant is not particularly limited as long as it is commonly used in vaccines, and examples thereof include ligands for innate immune receptors and cyclic dinucleotides such as cyclic diguanylate monophosphate (c-di-GMP). . As used herein, the term "ligand" means a substance that specifically binds to a receptor, and in particular, substances that specifically bind to a receptor and exhibit various physiological actions can be used. Such substances are also called "agonists".
Examples of innate immune receptors include toll-like receptors (TLR), RIG-I-like receptors (RIG-I-like receptors; RLR), NOD-like receptors (NOD-like receptors; NLR ), C-type lectin receptor (CLR), and the like.

TLR ligands include, for example, at least one TLR selected from the group consisting of TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9 Anything that interacts with can be selected as appropriate.
Examples of TLR-2 ligands include Pam3CSK4 and the like.
Examples of TLR-3 ligands include poly ICLC, polyinosine:polycytidylic acid (poly I:C), and the like.
Examples of TLR-4 ligands include R-type lipopolysaccharide, S-type lipopolysaccharide, paclitaxel, lipid A, monophosphoryl lipid A and the like.
Examples of TLR-5 ligands include flagellin and the like.
Examples of TLR-2 and TLR-6 ligands include MALP-2 and the like.
TLR-7 and TLR-8 ligands include, for example, resiquimod (R848), imiquimod (R837), gardiquimod, loxoribine, and the like.
Examples of TLR-9 ligands include CpG oligodeoxynucleotides and the like. CpG oligodeoxynucleotides include A-class TLR-9 ligand D35, B-class TLR-9 ligand K3, and the like.
Among them, CpG oligodeoxynucleotides are preferred as adjuvants.

<Other ingredients>
The vaccine composition for pulmonary or nasal administration of the present embodiment can be administered alone, or can be administered together with a pharmacologically acceptable carrier by conventional means for pulmonary or nasal administration. It can be used as a pharmaceutical composition. When used as a pharmaceutical composition for pulmonary or nasal administration, for example, the above nucleic acid-containing carrier and water or other physiologically acceptable liquids (e.g., physiological saline, phosphate-buffered saline ( PBS), etc., and may also contain physiologically acceptable excipients, vehicles, preservatives, stabilizers, binders, lyophilization aids, and the like.

<Method for producing a vaccine composition for pulmonary or nasal administration>
The vaccine composition for pulmonary or nasal administration of the present embodiment can be prepared by preparing the above nucleic acid-containing carrier and then mixing it with an adjuvant or a pharmacologically acceptable carrier, if necessary.

For the nucleic acid-containing carrier, first, a nucleic acid and a cationic molecule are mixed to form a complex (first step), and then the complex is mixed with γ-polyglutamic acid or a salt thereof to form a complex. It is obtained by coating with γ-polyglutamic acid (second step).

In the first step, the charge ratio between the nucleic acid and the cationic molecule (the phosphate group of the nucleic acid and the cationic group (eg, amino group) of the cationic molecule) is preferably 1:2 to 1:8, more preferably is 1: 2 to 1: 6, more preferably 1: 4, and incubated at 15 ° C. or higher and 25 ° C. or lower for 30 seconds or more and 300 minutes or less, preferably 10 minutes or more and 180 minutes or less. Assembled to create a composite. The concentration of the nucleic acid in the mixture can be appropriately set in consideration of the application, size (molecular weight), etc., and can be, for example, 0.01 ng/μL or more and 1000 ng/μL or less.

In the second step, the complex prepared in the first step and γ-polyglutamic acid are combined with the nucleic acid, the cationic molecule, and the charge ratio of γ-polyglutamic acid (the phosphate group of the nucleic acid, the cationic group of the cationic molecule (for example, , amino groups) and γ-polyglutamic acid carboxy groups) is preferably 1:2 or more and 8 or less: 4 or more and 16 or less, more preferably 1:2 or more and 6 or less: 6 or more and 10 or less, still more preferably is mixed at a ratio of 1:4:8 and incubated at 15 ° C. or higher and 25 ° C. or lower for 30 seconds or longer and 300 minutes or shorter, preferably 10 minutes or longer and 180 minutes or shorter to self-assemble the complex to form γ- Coat with polyglutamic acid. Thereby, a nucleic acid-containing carrier having a negative surface charge can be obtained.

When the adjuvant is a nucleic acid, in the first step, the nucleic acid may be mixed with the cationic molecule and encapsulated in the nucleic acid-containing carrier. Alternatively, the adjuvant may be appropriately mixed with the produced nucleic acid-containing carrier to obtain the vaccine composition for pulmonary or nasal administration of the present embodiment.

Subjects to whom the vaccine composition for pulmonary or nasal administration of the present embodiment is administered are animals classified as mammals including humans (monkeys, marmosets, mice, rats, cows, horses, cats, dogs, pigs, sheep, goats, rabbits, etc.).

The dose of the vaccine composition for pulmonary or nasal administration of the present embodiment can be appropriately selected in consideration of the type of administration subject (including age, sex, etc.), the type of nucleic acid, etc., but generally For example, in humans (with a body weight of 60 kg), the nucleic acid amount can be about 0.1 μg or more and 3000 mg or less per administration.

The specific form of transpulmonary or nasal administration of the vaccine composition for transpulmonary or nasal administration of the present embodiment is not particularly limited. Examples include direct administration of the vaccine composition for pulmonary administration of the present embodiment, inhalation using an aerosol, dry powder, or nebulizer, and administration using a humidifier. Examples of nasal administration include methods such as direct dripping of the vaccine composition for nasal administration of the present embodiment, aerosol, dry powder, and intranasal spraying using a nebulizer. In particular, these forms do not require injections and can play an important role in unmedicated villages and developing countries.
The frequency of administration may be a single administration of the above doses, and the above doses are given once every 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or every six months. It may be administered multiple times, such as two times or more.

Diseases to which the vaccine composition for pulmonary or nasal administration of the present embodiment is applied include, for example, influenza virus, respiratory syncytial virus, adenovirus, human metapneumovirus, cytomegalovirus, MERS coronavirus, and SARS coronavirus-2. Viral pneumonia caused by known viruses such as measles virus, varicella virus, etc., or viruses of emerging infectious diseases that will occur in the future; Examples include infectious respiratory diseases.
The vaccine composition for pulmonary or nasal administration of the present embodiment can effectively prevent the infectious respiratory diseases described above. That is, in one embodiment, the present invention provides a method for preventing infectious respiratory diseases, comprising pulmonary or nasal administration of the vaccine composition for pulmonary or nasal administration to a subject animal.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Reference example 1]
(Study of conditions for nucleic acid-containing carrier 1)
Using luciferase-encoding mRNA (SEQ ID NO: 5) as a model nucleic acid, the optimum type of cationic molecule and the molecular weight of γ-polyglutamic acid were investigated.

(1) Materials Synthetic mRNA containing mRNA encoding luciferase (SEQ ID NO: 5) was obtained from Trilink.
Examples of cationic molecules include polyethyleneimine (PEI) (manufactured by Sigma-Aldrich), dendrigraft poly-L-lysine (DGL) (manufactured by COLCOM, trade name “dendrigraft poly-L-lysine (G5)”, and 1 , 2-dioleoyl-3-trimethylammonium propane (DOTAP) (manufactured by NOF CORPORATION) was used.
Also, γ-polyglutamic acid (γ-PGA: weight average molecular weight 2,500, 5,000, and 7,500) (manufactured by Peptide Institute) was used.

(2) Preparation of a complex with an mRNA encoding luciferase and PEI, DGL, or DOTAP at a charge ratio (molar ratio of the phosphate group of the mRNA to the amino group of the cationic molecule) of 1:4 or 1:8. and allowed to stand at room temperature (about 25° C.) for 15 minutes to construct a complex composed of mRNA encoding luciferase and PEI, DGL, or DOTAP.

Then, these complexes were pulmonally administered to mice (5 weeks old) so that the amount of mRNA was 10 μg/mouse (50 μL/mouse as solution amount). Six hours after administration, the lungs were excised, homogenized in a buffer for cell disruption, the homogenate was centrifuged, and the supernatant was collected. A substrate (Picagene luminescence kit, manufactured by Toyo Benet Co., Ltd.) was added, and luciferase activity in the supernatant was measured as luminescence units (RLU) using a luminometer (Lumat LB 9507; manufactured by Berthold). The results are shown in FIG.

As shown in Figure 2, luciferase activity was particularly high in complexes using DOTAP as the cationic molecule. Therefore, it was decided to use DOTAP as the cationic molecule in subsequent tests.

(3) Production of nucleic acid-containing carrier 1
Next, a complex consisting of mRNA encoding luciferase and DOTAP was charged with the charge ratio of the mRNA, DOTAP and γ-PGA (the molar ratio of the phosphate group of mRNA, the amino group of the cationic molecule and the carboxyl group of γ-PGA γ-PGA with a weight average molecular weight of 2,500, 5,000, or 7,500 is mixed so that the ratio) is 1:4:8, and left to stand at room temperature (about 25° C.) for 15 minutes. , constructed a nucleic acid-containing carrier in which a complex consisting of mRNA encoding luciferase and DOTAP was coated with γ-PGA.

Then, these nucleic acid-containing carriers were pulmonally administered to mice (5 weeks old) so that the mRNA amount was 10 μg/mouse (the solution amount was 50 μL/mouse). Six hours after administration, the lungs were excised, homogenized in a buffer for cell disruption, the homogenate was centrifuged, and the supernatant was collected. A substrate (Picagene luminescence kit, manufactured by Toyo Benet Co., Ltd.) was added, and the luciferase activity in the supernatant was measured as luminescence units (RLU) using a luminometer. The results are shown in FIG.

As shown in FIG. 3, luciferase activity was particularly high in nucleic acid-containing carriers using γ-PGA with a weight average molecular weight of 2,500. Therefore, γ-PGA with a weight average molecular weight of 2,500 was used in subsequent tests.

In addition, a complex composed of mRNA encoding luciferase and DOTAP was obtained by adding a small amount of rhodamine-labeled phospholipid to a nucleic acid-containing carrier coated with γ-PGA having a weight average molecular weight of 2,500, and the amount of mRNA was It was pulmonary administered to mice (5 weeks old) at a concentration of 10 µg/mouse (50 µL/mouse as solution volume). Six hours after administration, various organs were excised from the mice, and fluorescence in the excised liver, kidney, spleen, heart, and lung was measured using an in vivo imaging device (IVIS Lumina II; manufactured by Caliper Life Sciences Inc.). The results are shown in FIG. 4 (left).
In addition, a complex consisting of mRNA encoding luciferase and DOTAP was coated with γ-PGA having a weight average molecular weight of 2,500, and the amount of mRNA was 10 μg/mouse (the solution amount was 10 μg/mouse). 50 μL/mouse) was pulmonary administered to mice (5 weeks old). Six hours after the administration, the substrate luciferin was administered intraperitoneally to 12 mg/mouse, various organs were excised from the mice, and the excised liver, kidney, spleen, heart, and lung were examined for luciferase expression levels. It was measured using a vivo imaging device (IVIS Lumina II; manufactured by Caliper Life Sciences Inc.). The results are shown in FIG. 4 (right side).

As shown in Figure 4, accumulation of nucleic acid-containing carriers and expression of luciferase were observed only in the lungs.

In addition, a complex consisting of mRNA encoding luciferase and DOTAP was coated with γ-PGA having a weight average molecular weight of 2,500, and the amount of mRNA was 10 μg/mouse (the solution amount was 10 μg/mouse). 50 μL/mouse) was pulmonary administered to mice (5 weeks old). Six hours after administration, the liver, kidney, spleen, heart and lung were excised, each homogenized in a cell disruption buffer, the homogenate was centrifuged, and the supernatant was collected. A substrate (Picagene luminescence kit, manufactured by Toyo Benet Co., Ltd.) was added, and luciferase activity in the supernatant was measured as luminescence units (RLU) using a luminometer (Lumat LB 9507; manufactured by Berthold). The results are shown in FIG.

As shown in Figure 5, luciferase activity was below the detection limit in organs other than the lung.

(4) Investigation of dosage of nucleic acid 0.5 μg, 5.0 μg, or 10 μg/mouse (50 μL/mouse as solution volume) was pulmonary administered to mice (5 weeks old). Six hours after administration, the lungs were excised, homogenized in a buffer for cell disruption, the homogenate was centrifuged, and the supernatant was collected. A substrate (Picagene luminescence kit, manufactured by Toyo Benet Co., Ltd.) was added, and the luciferase activity in the supernatant was measured as luminescence units (RLU) using a luminometer. The results are shown in FIG.

As shown in Figure 6, luciferase activity tended to improve as the amount of mRNA increased. Based on this result, the dose of mRNA was set at 10 μg/mouse (50 μL/mouse as solution volume) in subsequent tests.

(5) Investigation of protein expression duration Next, a complex consisting of mRNA encoding luciferase and DOTAP is coated with γ-PGA having a weight average molecular weight of 2,500. It was pulmonary administered to mice (5 weeks old) at a concentration of 10 µg/mouse (50 µL/mouse as solution volume). 6, 24, 48, or 72 hours after administration, lungs were excised, homogenized in buffer for cell disruption, the homogenate was centrifuged, and the supernatant was collected. A substrate (Picagene luminescence kit, manufactured by Toyo Benet Co., Ltd.) was added, and the luciferase activity in the supernatant was measured as luminescence units (RLU) using a luminometer. The results are shown in FIG.

As shown in FIG. 7, it was revealed that the expression of luciferase in the lungs of the nucleic acid-containing carrier persisted for about 48 hours.

[Reference example 2]
(Immune Induction Confirmation Test Using mRNA-Containing Carrier Encoding Ovalbumin)
Using ovalbumin as a model antigen protein, the immunity-inducing effect of nucleic acid-containing carriers was examined. Synthetic mRNA containing mRNA (SEQ ID NO: 6) encoding ovalbumin (OVA) was obtained from Trilink.

(1) Preparation of Nucleic Acid-Containing Carrier OVA-encoding mRNA and DOTAP were mixed so that the charge ratio (molar ratio between the phosphate group of the mRNA and the amino group of the cationic molecule) was 1:4. 25° C.) for 15 minutes to construct a complex consisting of mRNA encoding OVA and DOTAP.

Next, a complex consisting of mRNA encoding OVA and DOTAP was added to the charge ratio of mRNA, DOTAP and γ-PGA (the molar ratio of the phosphate group of mRNA, the amino group of the cationic molecule and the carboxyl group of γ-PGA γ-PGA (weight average molecular weight: 2,500) was mixed so that the ratio) was 1:4:8 and allowed to stand at room temperature (about 25°C) for 15 minutes to mix OVA-encoding mRNA and DOTAP. A nucleic acid-containing carrier coated with γ-PGA was constructed.

Next, the resulting nucleic acid-containing carrier was fed to mice (5 weeks old) at 2-week intervals for a total of 4 times so that the amount of mRNA was 10 μg/mouse (the amount of solution was 50 μL/mouse). It was administered pulmonary. As a control group, mice were administered with a nucleic acid-containing carrier in which only mRNA encoding OVA and a complex consisting of mRNA encoding luciferase and DOTAP were coated with γ-PGA having a weight average molecular weight of 2,500. Groups were also prepared.
Serum and bronchoalveolar lavage fluid were collected from mice two weeks after the last dose. OVA-specific IgG and IgA antibodies in serum and bronchoalveolar lavage fluid were measured by ELISA. Specifically, OVA was added to the immunoplate, incubated for a certain period of time, and then blocked. Mouse serum was added to this plate, incubated for a certain period of time, and then washed with PBS containing a surfactant. After that, HRP-modified anti-mouse IgG or IgA antibody was added and incubated for a certain period of time. After washing, HRP substrate was added and the amount of each antibody was measured. The results are shown in FIG.

As a result, as shown in FIG. 8, the production of antibodies specific to OVA was not observed in either the mRNA encoding OVA alone or the nucleic acid-containing carrier (carrier containing luciferase-encoding mRNA) containing no OVA-encoding mRNA. I didn't. On the other hand, carriers containing OVA-encoding mRNA increased not only IgG in serum but also IgG and IgA antibodies in bronchoalveolar lavage fluid. That is, it was revealed that both pulmonary and systemic cell-mediated immunity and humoral immunity can be induced by using the vaccine composition for transpulmonary or nasal administration of the present embodiment.

[Example 1]
(Immune Induction Confirmation Test Using mRNA-Containing Carrier Encoding Viral Antigen Protein)
Next, using the SARS coronavirus-2 spike protein as a virus antigen protein, the immunity-inducing effect of the nucleic acid-containing carrier was examined. The amino acid sequence of the spike protein of SARS coronavirus-2 is shown in SEQ ID NO:3, and the nucleotide sequence of mRNA encoding the spike protein of SARS coronavirus-2 is shown in SEQ ID NO:4. Synthetic mRNA containing the mRNA encoding the spike protein of SARS coronavirus-2 (SEQ ID NO: 4) was obtained from Trilink.

(1) Design of mRNA Encoding Antigen Proteins Synthetic mRNAs containing mRNAs encoding antigen proteins shown below were designed (see FIG. 9). For 3) to 5), sequences of various signal peptides are added.
1) S1 mRNA (amino acid sequence of S1: SEQ ID NO: 7, nucleotide sequence of S1 mRNA: SEQ ID NO: 8)
2) RBD (receptor binding domain) mRNA (amino acid sequence of RBD: SEQ ID NO: 1, base sequence of RBD mRNA: SEQ ID NO: 2)
3) IgE signal peptide (IgE)-RBD mRNA (amino acid sequence of IgE: SEQ ID NO: 9, base sequence of IgE mRNA: SEQ ID NO: 10, amino acid sequence of IgE-RBD: SEQ ID NO: 11, base sequence of IgE-RBD mRNA: sequence number 12)
4) RBD-diphtheria toxin T cell epitope (DTE) mRNA (amino acid sequence of DTE: SEQ ID NO: 13, base sequence of DTE mRNA: SEQ ID NO: 14, amino acid sequence of RBD-DTE: SEQ ID NO: 15, base of RBD-DTE mRNA Sequence: SEQ ID NO: 16)
5) Lucia (registered trademark) luciferase codon-optimized signal sequence (secretory Luc)-RBD mRNA (amino acid sequence of secretory Luc: SEQ ID NO: 17, base sequence of secretory Luc mRNA: SEQ ID NO: 18, secretory Luc-RBD Amino acid sequence of: SEQ ID NO: 19, base sequence of secretory Luc-RBD mRNA: SEQ ID NO: 20)

Next, the synthesized mRNAs of 1) to 5) above were introduced into HepG2 cells using Lipofectamine (registered trademark) messenger Max, and the expression of antigen proteins in the cell lysate was evaluated by Western blotting. In addition, the amount of protein in the culture supernatant of the secretory Luc-RBD was also measured by Western blotting. The results are shown in FIG.

As shown in FIG. 10, S1 protein and RBD-DTE showed low protein expression levels, whereas RBD and IgE-RBD showed distinct protein bands. Furthermore, in secretory Luc-RBD, expression of a large amount of antigenic proteins was observed, and antigenic proteins secreted in the culture supernatant were also detected.
From the above, it was confirmed that the antigen protein of SARS coronavirus-2 was synthesized from the designed mRNA.

(2) Preparation of Nucleic Acid-Containing Carrier Each of the mRNAs synthesized in (1) 1) to 5) and DOTAP at a charge ratio (molar ratio between the phosphate group of the mRNA and the amino group of the cationic molecule) of 1:4 and allowed to stand at room temperature (about 25° C.) for 15 minutes to construct a complex composed of each mRNA and DOTAP.

Next, in the complex composed of each mRNA and DOTAP, the charge ratio of mRNA, DOTAP and γ-PGA (the molar ratio of the phosphate group of mRNA, the amino group of the cationic molecule and the carboxyl group of γ-PGA) is γ-PGA (weight average molecular weight: 2,500) was mixed at a ratio of 1:4:8 and allowed to stand at room temperature (about 25°C) for 15 minutes to form a complex consisting of each mRNA and DOTAP. A nucleic acid-containing carrier coated with γ-PGA was constructed.

(3) Confirmation Test 1 for Immunity Induction Effect Using Nucleic Acid-Containing Carrier
Next, each of the nucleic acid-containing carriers obtained (S1 mRNA-containing carrier, RBD mRNA-containing carrier, IgE-RBD mRNA-containing carrier, and RBD-DTE mRNA-containing carrier) was added to an amount of mRNA of 10 μg/mouse (solution amount of 50 μL/mouse) was administered to mice (5 weeks old) by pulmonary administration a total of 4 times at 2-week intervals. Spleens and lungs were harvested from mice two weeks after the final dose. Lung cells and splenocytes were isolated from harvested lungs and spleens, respectively, and cultured in media containing fragment peptides of SARS coronavirus-2 spike protein. INF-γ secreted into the culture supernatant was measured as an index of cell-mediated immunity. The results are shown in FIG.

As shown in Figure 11, the administration of the nucleic acid-containing carrier partially activated cell-mediated immunity in the lungs and the whole body.

In addition, serum and bronchoalveolar lavage fluid were collected from the mice two weeks after the final administration. IgG antibodies specific for the SARS coronavirus-2 spike protein in serum and bronchoalveolar lavage fluid were measured by ELISA. Specifically, the SARS coronavirus-2 spike protein was added to the immunoplate, incubated for a certain period of time, and then blocked. Mouse serum was added to this plate, incubated for a certain period of time, and then washed with PBS containing a surfactant. After that, HRP-modified anti-mouse IgG antibody was added and incubated for a certain period of time. After washing, HRP substrate was added and the amount of antibody was measured. The results are shown in FIG.

As shown in Figure 12, administration of the nucleic acid-containing carrier did not increase IgG antibodies in serum and bronchoalveolar lavage fluid.

(4) Confirmation Test 2 for Immunity Induction Effect Using Nucleic Acid-Containing Carrier
Next, each of the obtained nucleic acid-containing carriers (RBD mRNA-containing carrier, RBD-DTE mRNA-containing carrier, and secretory Luc-RBD mRNA-containing carrier) was added to an amount of mRNA of 10 μg/mouse (50 μL/mouse as a solution amount). It was pulmonary administered to mice (5-week-old) for a total of 4 times at 2-week intervals (1 mouse). Spleens and lungs were harvested from mice two weeks after the final dose. Lung cells and splenocytes were isolated from harvested lungs and spleens, respectively, and cultured in media containing fragment peptides of SARS coronavirus-2 spike protein. INF-γ secreted into the culture supernatant was measured as an index of cell-mediated immunity. The results are shown in FIG.

As shown in Figure 13, the administration of the secretory Luc-RBD mRNA-containing carrier markedly increased local and systemic cell-mediated immunity in the lung.

In addition, serum and bronchoalveolar lavage fluid were collected from the mice two weeks after the final administration. IgG antibodies specific to the SARS coronavirus-2 spike protein in serum and bronchoalveolar lavage fluid were measured by ELISA using the same method as in (3) above. The results are shown in FIG.

As shown in Figure 14, administration of the secretory Luc-RBD mRNA-containing carrier increased 1gG antibody in only one case.

[Example 2]
(Humoral immunity induction confirmation test 1 using a carrier containing mRNA encoding a virus antigen protein and an adjuvant)
Next, the secretory Luc-RBD mRNA-containing carrier prepared in Example 1 was combined with various adjuvants to examine the inducibility of humoral immunity.

The secretory Luc-RBD mRNA-containing carrier obtained in Example 1 was mixed with the amount of mRNA of 10 μg/mouse (50 μL/mouse of solution) and polyinosine:polycytidylic acid (PolyI:C ) or 10 μg each of CpG oligodeoxynucleotides were simultaneously pulmonally administered to mice (5 weeks old) for a total of 4 times at 2-week intervals. Serum and bronchoalveolar lavage fluid were collected from mice two weeks after the last dose. IgG antibodies specific for the SARS coronavirus-2 spike protein in serum and bronchoalveolar lavage fluid were measured by ELISA using the same method as in Example 1 (3) above. The results are shown in FIG.

As shown in FIG. 15, the combined use of a secretory Luc-RBD mRNA-containing carrier and a CpG oligodeoxynucleotide as an adjuvant resulted in a reduction in IgG antibody production compared to administration of only a secretory Luc-RBD mRNA-containing carrier. It was found to increase more than 20 times.

From the above, it was clarified that both cell-mediated immunity and humoral immunity can be induced in the lung and the whole body by using the vaccine composition for transpulmonary or nasal administration of the present embodiment.

[Example 3]
(Acute Toxicity Test of mRNA-Containing Carrier Encoding Viral Antigen Protein)
Next, in order to confirm the safety of the nucleic acid-containing carrier to test animals, the RBD mRNA-containing carrier prepared in Example 1 was used to conduct an acute toxicity test by intratracheal administration to rats, which was commissioned to the Japan Food Research Laboratories. Carried out. Specifically, the contents of the test are as shown below.

A test group (3 groups) and a control group (1 group) were set. In the test groups, test solutions prepared so that the mRNA concentrations in the RBD mRNA-containing carrier were 0.2, 0.1 and 0.05 mg/mL were administered at doses of 0.2, 0.1 and 0. Each was administered as a single intratracheal dose of .05 mg/kg. A control group was similarly administered a 5 w/v % glucose solution containing Tris as a solvent control. The observation periods were 1 and 14 days, and 4 males and 4 females/group were used for the 1-day observation period, and 6 males and 6 females/group were used for the 14-day observation period.
General condition observation and weight measurement were performed during the test period. At the end of the observation period, hematological and blood biochemical tests will be performed during the 1-day observation period, and organ weight measurement, bronchoalveolar lavage fluid test, macroscopic examination of systemic organs, and pathological tissue will be performed during the 14-day observation period. A medical examination was performed.

As a result, no deaths or abnormal general conditions were observed in any of the test animals. In histopathological examination, no changes suggestive of toxicity of the RBD mRNA-containing carrier were observed.

Based on the above, under the conditions of this test, the median lethal dose (LD50 value) of the nucleic acid-containing carrier was evaluated as exceeding 0.2 mg/kg as the amount of mRNA for both males and females.

[Example 4]
(Humoral immunity induction confirmation test 2 using a carrier containing mRNA encoding a virus antigen protein and an adjuvant)
Next, the secretory Luc-RBD mRNA-containing carrier prepared in Example 1 was combined with an adjuvant to examine the effect of inducing immunity, and spike proteins introduced with two mutations used in commercially available mRNA vaccines. Comparison with full-length mRNA was performed.

SEQ ID NO: 23 shows the base sequence of the spike protein full-length mRNA with two mutations introduced. Using the full-length spike protein mRNA into which two mutations had been introduced, a carrier containing full-length spike protein mRNA into which two mutations had been introduced was produced in the same manner as in (2) of Example 1 above.

The secretory Luc-RBD mRNA-containing carrier obtained in Example 1, or the spike protein full-length mRNA-containing carrier into which the two mutations were introduced, was 10 μg/mouse as the amount of mRNA (50 μL/mouse as the solution amount). 1 mouse) and 10 µg of CpG oligodeoxynucleotide (D35) as an adjuvant were simultaneously administered to mice (6 weeks old) via the lungs for a total of 4 times at 2-week intervals. Serum and bronchoalveolar lavage fluid were collected from mice two weeks after the last dose. IgG antibodies specific for the SARS coronavirus-2 spike protein in serum and bronchoalveolar lavage fluid were measured by ELISA using the same method as in Example 1 (3) above. The results are shown in FIG.

As shown in FIG. 16, particularly remarkable antibody induction (humoral immunity induction) was observed only when the secretory Luc-RBD mRNA-containing carrier was used in combination with CpG oligodeoxynucleotide (D35) as an adjuvant.

[Example 5]
(Test to confirm the effect of neutralizing antibody on suppressing virus proliferation)
The effect of neutralizing antibody contained in the serum of mice inoculated with the vaccine composition for pulmonary or nasal administration of the present embodiment to suppress virus proliferation was examined.

The secretory Luc-RBD mRNA-containing carrier obtained in Example 1 was used alone or as an adjuvant with CpG oligodeoxynucleotide at 10 μg/mouse as the amount of mRNA (50 μL/mouse as the amount of solution). (D35) 10 µg was simultaneously administered to mice (6 weeks old) by pulmonary administration every 2 weeks for a total of 4 times. Serum was collected from mice two weeks after the last dose. ACE2 and FcγR co-expressing cells were infected with SARS-CoV-2 in 10-fold, 40-fold, 160-fold, or 640-fold dilutions of serum and neutralizing activity was assessed by the plaque assay. The results are shown in Table 1 below. In Table 1, + means an increase in plaques, +/- means no change, and - means a decrease in plaques. The control is a group with serum from mice that received vehicle only.

As shown in Table 1, viral plaque reduction (neutralizing activity) was observed in the sera of 3 out of 4 mice that used a secretory Luc-RBD mRNA-containing carrier in combination with CpG oligodeoxynucleotide (D35) as an adjuvant. Admitted. In addition, no increase in plaque (antibody-dependent enhancement of infection) was observed.

[Example 6]
(Humoral immunity induction confirmation test 3 using a carrier containing mRNA encoding a virus antigen protein and an adjuvant)
Next, the secretory Luc-RBD mRNA-containing carrier prepared in Example 1 was combined with three types of nucleic acid-based adjuvants to examine the effect of inducing immunity. Three adjuvants include D35, a CpG oligodeoxynucleotide and A-class TLR-9 ligand, K3, a CpG oligodeoxynucleotide and B-class TLR-9 ligand, and cyclic diguanylate monophosphate. (c-di-GMP) was used. It is known that D35 has a strong effect of stimulating cell-mediated immunity, and K3 has a strong effect of stimulating humoral immunity. In addition, c-di-GMP is known to act on nucleic acid receptors in cells to induce immunity.

The secretory Luc-RBD mRNA-containing carrier obtained in Example 1 was mixed with the amount of mRNA of 10 μg/mouse (50 μL/mouse of solution) and CpG oligodeoxynucleotide (D35 or K3 ), or 10 μg each of c-di-GMP was simultaneously pulmonally administered to mice (6 weeks old) for a total of 4 times at 2-week intervals. Serum and spleens were collected from mice two weeks after the last dose. IgG antibodies specific to the spike protein of SARS coronavirus-2 in serum were measured by ELISA using the same method as in (3) of Example 1 above. Splenocytes were isolated from harvested spleens and cultured in medium containing fragment peptides of SARS-CoV-2 spike protein. INF-γ secreted into the culture supernatant was measured as an index of cell-mediated immunity. The results are shown in FIG.

As shown in FIG. 17, when K3 was used as an adjuvant, serum IgG antibody showed the highest level (humoral immunity was particularly significantly induced), and INF-γ was the highest when D35 was used. (cell-mediated immunity is especially markedly induced).

[Example 7]
(Administration method and adjuvant effect confirmation test on immune induction effect)
The effects of pulmonary or nasal administration methods and adjuvants on cell-mediated immunity induction were investigated.

The secretory Luc-RBD mRNA-containing carrier obtained in Example 1 was mixed with the amount of mRNA of 10 μg/mouse (50 μL/mouse of solution) and CpG oligodeoxynucleotides (D35, K3 , or a mixture thereof) was simultaneously administered to mice (6 weeks old) by pulmonary or nasal administration for a total of 4 times at 2-week intervals. Two weeks after the last dose, spleens and lungs in the case of pulmonary administration were harvested from the mice. Splenocytes and lung cells were isolated from the harvested spleen and lung, respectively, and cultured in a medium containing fragment peptides of SARS-CoV-2 spike protein. INF-γ secreted into the culture supernatant was measured as an index of cell-mediated immunity. The results are shown in FIG.

As shown in FIG. 18, when a secretory Luc-RBD mRNA-containing carrier was used in combination with CpG oligodeoxynucleotide (D35) as an adjuvant, systemic and pulmonary cell-mediated immunity was significantly increased after transpulmonary administration.
On the other hand, when the secretory Luc-RBD mRNA-containing carrier was used in combination with CpG oligodeoxynucleotide (K3) as an adjuvant, no significant increase in cell-mediated immunity was observed after transpulmonary administration.

In addition, when a secretory Luc-RBD mRNA-containing carrier and one type of CpG oligodeoxynucleotide (D35) as an adjuvant are used in combination, and when a secretory Luc-RBD mRNA-containing carrier and two types of CpG oligodeoxynucleotides are used as adjuvants (mixture of D35 and K3) significantly increased systemic cell-mediated immunity after intranasal administration.

According to the vaccine composition for transpulmonary or transnasal administration of the present embodiment, immunity can be effectively induced in the lungs or the whole body.

1... nucleic acid encoding antigen protein, 2... cationic molecule, 3... complex, 4... γ-polyglutamic acid or its salt, 10... nucleic acid-containing carrier.

Claims (12)

1. A vaccine composition for pulmonary or nasal administration, comprising a nucleic acid-containing carrier in which a complex consisting of a nucleic acid encoding an antigen protein and a cationic molecule is coated with γ-polyglutamic acid or a salt thereof.
2. The vaccine for pulmonary or nasal administration according to claim 1, wherein the nucleic acid, the cationic molecule, and the γ-polyglutamic acid or its salt have a charge ratio of 1:2 or more and 8 or less:4 or more and 16 or less. Composition.
3. The vaccine composition for pulmonary or nasal administration according to claim 1 or 2, wherein the cationic molecule is 1,2-dioleoyl-3-trimethylammonium propane.
4. The vaccine composition for pulmonary or nasal administration according to any one of claims 1 to 3, wherein the γ-polyglutamic acid or its salt has a weight average molecular weight of 2,000 or more and 3,000 or less.
5. The vaccine composition for pulmonary or nasal administration according to any one of claims 1 to 4, wherein the nucleic acid is mRNA.
6. The vaccine composition for pulmonary or nasal administration according to any one of claims 1 to 5, wherein the nucleic acid is an mRNA encoding a viral antigen protein.
7. The vaccine composition for pulmonary or nasal administration according to any one of claims 1 to 6, wherein the nucleic acid is mRNA encoding the SARS coronavirus-2 spike protein.
8. The transpulmonary or according to any one of claims 1 to 7, wherein the nucleic acid comprises an mRNA encoding the receptor-binding domain of the SARS coronavirus-2 spike protein consisting of the amino acid sequence represented by SEQ ID NO: 1. A vaccine composition for nasal administration.
9. The vaccine composition for pulmonary or nasal administration according to any one of claims 1 to 8, wherein the nucleic acid has a secretory signal sequence operably linked to the 5' end or 3' end.
10. The vaccine composition for pulmonary or nasal administration according to claim 9, wherein the secretory signal sequence is a sequence encoding a signal peptide derived from secretory luciferase.
11. The vaccine composition for pulmonary or nasal administration according to any one of claims 1 to 10, further comprising an adjuvant.
12. The vaccine composition for pulmonary or nasal administration according to claim 11, wherein the adjuvant is a CpG oligodeoxynucleotide.

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