Immune adjuvant comprising imiquimod and nod2 receptor-stimulating component or bacterium

Abstract

The present invention relates to a vaccine immune adjuvant that utilizes the synergy between imiquimod and a Nod2 receptor-stimulating component or a Nod2 receptor-stimulating bacterium. According to one aspect of the present invention, a vaccine immune adjuvant composition comprising, as active ingredients, imiquimod and a Nod2 receptor-stimulating component or a Nod2 receptor-stimulating bacterium can enhance the efficacy of a vaccine to be greater than that of the administration of imiquimod alone, and thus can be effectively used as a complex immune adjuvant for increasing immune responses of various vaccines such as an SARS-CoV-2 virus vaccine, an influenza A virus vaccine and an anticancer vaccine against melanoma.

Description

Immune adjuvants containing imiquimod and NOD2 receptor stimulating components or bacteria

[0001] The present invention relates to a vaccine immune adjuvant utilizing the synergy of imiquimod and a Nod2 receptor stimulating component or a Nod2 receptor stimulating bacterium.

[0002] This application was carried out with the support of the following tasks.

[0003] [Ministry Name] Defense Acquisition Program Administration

[0004] [Research Management Specialist Agency] Agency for Defense Development

[0005] [Research Project Name] Future Challenge Defense Technology Research and Development Project

[0006] [Research Project Title] Development of an Innovative Vaccine Platform Technology for Rapid Response to Future Biological Weapons

[0007] [Contribution rate] 1 / 2

[0008] [Host Organization] Seoul National University Industry-Academic Cooperation Foundation

[0009] [Research Period] January 1, 2024 - December 31, 2024

[0010]

[0011] [Ministry Name] Seoul National University

[0012] [Research Management Specialist Organization] Seoul National University Industry-Academic Cooperation Foundation

[0013] [Research Project Name] In-school Research Project (Seoul National University Creative Leading Young Researcher Support Project)

[0014] [Research Project Name] Development of a Symbiotic Bacteria-Based Composite Mucosal Immunopotentiator and Vaccine Platform

[0015] [Contribution rate] 1 / 2

[0016] [Host Organization] Seoul National University Industry-Academic Cooperation Foundation

[0017] [Research Period] January 1, 2024 - June 30, 2024

[0018] An adjuvant is a general term for substances that enhance the efficacy of a vaccine. These adjuvants are essential for enhancing the immunogenicity and duration of low-immunogenicity antigens, improving immune responses in populations with poor responses, and reducing the amount of vaccine antigen required.

[0019] Within living organisms, the immune system refers to the biological structures and processes that protect organisms from disease by detecting and eliminating pathogens and tumor cells. The immune system can be broadly divided into the innate immune system (intrinsic immune system) and the adaptive immune system (adaptive immune system).

[0020] The innate immune system is a non-specific defense mechanism that protects the host from infection. It responds immediately to specific pathogens without remembering them. The innate immune system includes complement, found in the skin, mucous tissue, stomach acid, and blood, which block the invasion of antigens. Cells include macrophages, which are responsible for phagocytosis, polymorphonuclear leukocytes, and natural killer (NK) cells, which kill infected cells. In fact, most infections by invading pathogens are initially protected by this innate immune system.

[0021] In contrast, the adaptive immune system is antigen- or pathogen-specific. To induce a specific immune response, it must recognize non-self antigens through antigen presentation. The adaptive immune system is capable of specific immune responses to specific antigens or antigen-infected cells. Furthermore, memory cells within the adaptive immune system can recall previously performed immune responses, enabling rapid elimination of the same pathogen when it invades the body multiple times.

[0022] Meanwhile, microorganisms possess pathogen-associated molecular patterns (PAMPs) composed of glycoproteins such as lipopolysaccharides (LPS), β-1,3-glucan, and peptidoglycans (PGN). These PAMPs are recognized by specific proteins of the host immune system, such as pattern recognition receptors (PRRs). Each PRR recognizes the appropriate PAMPs present on the pathogen surface and forms a complex, which triggers a series of immune responses, including phagocytosis, nodule formation, encapsulation, activation of the proteinase cascade, and synthesis of antimicrobial peptides.

[0023] Toll-like receptors (TLRs) are representative PRRs, and TLR agonists are being developed as various vaccine adjuvants due to their strong activity against immune cells. Another type of PRR, Nod-like receptors (NLRs), contribute significantly to immune responses in vivo alongside TLRs. Nod (nucleotide-binding oligomerization domain-containing protein) 1 and Nod2 are known to play a crucial role in the innate response by recognizing fragments of peptidoglycan present on the surface of most bacterial cell walls. Nod1 is expressed in epithelial cells of the stomach and large intestine, macrophages of the pancreas, lungs, kidneys, spleen, dendritic cells, etc., and is known to recognize dipeptides or tripeptides, which are stem peptides of DAP (diaminopimelic acid)-type peptidoglycan of Gram-negative bacteria and some Gram-positive bacteria (Hisamatsu T et al., J. Biol. Chem., 278:32962, 2003). Nod2 is a protein distributed in bone marrow-derived macrophages, neutrophils, dendritic cells, and Paneth cells of the small intestine, and has the characteristic of being expressed in a more limited area than Nod1, and its expression is further induced by inflammatory cytokines such as TNF-α and IFN-γ, so its expression is induced more during infection than in normal times.

[0024] The most widely known agonist (ligand) recognized by Nod2 is muramyl dipeptide (MDP), a structure common to the peptidoglycan components of both Gram-negative and Gram-positive bacteria. Detection of these agonists by the Nod2 receptor leads to the expression and secretion of inflammatory cytokines and chemokines, which also contribute to the initiation of adaptive immunity. Specific stimulation of Nod2 is known to lead to the induction of an immune profile dominated by Th2 cells, and when stimulated together with TLR agonists, it is known to have a synergistic effect inducing Th1, Th2, and Th17 immune responses.

[0025] The vast majority of the microbiome inhabiting our bodies resides in mucosal pathways, including the nasal and gastrointestinal tracts. They play a beneficial role in the body, including strengthening mucosal barrier function, stimulating immune responses, preventing pathogens from establishing themselves through competition, and producing beneficial metabolites. Furthermore, it has been reported that the microbiome can influence the efficacy of parenteral and mucosal vaccines.

[0026] Commercially available nasal mucosal vaccines are limited to certain age groups with established immunity and have lower preventive efficacy compared to injectable vaccines. While immune adjuvants can be used to address these issues, most current vaccine adjuvants are limited to intramuscular injections.

[0027] Accordingly, the present invention proposes an immune adjuvant that can increase the usability of a mucosal vaccine by using a Nod2 receptor stimulating component or bacteria with high Nod2 receptor stimulating activity residing in the mucosa together with imiquimod, an immune adjuvant, to enhance the mucosal immune enhancement effect.

[0028] One aspect is to provide an immune adjuvant composition comprising, as a first active ingredient, an immune adjuvant, and as a second active ingredient, at least one selected from the group consisting of a Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacteria; and comprising the first active ingredient or the second active ingredient, wherein the first active ingredient or the second active ingredient is administered in combination with the second active ingredient or the first active ingredient, respectively.

[0029] Another aspect is to provide a vaccine composition comprising an antigen and the above-described immune adjuvant composition.

[0030] Another aspect provides a method for boosting the immunity of a subject, comprising the steps of: administering to the subject an effective amount of an immune adjuvant; and administering to the subject in need thereof at least one selected from the group consisting of a Nod2 receptor stimulating component and a Nod2 receptor stimulating bacteria.

[0031] Another aspect provides a use of an immune adjuvant composition comprising, as a first active ingredient, an immune adjuvant, and, as a second active ingredient, at least one selected from the group consisting of a Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacterium.

[0032] Another aspect provides a use of at least one selected from the group consisting of an immune adjuvant as a first active ingredient for the manufacture of a vaccine composition, and a Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacterium as a second active ingredient.

[0033] The terminology used in this specification is intended to appropriately express preferred embodiments of the present invention and may vary depending on the intent of the user or operator, or the customary practices in the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents of this specification as a whole. Throughout this specification, when a part is said to "include" a certain component, unless otherwise specifically stated, this does not mean that other components are excluded, but rather that other components may be included.

[0034] Unless otherwise defined, all technical terms used in this invention may be used with the same meanings as commonly understood by those skilled in the art in the relevant fields of the present invention. While preferred methods and samples are described herein, similar or equivalent methods may also be included within the scope of the present invention.

[0035] One aspect provides an immune adjuvant composition comprising, as a first active ingredient, an immune adjuvant, and as a second active ingredient, at least one selected from the group consisting of a Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacteria; and comprising the first active ingredient or the second active ingredient, wherein the first active ingredient or the second active ingredient is administered in combination with the second active ingredient or the first active ingredient, respectively.

[0036] The above immune adjuvants include imiquimod, resiquimod (R848), dactolisib (RTB101), gardiquimod, sumanirole (PNU-95,666), lipopolysaccharide (LPS), monophosphoryl Lipid A (MPL), GLA, AS04 (a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)), Poly I:C, CpG oligonucleotides, C-type lectin ligands (TDB), aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS03 (a mixture of DL-αtocopherol, squalene, and polysorbate 80, an emulsifier), flagellin, AS01, AS02, ISCOMs, ISCOMMATRIX, IL-12, GM-CSF (Granulocyte-macrophage colony-stimulating factor), saponins (QS-21), α-galactosylceramide, quil A, IC31 (oligo nucleotide and cationic peptide), CFA01 (cationic liposome), and GLA-SE (oil-in-water emulsion of MPL and glucopyranosyl lipid) may be used, but are not limited thereto. In a preferred embodiment, the immune adjuvant may be imiquimod.

[0037] In one specific example, the imiquimod may be represented by the following chemical formula 1:

[0038] [Chemical Formula 1]

[0039] .

[0040]

[0041] In one specific example, the Nod2 receptor stimulating component may be a bacterial cell wall or a cell wall-derived substance.

[0042] The above bacteria may be gram-positive or gram-negative bacteria.

[0043] The cell wall-derived material may be, but is not limited to, teichoic acid, lipoteichoic acid, or peptidoglycan.

[0044] The above peptidoglycan may include MDP (muramyl dipeptide) as a component.

[0045] In one specific example, the Nod2 receptor stimulating bacteria may be, but is not limited to, Staphylococcus spp., Klebsiella spp., or Corynebacterium spp.

[0046] In one specific example, the strain of the genus Staphylococcus may be, but is not limited to, Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), or Staphylococcus capitis (S. capitis). In addition, the strain of the genus Klebsiella may be, but is not limited to, Klebsiella aerogenes (K. aerogenes), Klebsiella pneumoniae (K. pneumoniae), or Klebsiella oxytoca (K. oxytoca).

[0047] As used herein, “immune adjuvant” or “vaccine adjuvant” refers to a substance or composition added to a vaccine or pharmaceutically active ingredient to increase and / or influence the immune response, and includes a wide range of substances or strategies that can enhance the immunogenicity of an antigen incorporated into or co-administered with the immune adjuvant.

[0048] The above Nod2 receptor stimulating component or Nod2 receptor stimulating bacteria can promote the activity of imiquimod.

[0049] The above-described immune adjuvant composition can enhance humoral immunity, cellular immunity, mucosal immunity, and / or systemic immunity. For example, it can simultaneously enhance humoral immunity and cellular immunity, or simultaneously enhance mucosal immunity and systemic immunity, but is not limited thereto.

[0050] The above immunity may include, but is not limited to, immunity against infection by any one of the pathogens of viruses, fungi, bacteria and parasites, or immunity against cancer.

[0051] As used herein, "combination administration," "combination therapy," or "in combination" refers to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of the combination therapy may be administered simultaneously, sequentially, or in any order. The components may be administered in different dosages, at different frequencies, or via different routes, as appropriate.

[0052] Specifically, the combination administration may be by administering the first active ingredient and the second active ingredient simultaneously, or by administering the first active ingredient followed by the second active ingredient. The combination therapy according to the present invention may be defined as being capable of providing a synergistic effect if the efficacy, as measured by, for example, the degree of response, the rate of response, the time until disease progression, or the duration of survival, is therapeutically superior to the efficacy that can be obtained by administering one or the other of the components of the combination therapy at the usual dose. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the above alone. In particular, a synergistic effect is considered to exist if the usual doses of the first active ingredient and the second active ingredient can be reduced without compromising one or more of the degree of response, the rate of response, the time until disease progression, and the survival data, particularly without compromising the duration of response, and with reduced and / or fewer problematic side effects than when each component is used at the usual dose.

[0053] As used herein, “administered simultaneously” is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

[0054] As used herein, the term "administered sequentially" is not particularly limited and means that the components of the combination therapy are administered one after another or in clusters with a specific time interval between administrations, rather than simultaneously. The time intervals may be the same or different between the administrations of each of the components of the combination therapy, and may be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

[0055] In one specific example, the first and second effective ingredients may be administered via independent routes. Each effective ingredient may be administered independently by a person skilled in the art with a suitable dosage and administration method. Specifically, the first and second effective ingredients may be administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, stereotactically, orally, by direct injection or perfusion. Specifically, the first and second effective ingredients may be administered orally, intravenously, or subcutaneously. More specifically, the first and second effective ingredients may be administered to, but are not limited to, the nasal mucosa, the oral mucosa, the sublingual, the rectal, or the vaginal mucosa.

[0056] As used herein, the “immune adjuvant composition” and / or “vaccine composition” may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the composition is one commonly used in the manufacture of pharmaceuticals, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, current edition.

[0057] The above-mentioned immune adjuvant composition and / or vaccine composition may be formulated using a method known in the art so as to enable rapid release, or sustained or delayed release, of the active ingredient upon administration. In addition, various bases and / or additives necessary and appropriate for the formulation of the formulation may be included, and, within a range that does not reduce the effect thereof, the composition may further include known compounds such as nonionic surfactants, silicone polymers, preservatives, flavoring agents, preservatives, bactericides, oxidation stabilizers, organic solvents, ionic or nonionic thickeners, softeners, antioxidants, free radical scavengers, opacifiers, stabilizers, emollients, silicones, α-hydroxy acids, antifoaming agents, moisturizers, vitamins, insect repellents, flavoring agents, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, alkalizing or acidifying agents, or colorants. These formulations may include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders, and may be oral or parenteral dosage forms.

[0058] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more active substances of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0059] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0060] The above administration can be administered by any method known in the art. Administration can be administered directly to the subject by any means, including intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The above administration can be systemic or local.

[0061] The above mucosa may include, but is not limited to, the nasal mucosa, the oral mucosa, the sublingual, the rectal, or the vaginal.

[0062] The subject may include, but is not limited to, a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat.

[0063] A suitable dosage of the above-mentioned immune adjuvant composition and / or vaccine composition is 0.001 to 1000 mg, for example, 0.1 to 500 mg or 0.1 to 300 mg per kg of body weight per day, which may be administered once or several times in divided doses. However, the dosage may vary depending on the formulation method, administration method, administration time, and / or administration route of the immune adjuvant composition and / or vaccine composition. In addition, the dosage may vary depending on various factors including the type and degree of the response to be achieved by administration of the composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the pharmaceutical field, and a person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment.

[0064] Another aspect provides a vaccine composition comprising an antigen and the above-described immune adjuvant composition.

[0065] In one specific example, the antigen is not particularly limited thereto as long as it can induce an immune response in vivo, but may be, for example, a bacterial antigen, a viral antigen, a cancer antigen, or a combination thereof, and may include, for example, antigens of Bacillus anthracis, Yersinia pestis, Vibrio cholerae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Salmonella, and Escherichia coli and toxins produced by them, and other examples include, but are not limited to, Coronavirus, influenza virus, Epstein-Barr virus, HAV (hepatitis A virus), HBV (hepatitis B virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), Hantaan virus, cytomegalovirus (CMV), human immunodeficiency virus (HIV), human papilloma virus (HPV), poliovirus, Ebola virus, rotavirus, dengue virus, West Nile virus, yellow fever virus, adenovirus, Japanese encephalitis virus, BK virus, smallpox virus, Zika virus,It may include severe fever with thrombocytopenia syndrome virus (SFTS virus) or herpes simplex virus (HSV) itself or antigens of said virus, and as other examples, it may include, but is not limited to, antigens derived from human papillomavirus (HPV), carcinoembryonic antigen, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), Her2 / neu, MUC-1, BCR / ABL, alpha-fetoprotein (AFP), antigens derived from Epstein-Barr virus (EBV), antigens derived from human hepatitis virus B (HBV), antigens derived from human hepatitis virus C (HCV), carcinoma-125 (CA-125), carcinoma-72-4 (CA-72-4), carcinoma-15-3 (CA-15-3), or carcinoma-19-9 (CA-19-9).

[0066] In one specific example, the antigen may be, but is not limited to, a coronavirus or an influenza virus.

[0067] The coronavirus may be an RNA virus belonging to the Coronavirinae subfamily, and specifically, may include, but is not limited to, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, MERS-CoV, and SARS-CoV-2. In a preferred embodiment, the coronavirus may be a SARS-CoV-2 virus.

[0068] The above influenza virus may be a virus belonging to the Orthomyxoviridae family, which is an RNA virus, and specifically, may be a virus of the genus Influenza A, Influenza B, and / or Influenza C, and may include, but is not limited to, a virus having glycoproteins called hemagglutinin (HA) and neuraminidase (NA) on the surface of the virus particle, such as H1N1 subtype, H3N2 subtype, H5N1 subtype, and H7N7 subtype, etc. In a preferred specific example, the influenza virus may be an influenza A virus.

[0069] In one specific example, the antigen may be, but is not limited to, a melanoma-specific antigen.

[0070] The melanoma described above may be a type of skin cancer, and may be a solid cancer arising from the skin, or a metastatic cancer that spreads from the skin to other organs, but is not limited thereto. In a preferred embodiment, the melanoma may be a type of skin cancer that is prone to lung metastasis.

[0071] The viral antigen of the present specification may be used without limitation as long as it is a substance capable of inducing an immune response to coronavirus and / or influenza virus infection or a substance capable of inducing an immune response to melanoma, and can be easily selected by a person skilled in the art.

[0072] As used herein, the “vaccine composition” may further comprise one or more pharmaceutically acceptable vaccine immune adjuvants. For example, aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (mixture of DL-αtocopherol, squalene, and polysorbate 80 as an emulsifier), CpG, flagellin, Poly I:C, AS01, AS02, ISCOMs, ISCOMMATRIX, IL-12, GM-CSF (Granulocyte-macrophage colony-stimulating factor), MPL (Monophosphoryl Lipid A), GLA, R848, saponins (QS-21), C-type lectin ligands (TDB), α-galactosylceramide, quil A, IC31 (oligo nucleotide and cationic peptide), CFA01 (cationic liposome), and It may be, but is not limited to, GLA-SE (oil-in-water emulsion of MPL and glucopyranosyl lipid).

[0073] The dosage of the above vaccine composition may be 0.001 to 1000 mg / kg for adults, but is not limited thereto, and can be easily determined by a person skilled in the art in consideration of the purpose of use, target disease (type thereof, etc.), patient age, weight, medical history, etc., and the frequency of administration to the subject (or patient) can also be easily determined by a person skilled in the art in consideration of the purpose of use, target disease (type thereof, severity thereof, etc.), patient age, weight, medical history, progress, etc.

[0074] The frequency of administration of the above vaccine composition may be, for example, daily or every few months, or once or twice before each epidemic, but is not limited thereto, and a person skilled in the art can easily determine the interval between additional immunizations while observing the course of maintaining immunogenicity.

[0075] Another aspect provides a method of boosting the immunity of a subject comprising the steps of: administering to the subject an effective amount of an immune adjuvant; and administering to the subject in need thereof at least one selected from the group consisting of a Nod2 receptor stimulating component and a Nod2 receptor stimulating bacteria.

[0076] In this specification, “boosting immunity” means activating the activity of immune cells of an individual to enhance the immune response, and can be used interchangeably with “increasing immunity” or “increasing immunity.”

[0077] Another aspect provides a use of an immune adjuvant composition comprising, as a first active ingredient, an immune adjuvant, and, as a second active ingredient, at least one selected from the group consisting of a Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacterium.

[0078] Another aspect provides the use of a vaccine composition comprising, as a first active ingredient, an immune adjuvant, and as a second active ingredient, at least one selected from the group consisting of a Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacterium.

[0079] An immune adjuvant composition for a vaccine comprising imiquimod and a Nod2 receptor stimulating component or a Nod2 receptor stimulating bacterium as active ingredients according to one aspect can enhance the effectiveness of the vaccine compared to when imiquimod is administered alone, and thus can be effectively used as a complex immune adjuvant to increase the immune response of various vaccines, such as a SARS-CoV-2 virus vaccine, an influenza A virus vaccine, and an anti-cancer vaccine for melanoma.

[0080] Figure 1a is a graph measuring blood antigen (Human serum albumin, HSA)-specific IgG (antibody) produced two weeks after nasal immunization using immune adjuvants (MALP-2, Poly(I:C), 3D-PHAD, Flagellin, imiquimod, resiquimod, CpG) in groups divided according to antibiotic treatment.

[0081] Figure 1b is a graph showing the dependence of the activity of immune adjuvants (MALP-2, Poly(I:C), 3D-PHAD, flagellin, imiquimod, resiquimod, CpG) on nasal commensal bacteria through the amount of interferon (IFN)-γ secretion, one of the T cell cytokines. The graph measures the amount of IFN-γ secreted when splenocytes obtained in the same experiment as Figure 1a are re-stimulated with antigen (HSA).

[0082] Figure 2a is a graph measuring the production of IL-12p40 and IL-6 cytokines, which are important for T cell differentiation, after sequential treatment of bone marrow derived dendritic cells (BMDCs) in vitro with bacterial components (P3C (Pam3CSK4), LPS, FLA (flagellin), CpG, iE-DAP, MDP) and 3D-PHAD, an immune adjuvant candidate.

[0083] Figure 2b is a graph measuring the production of IL-12p40 and IL-6 cytokines, which are important for T cell differentiation, after sequential treatment of bone marrow-derived dendritic cells with bacterial components (particularly MDP) and imiquimod, an immune adjuvant candidate.

[0084] Figures 2c to 2e are graphs showing the secretion of IL-12p40 and IL-6 cytokines after treatment with 3D-PHAD, MPLA, and imiquimod at different concentrations in cells pretreated with and without MDP in bone marrow-derived dendritic cells.

[0085] Figure 2f shows wild type and Nod2 - / - This graph compares the secretion levels of IL-12p40 and IL-6 during continuous treatment with MDP and imiquimod in dendritic cells.

[0086] Figure 3a is a graph comparing the production of antigen (HSA)-specific IgG in a group of mice immunized with a combination of 3D-PHAD and MDP.

[0087] Figure 3b is a graph comparing the production of antigen (HSA)-specific IgG in groups of mice immunized with a combination of imiquimod and MDP.

[0088] Figure 3c is a graph comparing the production of cytokines (IFN-γ, IL-5, and IL-21) after re-stimulation of spleen cells collected after immunization with an antigen (HSA) according to the combination of imiquimod and MDP.

[0089] Figure 3d is a graph measuring the production of NP-OVA-specific IgG in the serum of mice immunized with a combination of imiquimod and MDP using NP-OVA (3-nitrophenylacetyl-ovalbumin) as an antigen during immunization (left) and a graph measuring IgG with high affinity to NP (right).

[0090] Figure 4a is a graph comparing the antigen (iPR8)-specific IgG production in serum obtained 14 days after vaccination with an inactivated viral antigen (inactivated PR8, iPR8) of IAV in mice together with a combination of imiquimod and MDP.

[0091] Figure 4b is a graph comparing the production of T cell-derived cytokines (IFN-γ and IL-5) secreted after antigen re-stimulation for spleen cells obtained at the same time as Figure 4a.

[0092] Figure 4c is a graph showing the survival rate for 14 days after IAV infection in mice 2 weeks after vaccination.

[0093] Figure 4d is a graph showing the change in body weight over 14 days after IAV infection in mice 2 weeks after vaccination (in the same experiment as Figure 4c).

[0094] Figure 4e is a graph showing representative photographs and numerical results of histological analysis of mouse lung tissues obtained 1, 3, and 5 days after IAV infection 2 weeks after vaccination in mice, stained with H&E.

[0095] Figure 4f is a graph measuring the secretion level of antigen (iPR8)-specific IgA in bronchoalveolar lavage fluid (BALF) of mice obtained 1, 3, and 5 days after IAV infection 2 weeks after vaccination.

[0096] Figure 4g is a graph measuring the gene expression levels of hemagglutinin (HA) and nucleoprotein (NP) of influenza virus detected in the lungs of mice infected with IAV 2 weeks after vaccination, 1 day, 3 days, and 5 days later, using Real-Time qPCR.

[0097] Figure 4h is a graph showing the infectivity of virus titers obtained through plaque analysis in bronchoalveolar lavage fluid of mice obtained 1, 3, and 5 days after IAV infection 2 weeks after vaccination.

[0098] Figure 5a is a graph comparing the antigen (spike protein) specific IgG levels in serum collected on day 14 after vaccination with the SARS-CoV-2 spike protein in mice in combination with imiquimod and MDP.

[0099] Figure 5b is a graph showing the production of secreted T cell cytokines (IFN-γ and IL-5) after antigen (spike protein) re-stimulation in spleen cells collected in the same experiment as Figure 5a.

[0100] Figure 5c is a graph (left) showing how much the serum can neutralize the infection of the pseudovirus by mixing the serum with the SARS-CoV-2 pseudovirus for a certain period of time to evaluate the neutralization ability of the serum obtained in Figure 5a, then infecting a cell line and measuring the relative luminescence ratio of luciferase in the infected cells, and using this to determine the neutralizing antibody titer (NT 50 ) is a graph (right) calculated and displayed.

[0101] Figure 5d shows spike-specific IgG and logNT of serum obtained from vaccination (in the same experiment as Figure 5a). 50 This graph shows that there is a significant correlation between the two.

[0102] Figure 6a is a graph showing the relative proportions of bacteria identified by species and cultured from the nasal cavity of a healthy person.

[0103] Figure 6b is a graph showing the Nod2 stimulating activity of some live bacteria (top) or bacteria inactivated by ultraviolet (UV) irradiation (bottom) among the isolated single bacterial strains.

[0104] Figure 6c is a graph showing the degree of induction of IL-12p40 and IL-6 production in bone marrow-derived dendritic cells to compare the synergistic effect between isolated bacteria and imiquimod.

[0105] Figure 6d is a graph comparing the production of antigen (HSA)-specific IgG in serum obtained 14 days after vaccination with antigen and imiquimod in mice pretreated with Staphylococcus aureus (S. aureus) or Staphylococcus epidermidis (S. epidermidis) in the nasal cavity.

[0106] Figure 6e is a graph comparing the production of antigen (HSA)-specific IgG in serum obtained on day 14 after priming mice pretreated with S. aureus or S. epidermidis inactivated by ultraviolet (UV) irradiation with antigen and imiquimod, and in serum obtained on day 14 after one additional vaccination (boosting).

[0107] Figure 6f is a graph comparing the production of antigen (HSA)-specific IgG in serum obtained 14 days after pretreatment of mice nasally with M1, which has relatively high Nod2 stimulating activity, and FM5, which has relatively low Nod2 stimulating activity among isolated Staphylococcus epidermidis, and vaccination with antigen and imiquimod.

[0108] Figure 6g shows the results of pretreatment of wild-type (WT) mice with high Nod2 stimulating activity with Staphylococcus aureus (S. aureus) and Nod2 - / - This graph shows the production of antigen (HSA)-specific IgG induced by HSA and imiquimod immunization in serum obtained 14 days after vaccination with antigen and imiquimod in mice.

[0109] Figure 6h is a graph showing the production of T cell-derived cytokines (IFN-γ, IL-5, and IL-21) after re-stimulation with antigen of splenocytes obtained in the same experiment as Figure 6g.

[0110] Figure 6i shows the Nod2 stimulating activity of wild-type (WT) mice pretreated with S. epidermidis, which has relatively low Nod2 stimulating activity. - / - This graph compares the antigen (HSA)-specific IgG production in serum obtained 14 days after vaccination with antigen and imiquimod in mice.

[0111] Figure 6j is a graph showing the production of T cell-derived cytokines (IFN-γ, IL-5, and IL-21) after re-stimulation with antigen of spleen cells obtained in the same experiment as Figure 6i.

[0112] Figure 7a is a graph comparing the production of antigen (iPR8)-specific IgG in serum obtained on day 14 after vaccination with iPR8 antigen and imiquimod in mice pretreated with either Staphylococcus aureus (S. aureus), which has relatively high Nod2 stimulating activity, or Staphylococcus epidermidis (S. epidermidis), which has relatively low Nod2 stimulating activity.

[0113] Figure 7b is a graph showing the secretion amounts of IFN-γ and IL-5 cytokines after re-stimulation of spleen cells collected in the same experiment as Figure 7a with HA antigen.

[0114] Figure 7c is a graph showing the survival rate measured for 11 days after inoculation of mice with IAV on the 14th day, similar to Figure 7a.

[0115] Figure 7d is a graph showing the change in body weight over 11 days after infection with IAV in the same experiment as Figure 7c.

[0116] Figure 7e is a graph that quantifies the representative photographs and histological analysis results taken after staining mouse lung tissue with H&E 5 days after infection with IAV following immunization, similar to Figure 7c.

[0117] Figure 7f is a graph showing the results of measuring antigen-specific IgA in mouse bronchoalveolar lavage fluid (BALF) 5 days after infection with IAV following immunization, similar to Figure 7e.

[0118] Figure 7g is a graph showing the gene expression levels of influenza hemagglutinin (HA) and nucleoprotein (NP) in the lung tissue of mice 5 days after infection, measured using Real-Time qPCR, similar to Figure 7f.

[0119] Figure 7h is a graph measuring the infectious virus titer through plaque analysis of mouse bronchoalveolar lavage fluid (BALF) 5 days after infection, similar to Figure 7f.

[0120] Figure 8 shows the wild type or Nod2 strain of Staphylococcus aureus (S. aureus). - / - This graph shows the survival rate of mice for 41 days after pretreatment of the nasal cavity with B16F10 cancer cell lysate antigen and imiquimod, intranasal inoculation, and subcutaneous injection of B16F10 melanoma cancer cells 14 days later.

[0121] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0122] Terms or words used in the specification and claims of the present invention are not to be construed as limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0123] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0124] Throughout the specification of the present invention, “A and / or B” means A or B, or A and B.

[0125]

[0126] Reference Example 1. Mouse

[0127] Age- and sex-matched C57BL / 6 mice were purchased from OrientBio (Seongnam, Korea). C57BL / 6 Nod2 - / - Mice were obtained from Professor Jong-Hwan Park (Chonnam National University, Korea). All experiments were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University.

[0128]

[0129] Reference Example 2. Cell Culture

[0130] Madin-Darby Canine Kidney (MDCK) cells were cultured in complete MEM (Minimal Essential Medium, Gibco) containing 10% FBS (Fetal Bovine Serum, Gibco, Waltham, MA) and 1% penicillin / streptomycin (Gibco) at 37°C in a 5% CO2 incubator. Huh-7, B16F10, HEK293FT, and HEK-Nod2 reporter cell lines were cultured in complete Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% FBS and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator.

[0131]

[0132] Reference Example 3. Virus propagation and infection

[0133] Influenza A virus (IAV; A / PR / 8 / 34) was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and propagated according to the manufacturer's instructions. Virus inoculation was performed when MDCK cells were 80–90% confluent. For virus inoculation, cells were infected with a multiplicity of infection (MOI) of 0.01 for 1–2 hours. One to two hours after infection, virus growth medium was added and the cells were incubated in an incubator at 37°C and 5% CO2 for 2–4 days, until 80% of the cell monolayer developed cytopathic effect (CPE). The supernatant was centrifuged at 1500 g for 30 minutes to separate cell debris and filtered through an Amicon Ultracentrifugal-15 (Millipore). Virus concentrations were determined by plaque assay and stored in a -80°C deep freezer.

[0134] Mice were challenged with IAV (2xLD) via the intranasal route 50 pfu / 30㎕ in PBS). Infected mice were monitored for 14 days to determine body weight change (%) and survival rate (%). In addition, mice were euthanized on days 1, 3, and 5 after infection for analysis, and whole lung tissue and bronchoalveolar lavage fluid (BALF) were collected, respectively.

[0135]

[0136] Reference Example 4. Reagent

[0137] Muramyl dipeptide (MDP) and MALP-2 were purchased from Bachem (Heidelberg, Germany) and Enzo Life Science (Farmingdale, NY, USA), respectively. Pam3CSK4 (P3C), LPS, Poly(I:C), Flagellin, CpG (ODN-1826), and iE-DAP were purchased from InvivoGen (San Diego, CA, USA). Imiquimod and Resiquimod were purchased from Tocris Bioscience (Ellisville, MO, USA). 3D-PHAD and MPLA were purchased from Avanti polar lipids (Birmingham, AL, USA).

[0138]

[0139] Reference Example 5. Mouse Immunization

[0140] Female mice aged 7–10 weeks were anesthetized with isoflurane and immunized intranasally with antigen alone, antigen plus imiquimod (10 μg), MDP (100 ng), or imiquimod plus MDP. Human serum albumin (HSA, 30 μg), inactivated-PR8 (iPR8, 1 × 10 6pfu) and recombinant spike protein (SARS-CoV-2, 5 μg) were used as antigens. To screen for immune adjuvants with synergistic effects with commensal bacteria, mice were intranasally immunized with MALP-2 (10 μg), Poly(I:C) (10 μg), 3D-PHAD (10 μg), flagellin (1 μg), imiquimod (10 μg), resiquimod (10 μg), or CpG (10 μg), respectively. Two weeks after immunization, the mice were euthanized, and serum and spleen were collected for analysis. To deplete the commensal bacteria in the nasal cavity, 30 μl of an antibiotic cocktail containing ampicillin (1 mg / ml), neomycin (1 mg / ml), metronidazole (1 mg / ml), and vancomycin (0.5 mg / ml) was administered intranasally once, and the same antibiotic cocktail was added to the drinking water and allowed to drink ad libitum for one week before immunization. To evaluate the synergistic effect of isolated bacteria, bacteria (5 x 10 5 cfu) was pretreated intranasally, and a combination of antigen (HSA, iPR8, etc.) and imiquimod was administered intranasally.

[0141]

[0142] Reference Example 6. ELISA (IgG, IgA)

[0143] To measure antibody production in serum and bronchoalveolar lavage fluid (BALF), antigen-specific IgG and IgA levels were measured in serially diluted serum and BALF, respectively, using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (SouthernBiotech, Birmingham, AL, USA). ELISA plates (Corning) were prepared by incubating HSA (30 μg / ml), iPR8 (1 × 10 5pfu / well) or SARS-CoV-2 spike protein (3 μg / ml, SinoBiological) overnight at 4°C, then serum and BALF were diluted in PBS containing 1% BSA and added to each well for reaction. After washing the plate, AP-conjugated anti-IgG1, IgG 2b , IgG 2c And the plate was reacted with a combination of IgG3 antibodies or AP-conjugated anti-IgA antibodies, and after the reaction was completed, the plate was washed five times and reacted with pNPP substrate solution (SouthernBiotech) until pNPP turned yellow, and then the absorbance at a wavelength of 405 nm was measured using a GloMax Discover Microplate Reader (Promega, Madison, WI).

[0144]

[0145] Reference Example 7. ELISA (Cytokine)

[0146] Secretion levels of IFN-γ (DY485), IL-5 (DY405), IL-21 (DY594), IL-12p40 (DY2398), and IL-6 (DY406) were measured using Duoset ELISA kits (R&D Systems) according to the manufacturer's instructions.

[0147]

[0148] Reference Example 8. Re-stimulation of spleen cells

[0149] Spleens were harvested from mice and dissociated into single cells using a 70 μm cell strainer (Falcon). Splenocytes were rinsed in red blood cell lysis buffer to remove red blood cells, and then centrifuged at 1,500 rpm for 5 min at 4°C. The supernatant was removed, and the pellet was resuspended in complete RPMI1640 medium containing 10% FBS, 1% penicillin-streptomycin, L-glutamine (2 mM), sodium pyruvate (1 mM), MEM non-essential amino acids (Gibco), and 2-β-mercaptoethanol (50 μM). Spleen cells were seeded at 1 × 10 in round-bottom 96-well plates. 6 Cells were seeded at 1 / well and restimulated with HSA, iPR8, or Spike protein for 4 days in a 37°C, 5% CO2 incubator. To analyze cytokine secretion levels, cell culture supernatants were transferred to new plates and measured by ELISA.

[0150]

[0151] Reference Example 9. In vitro stimulation of bone marrow-derived dendritic cells (BMDCs)

[0152] To obtain bone marrow-derived dendritic cells (BMDCs), bone marrow cells were first isolated from the femurs and tibias of mice. For differentiation into dendritic cells, bone marrow cells were cultured in complete RPMI1640 medium (cRPMI1640) containing 10% FBS, 1% penicillin-streptomycin, 2-β-mercaptoethanol (50 μM), and recombinant GM-CSF (20 ng / ml, PeproTech). Fresh RMMI1640 complete medium was added on days 3 and 5 of culture, and non-adherent cells obtained on day 7 of culture were bone marrow-derived dendritic cells. Differentiated bone marrow-derived dendritic cells were seeded at 4 × 10 in 48-well plates. 5Cells were seeded at 100 cells / well and then pretreated with bacterial components such as Pam3CSK4 (100 ng / ml), LPS (100 ng / ml), flagellin (100 ng / ml), CpG (40 ng / ml), iE-DAP (1 μg / ml), or MDP (10 μg / ml) for 30 min. Subsequently, immune adjuvant candidates (3D-PHAD (1 μg / ml), MPLA (1 μg / ml), or imiquimod (1 μg / ml)) were treated and cultured in a 37°C, 5% CO2 incubator for 24 h. Cell culture supernatants were harvested, and cytokine secretion levels were measured by ELISA.

[0153]

[0154] Reference Example 10. Isolation of bacteria from humans

[0155] Mucus was collected from both nostrils of 12 healthy adults using sterile 3M Quick swabs (3M Microbiology Products, Maplewood, MN) (IRB no. C-2005-095-1124). The swabs were smeared directly onto Brain-Heart Infusion (BHI) agar plates and incubated in a 37°C incubator for 1–2 days. After 1–2 days of incubation, bacterial colonies were obtained from the plates and isolated based on morphology. For bacterial identification, individual bacterial DNA was extracted using the EZNA bacterial DNA kit (Omega Bio-tek, Norcross, GA), and then each bacterial species was confirmed by 16S rRNA sequencing using the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO: 1) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO: 2).

[0156]

[0157] Reference Example 11. Nod2 Stimulated Activation

[0158] Nod2-stimulated activity was measured using HEK293 cells expressing human Nod2 and an NF-κB-responsive luciferase reporter. Cells were cultured at 4 × 10 in DMEM supplemented with 10% FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), and 1% penicillin / streptomycin. 4 Cells were seeded at 1 cell / well in 96-well white plates. The following day, live or UV-inactivated bacteria were treated at a multiplicity of infection (MOI) of 1. For live bacteria, gentamicin (50 μg / ml, Gibco) was added to the medium 1 hour after inoculation. After 24 hours, reporter cells were lysed using cell lysis buffer, luciferase substrate (Promega) was added to the lysate, and relative luminescence units (RLU) were measured using a GloMax Discover Microplate Reader (Promega).

[0159]

[0160] Reference Example 12. SARS-CoV-2 pseudovirus neutralization assay

[0161] To generate SARS-CoV-2 pseudoviruses, the lentiviral backbone plasmid pNL4-3-.Luc.RE- and the spike expression plasmid pCAGGS were co-transfected into HEK293T cells. After 72 h of co-transfection, the supernatant was collected and mixed with PEG-it virus precipitation solution (System Biosciences, Palo Alto, CA). The precipitated pseudoviruses were collected by centrifugation at 1,500 g for 30 min and lysed at a 50% tissue culture infectious dose (TCID). 50) was quantified. To evaluate the pseudovirus neutralizing activity of mouse serum, the serum was diluted with PBS and incubated with ~120 TCID for 1 hour at 37°C. 50 After incubation with the pseudovirus, the serum-pseudovirus mixture was added to Huh7 cell monolayers in 96-well white plates. After 48 h of incubation, cells were lysed using cell lysis buffer, luciferase substrate was added to the lysate, and relative RLU signal values ​​were measured using a GloMax Discover Microplate Reader.

[0162]

[0163] Reference Example 13. Real-time PCR

[0164] Total RNA was extracted from the lungs using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Complementary DNA was synthesized from total RNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA) according to the manufacturer's instructions. Real-time qPCR was performed using GoTaq®qPCR Master Mix (Promega) on a CFX96 system (BioRad, Hercules, CA). Primer sequences for target genes are as follows: GAPDH forward (5'-TGCGACTTCAACAGCAACTC-3') (SEQ ID NO: 3), GAPDH reverse (5'-GCCTCTCTTGCTCAGTGTCC-3') (SEQ ID NO: 4), HA forward (5'-AAGGCAAACCTACTGGTCCTGTT-3') (SEQ ID NO: 5), HA reverse (5'-AATTGTTCGCATGGTAGCCTATAC-3') (SEQ ID NO: 6), NP forward (5'-AGGCACCAAACGGTCTTACG-3') (SEQ ID NO: 7), NP reverse (5'-TTCCGACGGATGCTCTGATT-3') (SEQ ID NO: 8). mRNA expression values ​​were corrected with GAPDH as an internal control.

[0165]

[0166] Reference Example 14. Plaque assay

[0167] Bronchoalveolar lavage fluid (BALF) collected from infected mice was serially diluted with PBS. MDCK cells were cultured to fill 90–95% of a 6-well plate, washed with PBS, and inoculated with 500 μl of each sample dilution for 2 h at room temperature. After virus adsorption, the infected cell monolayers were overlaid with regular MEM containing 1.5% agarose, TPCK-trypsin (2 μg / ml), and 1% penicillin / streptomycin at 37°C for 72 h. Plaques were observed and counted to calculate the titer of the BALF samples.

[0168]

[0169] Reference Example 15. Histopathology

[0170] Lung tissues were fixed in 4% paraformaldehyde solution at 4°C for one day and embedded in paraffin. Paraffin-embedded tissue blocks were then sectioned at 4 μm thickness for hematoxylin and eosin (H&E) staining. An average of three different sections were used for each lung specimen, and representative non-overlapping fields were selected. Histological analysis was performed using a semiquantitative scoring system. The lung histological scores were evaluated using three parameters, which are presented in Table 1.

[0171]

[0172]

[0173] Reference Example 16. Preparation of cancer cell lysate, anticancer vaccine immunization, and cancer cell injection.

[0174] B16F10 mouse melanoma cell line was cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS and 1% penicillin / streptomycin, 10 cm 2The cells were cultured until they were completely filled in a cell culture plate. The cells were detached with 0.25% Trypsin-EDTA (ethylenediaminetetraacetic acid), centrifuged at 1,200 rpm for 5 minutes to separate the supernatant, and the pellet was resuspended in 200 μl of PBS (phosphate buffer saline). The freeze-thawing process was repeated five times in liquid nitrogen and a 37°C water bath to prepare a lysate. The protein concentration of the cancer cell lysate was measured using the BCA (bicinchoninic acid) assay, and the cancer cell lysate (30 μg / 30 μl) was mixed with an immune adjuvant and administered intranasally as an antigen.

[0175] Cultured B16F10 cell line (1ⅹ10 6 Cells were injected subcutaneously into the dorsal area of ​​the mouse. After the cells were injected, the tumor size was measured using Vernier calipers from the time when a small tumor appeared (5-6 days), and the survival rate was recorded.

[0176]

[0177] Reference Example 17. Statistical Analysis

[0178] Statistical analysis was performed using GraphPad Prism 9.01 software (GraphPad), and results are expressed as ±standard error of the mean (SEM) and ±standard deviation (SD). Significant differences between two groups were analyzed using the Mann-Whitney test and Student's t-test, and significant differences between multiple groups were analyzed using two-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. Survival analysis was performed using the log-rank test, and correlations were analyzed using Spearman's correlation coefficient.

[0179]

[0180] Experimental Example 1. Synergistic Effects of Multiple Nasal Immune Adjuvants and Commensal Bacteria

[0181] To identify immune adjuvants that have synergistic activity with the nasal microbiota, we intranasally inoculated mice depleted of bacteria through antibiotic treatment and bacteria-sufficient mice with antigens and various immune adjuvant candidates, and measured the production of antigen-specific IgG (antibody) in the serum and the secretion of T cell-derived cytokines from spleen cells.

[0182] Specifically, one group of mice was administered a single intranasal dose of an antibiotic cocktail, then consumed drinking water containing the same antibiotics for one week prior to vaccination. Both antibiotic-treated and non-antibiotic-treated mice were then intranasally immunized with each candidate adjuvant and antigen (HSA). Two weeks later, serum and spleens were collected to compare humoral and cellular immune responses. The results are shown in Figure 1.

[0183] As shown in Figure 1, although to varying degrees, the reduction of commensal bacteria suppressed the production of antigen (HSA)-specific IgG induced by several immunoadjuvants used (Figure 1a). Most immunizations induced interferon (IFN)-γ secretion from antigen-restimulated spleen cells, and some immunoadjuvants showed that IFN-γ induction was dependent on the presence of commensal bacteria (Figure 1b).

[0184] In summary, considering both antigen-specific antibody production and T cell cytokine production, the immune adjuvant properties of TLR7 agonists (imiquimod) and TLR4 agonists (MPLA and its analog 3D-PHAD) appeared to be most strongly dependent on the presence of commensal bacteria.

[0185]

[0186] Experimental Example 2. Interaction between bacterial components that produce synergistic effects in vitro and immune adjuvant candidates.

[0187] To assess which bacterial components were responsible for the synergistic effect with the selected immunoadjuvant, mouse bone marrow-derived dendritic cells (BMDCs) were pretreated with the microbial components for 30 minutes and then treated with 3D-PHAD or imiquimod for 24 hours. Cytokine levels in the cell culture supernatant were then measured, and the results are shown in Figures 2A and 2B.

[0188] As shown in Figures 2a and 2b, we confirmed that the production of cytokines IL-12p40 and IL-6, which promote T cell differentiation, was significantly increased in BMDCs stimulated with 3D-PHAD or imiquimod in the presence of MDP (muramyl dipeptide, Nod2 agonist) compared to imiquimod alone.

[0189] To determine the role of MDP in cytokine induction by 3D-PHAD or imiquimod, BMDCs were pretreated with MDP for 30 minutes and then treated with 3D-PHAD, MPLA, or imiquimod at different concentrations for 24 hours. The results are shown in Figures 2c to 2e.

[0190] As shown in Figures 2c to 2e, it was confirmed that the secretion of IL-12p40 and IL-6 increased in proportion to the concentration of 3D-PHAD, MPLA, and imiquimod used.

[0191] MDP is a component of bacterial cell walls and activates the Nod2 receptor, so Nod2 - / - Using DC (dendritic cells), we examined whether MDP contributed to the increased secretion of IL-12p40 and IL-6 induced by imiquimod, and the results are shown in Fig. 2f.

[0192] As shown in Figure 2f, unlike in wild-type DCs, Nod2 - / -In DCs, we confirmed that the increase in cytokine production induced by MDP was abolished. Consistently, these results indicate that MDP promotes the immunoadjuvant activity of 3D-PHAD and imiquimod in vitro.

[0193]

[0194] Experimental Example 3. Synergistic Effect of MDP-Imiquimod Interaction in Vivo

[0195] Since in vitro experiments using BMDCs demonstrated that MDP enhanced the immune adjuvant activity in combination with 3D-PHAD or imiquimod, we investigated using a mouse model whether MDP could enhance the immune response induced by 3D-PHAD or imiquimod in the presence of depletion of commensal bacteria.

[0196] Specifically, antibiotic-treated mice were intranasally primed with HSA alone, HSA plus 3D-PHAD or imiquimod, MDP, 3D-PHAD plus MDP, or imiquimod plus MDP, and then boosted once 2 weeks later. Serum was collected 2 weeks after the primary or booster immunization, and antigen-specific antibody responses were measured by ELISA. The results are shown in Figures 3A and 3B.

[0197] As shown in Figures 3a and 3b, there was no significant difference in the level of HSA-specific IgG in mice immunized with 3D-PHAD regardless of the presence or absence of MDP after boosting (Figure 3a), but the production of HSA-specific IgG was significantly increased in mice immunized with a combination of imiquimod and MDP compared to when immunized with imiquimod alone (Figure 3b).

[0198] To evaluate the enhancement of T cell responses by MDP, spleen cells were collected two weeks after the first immunization and stimulated ex vivo with HSA, and the results are shown in Fig. 3c.

[0199] As shown in Fig. 3c, after antigen re-stimulation, it was confirmed that the production of cytokines IFN-γ, IL-5, and IL-21 increased in spleen cells of mice immunized with a combination of imiquimod and MDP.

[0200] To determine whether the antibodies increased upon immunization with the combination of imiquimod and MDP were high-affinity antibodies, NP-OVA was used as an antigen for immunization, and serum obtained from the immunization was used to measure the production of antigen-specific IgG and IgG with high affinity for NP using ELISA plates coated with NP-OVA or NP9. The results are shown in Fig. 3d.

[0201] As shown in Figure 3d, the combination of imiquimod and MDP was confirmed to increase the production of not only antigen-specific IgG but also high-binding antibodies.

[0202] Therefore, co-administration of MDP and imiquimod through the nose of antibiotic-treated mice restored both humoral and cellular immune responses, whereas this effect was not observed in mice immunized with 3D-PHAD, confirming that other microbial substances are likely to be involved in the 3D-PHAD case.

[0203]

[0204] Experimental Example 4. Improved Efficacy of Influenza A Virus (IAV) Vaccine by Combining Imiquimod and MDP

[0205] To investigate the efficacy of combined administration of imiquimod and MDP as an intranasal vaccine against IAV, mice were immunized intranasally with iPR8 antigen alone, imiquimod, MDP, or imiquimod and MDP. Serum and spleen cells were collected 14 days after vaccination to assess the immune response induced by the IAV vaccine, and the results are shown in Figures 4a and 4b.

[0206] As shown in Figures 4a and 4b, mice co-vaccinated with iPR8, imiquimod, and MDP induced significantly higher levels of iPR8-specific IgG compared to mice immunized with imiquimod alone, MDP alone, or the control group (Figure 4a). After ex vivo restimulation of spleen cells, mice co-vaccinated with imiquimod and MDP showed a significant increase in the production of the cytokines IFN-γ and IL-5 (Figure 4b).

[0207] Two weeks after vaccination, mice were LD 50 (50% lethal dose) of IAV was infected, and body weight loss and survival rate were observed for 14 days after infection, and the results are shown in Figures 4c and 4d.

[0208] As shown in Figures 4c and 4d, all IAV-infected control mice began to rapidly lose weight and died 7 days after infection. Mice vaccinated with imiquimod alone continued to lose weight, with a survival rate of only 20%. Conversely, mice treated with imiquimod and MDP simultaneously prevented IAV-induced weight loss and all survived throughout the 14-day monitoring period.

[0209] Lungs and BALF were collected on days 1, 3, and 5 post-infection, and IAV infectivity was assessed and compared between mice immunized with imiquimod alone and those immunized with imiquimod plus MDP. Lung tissue sections were collected, stained with H&E, and photographed under a microscope. The histological analysis results are shown in Figure 4e.

[0210] As shown in Fig. 4e, the lungs of mice that received intranasal immunization with the iPR8 control group and imiquimod alone showed severe inflammation with extensive inflammatory cell infiltration around the bronchial tubes and perivascular areas, whereas the lungs of mice that received imiquimod and MDP in combination showed almost no histopathological findings and significantly lower histological scores.

[0211] To assess the antibody response in the mucosal compartment, IgA secretion levels in bronchoalveolar lavage fluid were measured. In the absence of MDP, mice immunized with the control or imiquimod alone induced low levels of IgA in the BALF. The results are shown in Figure 4f.

[0212] As shown in Figure 4f, mice co-vaccinated with imiquimod and MDP consistently showed the highest levels of IgA on days 1, 3, and 5 post-infection.

[0213] The gene expression levels of hemagglutinin (HA) and nucleoprotein (NP), which are involved in the intracellular entry and replication of IAV in the lungs, respectively, were measured using Real-Time qPCR, and the results are shown in Fig. 4g.

[0214] As shown in Fig. 4g, the levels of HA and NP mRNA were confirmed to be much lower in the lungs of mice immunized with a combination of imiquimod and MDP than with imiquimod alone.

[0215] The titer of IAV viruses capable of reinfection in bronchoalveolar lavage fluid was measured by plaque assay, and the results are shown in Fig. 4h.

[0216] As shown in Fig. 4h, compared to mice immunized with only imiquimod, all mice immunized with a combination of imiquimod and MDP showed almost no detectable infectious virus on days 1, 3, and 5 after infection.

[0217] These results indicate that the combination of imiquimod and MDP as a complex mucosal immune adjuvant can effectively suppress IAV infection by enhancing vaccine-induced immune responses when administered intranasally together with an IAV vaccine.

[0218]

[0219] Experimental Example 5. SARS-CoV-2 Neutralization Effect of the Combination of Imiquimod and MDP

[0220] To evaluate whether the combined administration of imiquimod and MDP could induce effective immune responses in vaccines against other respiratory diseases, we used SARS-CoV-2 spike protein antigen and SARS-CoV-2 pseudoviruses. Mice were intranasally immunized with the SARS-CoV-2 spike antigen alone or in combination with imiquimod, MDP, or imiquimod and MDP. Serum was collected from mice 14 days after immunization, and antibody production and neutralizing activity were measured. The results are presented in Figures 5A and 5B.

[0221] As shown in Figures 5a and 5b, spike-specific IgG levels were significantly higher in mice co-administered with imiquimod and MDP than in mice administered imiquimod alone (Figure 5a). Furthermore, ex vivo restimulation of spleen cells extracted from mice co-administered with imiquimod and MDP revealed that higher levels of IFN-γ and IL-5 were produced in cell culture supernatants compared to mice administered imiquimod alone (Figure 5b).

[0222] Next, to assess whether antigen-specific antibodies induced by a vaccine combining imiquimod and MDP could neutralize SARS-CoV-2 pseudoviruses, we constructed a reporter lentivirus-based pseudotyped SARS-CoV-2 expressing the spike protein on its surface but lacking infectivity. After co-incubation with serum and pseudoviruses, the cells were added to Huh-7 cells for 48 hours, and the neutralizing capacity of the vaccine-induced antibodies was assayed by measuring the decrease in relative luciferase units. The results are shown in Figures 5c and 5d.

[0223] As shown in Figures 5c and 5d, mice intranasally immunized with imiquimod and MDP showed a significant decrease in relative luminescence ratio and neutralizing antibody titer (NT 50 ) significantly increased, indicating that the antibody had neutralizing activity that could suppress pseudovirus infection (Fig. 5c). Spike-specific IgG and logNT due to immunity 50 There was a significant correlation between the two parameters, with higher levels observed in the imiquimod and MDP combination group (Fig. 5d). These results indicate that intranasal vaccination with imiquimod and MDP not only induces a strong antigen-specific antibody response, but also that these antibodies possess the ability to neutralize SARS-CoV-2.

[0224]

[0225] Experimental Example 6. Effect of imiquimod on the activation of nasal bacteria

[0226] The nasal cavity is home to bacteria with MDP structures recognized by the Nod2 receptor. To identify bacteria with high Nod2-stimulating activity, we assessed the Nod2-stimulating activity of human nasal bacteria using a reporter assay. Mucus was collected from both nostrils of 12 healthy adults (6 men, 6 women) using a swab. The swabs were spread on BHI agar and incubated at 37°C for one day. Species identification was performed using 16s rRNA sequence analysis, and Nod2-stimulating activity was measured in single colonies. The results are shown in Figures 6a and 6b.

[0227] As shown in Figures 6a and 6b, the three main bacteria isolated from the nasal cavity were Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and Klebsiella aerogenes (K. aerogenes). The distribution of culturable bacterial species varied among subjects (Figure 6a). Most S. aureus species showed strong Nod2-stimulating activity, whereas S. epidermidis and K. aerogenes, with the exception of S. epidermidis in subject M1, showed low activity (Figure 6b). These results were similar to the Nod2-stimulating activity of UV-inactivated bacteria (Figure 6b), indicating that bacteria of the same species have different Nod2 activities.

[0228] Additionally, to evaluate the synergistic effect between the isolated bacteria and imiquimod, BMDCs were pretreated with bacteria isolated from healthy individuals at various MOIs and then stimulated with imiquimod for 24 hours, and the results are shown in Fig. 6c.

[0229] As shown in Fig. 6c, when S. aureus isolated from FM5 subjects was sequentially treated with imiquimod, it was confirmed that IL-12p40 and IL-6 production was strongly induced in proportion to MOI.

[0230] To investigate whether bacteria with high Nod2 stimulating activity isolated from healthy individuals could enhance the immune response induced by imiquimod, two strains of bacteria with high and low Nod2 stimulating activity were selected from bacteria isolated from FM5 subjects in vitro based on their cytokine secretion levels and relative luciferase activities. To mimic the nasal cavity enrichment of bacteria with high or low Nod2 stimulating activity, mice were nasally colonized with S. aureus or S. epidermidis from FM5 subjects with high or low Nod2 activity for three consecutive days, and then immunized with HSA alone or in combination with imiquimod. The results are shown in Figs. 6d and 6e.

[0231] As shown in Fig. 6d, the production of HSA-specific IgG in the serum of mice co-inoculated with S. aureus and imiquimod was significantly higher than that of mice co-inoculated with S. epidermidis and imiquimod or imiquimod alone. This same trend was observed in bacteria inactivated by UV irradiation, as shown in Fig. 6e.

[0232] In addition, in order to compare the immune induction effect according to Nod2 stimulation activity within the same species, the strain derived from subject M1, which had relatively high Nod2 stimulation activity among S. epidermidis, and the strain isolated from subject FM5, which had low Nod2 stimulation activity, were combined with imiquimod and the results of immunization are shown in Figure 6f.

[0233] As shown in Figure 6f, S. epidermis derived from M1 subjects with high Nod2-stimulating activity tended to have a higher ability to induce antigen-specific IgG production than S. epidermis derived from FM5 subjects. Taken together, these results support the hypothesis that the synergistic effect of nasal commensal bacteria and imiquimod on immune induction is related to the Nod2-stimulating ability of the commensal bacteria.

[0234] To determine whether the Nod2 receptor is involved in promoting the immune adjuvant activity of imiquimod, bacteria isolated from FM5 subjects were cultured in mice deficient in Nod2 (Nod2 - / - ) mice were used. Wild-type (WT) and Nod2 colonized with S. epidermidis or S. aureus - / - Mice were intranasally immunized with HSA and imiquimod, and serum was collected two weeks later. The results are shown in Figures 6g to 6j.

[0235] As shown in Figures 6g to 6j, when S. aureus or S. epidermidis was colonized intranasally, HSA-specific IgG production induced by HSA and imiquimod immunization was mediated by Nod2. - / - In mice, the secretion of IFN-γ, IL-5, and IL-21 was decreased, but not in wild-type (WT) mice (Figs. 6g and 6i). In addition, the secretion of IFN-γ, IL-5, and IL-21 induced by restimulation of splenocytes with HSA was significantly reduced in the presence or absence of S. aureus or S. epidermidis, compared with Nod2 mice immunized with imiquimod. - / - It was rarely observed in mice (Figs. 6h and 6j). These results indicate that two species of bacteria in FM5 subjects promote the adjuvant activity of imiquimod through Nod2 in the nasal cavity.

[0236]

[0237] Experimental Example 7. Preventive Effect of Nasal Bacteria and Imiquimod on Influenza A Virus (IAV) Infection

[0238] Because we observed that bacteria with high Nod2 stimulating activity strongly enhanced the immune response induced by imiquimod, we next examined whether co-administration of bacteria and imiquimod with an IAV vaccine via the intranasal route could enhance the protective effect against IAV infection. Mice colonized with S. aureus or S. epidermidis were administered iPR8 alone or intranasally together with imiquimod. The results are shown in Figures 7A and 7B.

[0239] As shown in Figures 7a and 7b, 14 days after immunization, the production of iPR8-specific IgG was increased in the serum of mice immunized with imiquimod in the presence of S. aureus, which has high Nod2 stimulating activity, compared to mice immunized with imiquimod and S. epidermidis, which has low Nod2 activity (Figure 7a). In addition, splenocytes extracted from mice co-administered with imiquimod and S. aureus, which has high Nod2 stimulating activity, produced higher levels of IFN-γ and IL-5 when restimulated with HA antigen in vitro than splenocytes from mice co-administered with imiquimod and S. epidermidis, indicating a greater immune-enhancing effect (Figure 7b).

[0240] Next, LD on the mouse 50 A double dose of IAV was administered, and the body weight change rate and survival rate were continuously monitored for 14 days after infection, and the results are shown in Figures 7c to 7h.

[0241] As shown in Figures 7c to 7h, all mice intranasally immunized with S. aureus and imiquimod survived without weight loss, whereas mice immunized with S. epidermidis and imiquimod gradually lost weight, with a survival rate of nearly 60%. Weight loss was observed in mice administered only imiquimod, with only 40% surviving (Figures 7c and 7d). In addition, H&E-stained lung tissue sections showed severe lung damage with extensive inflammatory cell infiltration in the imiquimod alone group, whereas very mild histopathological findings were found in the group administered with S. aureus and imiquimod together (Figure 7e). On day 5 after infection, the gene expression levels of influenza HA and NP were significantly reduced in the lungs of mice administered with both S. aureus and imiquimod, and the viral titer in BALF was also reduced (Figures 7g and 7h). S. The combined treatment with S. epidermidis and imiquimod showed a decrease in infectivity compared to the imiquimod monotherapy group, but a higher infectivity than the combined treatment with S. aureus and imiquimod (Figs. 7g and 7h). This can be interpreted as a control of viral infection itself due to the increased production of antigen-specific IgA in alveolar lavage fluid (Fig. 7f). These results suggest that bacteria with high Nod2 stimulating activity can enhance the efficacy of imiquimod, effectively preventing IAV infection.

[0242]

[0243] Experimental Example 8. The Enhanced Effect of Nasal Bacteria and Imiquimod on Melanoma Treatment

[0244] We investigated whether the combination of bacteria with high Nod2 stimulating activity and imiquimod could enhance the therapeutic efficacy of anticancer vaccines by strongly enhancing the immune response to co-injected antigens. Mice colonized with S. aureus isolated from FM5 subjects were administered B16F10 cell line lysate alone or together with imiquimod, wild-type or Nod2 - / - The drug was administered intranasally to mice. After 14 days, the proliferating B16F10 melanoma cell line was injected subcutaneously into the dorsal side of the mice, and the survival rate was observed. The results are shown in Figure 8.

[0245] As shown in Figure 8, 20 or 21 days after immunization, Nod2 - / - All mice died, and the combination of S. aureus and imiquimod had little effect on survival. In contrast, all wild type mice, except one, died by day 25 when injected with antigen alone, and those that survived died by day 41. Imiquimod alone did not show any improvement in survival, except for one mouse that died on day 41. S. aureus alone had a 40% survival rate until day 28, but a 20% survival rate until day 41. The combination of S. aureus and imiquimod showed a 60% survival rate until day 40, with one additional mouse dying on day 41, for a final survival rate of 40%. These results suggest that, as in vaccines against infections, bacteria with high Nod2 stimulating activity can enhance the effect of imiquimod and effectively treat melanoma.

Claims

1. As the first active ingredient, imiquimod, and As a second effective ingredient, it comprises at least one selected from the group consisting of Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and Nod2 receptor stimulating bacteria; An immune adjuvant composition comprising the first effective ingredient or the second effective ingredient, wherein the first effective ingredient or the second effective ingredient is administered in combination with the second effective ingredient or the first effective ingredient, respectively.

2. In claim 1, The above imiquimod is an immune adjuvant composition represented by the following chemical formula 1: [Chemical Formula 1] .

3. In claim 1, An immune adjuvant composition wherein the Nod2 receptor stimulating component comprises a bacterial cell wall or a cell wall-derived substance.

4. In claim 3, An immune adjuvant composition, wherein the cell wall-derived material of the above bacteria is peptidoglycan or MDP (muramyl dipeptide).

5. In claim 1, An immune adjuvant composition, wherein the Nod2 receptor stimulating bacteria is at least one selected from the group consisting of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Klebsiella aerogenes (K. aerogenes), Staphylococcus capitis (S. capitis), and Corynebacterium spp.

6. In claim 1, An immune adjuvant composition wherein the Nod2 receptor stimulating component or Nod2 receptor stimulating bacteria promotes the activity of imiquimod.

7. In claim 1, The above composition is an immune adjuvant composition that enhances humoral immunity, cellular immunity, mucosal immunity, or systemic immunity.

8. In any one of claims 1 to 7, The above immune adjuvant composition is administered to the nasal mucosa, oral mucosa, sublingual, rectal or vaginal mucosa.

9. A vaccine composition comprising an antigen and an immune adjuvant composition of claim 1.

10. In claim 9, A vaccine composition wherein the antigen is a coronavirus or influenza virus.

11. In claim 9 or claim 10, A vaccine composition, wherein the vaccine composition is administered to the nasal mucosa, oral mucosa, sublingual, rectal or vaginal mucosa.

12. A step of administering an effective amount of imiquimod to the subject; and A method for boosting the immunity of an individual, comprising administering to an individual in need thereof at least one selected from the group consisting of a Nod2 (nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating component and a Nod2 receptor stimulating bacteria.

13. As a first effective ingredient for the preparation of an immune adjuvant composition, Imiquimod, and As a second effective ingredient, at least one use selected from the group consisting of Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating components and Nod2 receptor stimulating bacteria.

14. As a first effective ingredient for the manufacture of a vaccine composition, Imiquimod, and As a second effective ingredient, at least one use selected from the group consisting of Nod2 (Nucleotide-binding oligomerization domain-containing protein 2) receptor stimulating components and Nod2 receptor stimulating bacteria.

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