Mucosal vaccine composition and use thereof

Abstract

The present invention relates to a vaccine composition for mucosal immunity and use thereof. It has been identified that the vaccine composition of the present invention is in the form of a 2D patch and effectively induces nasal mucosal immunity despite nasal mucociliary clearance (MCC). Thus, the vaccine composition provided in the present invention can be variously utilized for mucosal immunity in which various antigenic substances are used.

Description

Mucosal vaccine composition and its use

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0030184, filed February 29, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a mucosal vaccine composition for mucosal immunity and its use.

[0003]

[0004] The nasal cavity is not only the first line of defense that physically blocks the invasion of foreign substances or pathogens (hereinafter referred to as “foreign antigens”), but also induces an immune response against antigens that have penetrated the mucosa through the nasopharynx-associated lymphoid tissue (NALT), an immune organ located beneath the nasal epithelium and the abundant blood vessels within the nasal cavity. The immune response that occurs through the NALT is called mucosal immunity, and the NALT corresponds to Peyer's patches, which are organs in the small intestine where immune responses occur.

[0005] When external antigens reach the NALT through the nasal mucosa, they induce an antigen-specific immune response through interaction with antigen-presenting cells (APCs) such as dendritic cells and macrophages, which not only produce IgA, which is a representative immunoglobulin of mucosal immunity, but also IgG and IgM immunoglobulins, and the activated immune cells can circulate through the bloodstream via lymphatic vessels to induce a systemic immune response.

[0006] Many pathogens, including viruses and bacteria, invade through the mucous membranes, making vaccines that can be absorbed through these tissues particularly useful. Furthermore, intranasal vaccines eliminate the need for needles, resulting in less pain and discomfort, and are safe and easy to administer. This makes them suitable for use in areas with limited medical personnel, potentially addressing vaccine imbalances. Furthermore, they offer the advantage of enabling rapid response to respiratory viral infections, such as influenza and SARS-CoV-2.

[0007] However, ciliated cells are arranged at regular intervals on the surface of the nasal mucosa, and these ciliated cells serve as our body's first line of defense by physically removing foreign substances and pathogens (antigens) attached to the mucosal surface through mucus secretion and ciliary movement. This phenomenon is called mucociliary clearance (MCC), and to successfully induce mucosal immunity in the nasal cavity, antigens must be delivered to the NALT located beneath the mucosa by avoiding the MCC. However, due to the anatomical / physiological structure of the nasal cavity, attaching antigens to the mucosa for a certain period of time and with a certain strength is a major obstacle in the development of mucosal vaccines for nasal administration.

[0008] Some researchers have attempted to overcome these obstacles by using various mucoadhesive materials to increase mucoadhesion and thus overcome MCC. These mucoadhesive materials include polysaccharide nanoparticles such as chitosan, poly(L-lactic-co-glycolic acid, PLGA), poly(acrylate), starch, or dextran, and their thiol functionalization. These mucoadhesive materials have primarily been attempted to form spherical nanoparticles that encapsulate active agents and facilitate controlled release. For example, chitosan nanoparticles encapsulating tetanus toxoid have an average size of 350 nm, and PLGA nanoparticles designed for hepatitis B vaccine delivery have a diameter of approximately 200 nm. Despite the diverse applications of these adhesive materials, these studies have not yet demonstrated satisfactory efficacy (immunogenicity). Furthermore, nanoemulsion vaccines are being studied as an alternative approach for nasal immunization. While nanoemulsions offer the advantage of being usable in formulations such as sprays, drops, and gels, they are not yet widely used.

[0009] Currently, the only FDA-approved nasal vaccine is AstraZeneca's attenuated influenza vaccine series (e.g., FluMist®), and no other vaccines using vaccine delivery materials have received FDA approval.

[0010] Accordingly, the inventors of the present invention studied a vaccine formulation effective for mucosal immunity and discovered that a vaccine containing tannic acid, a type of polyphenol, effectively induces mucosal immunity in a 2D patch structure that is adhesive to the nasal mucosa, thereby completing the present invention.

[0011] [Prior Art Literature]

[0012] [Patent Document]

[0013] US 2010-0092526 A1

[0014]

[0015]

[0016] An object of the present invention is to provide a mucosal vaccine composition comprising at least one type of antigen, tannic acid, and at least one type of cell-penetrating peptide.

[0017] Additionally, the present invention is to use the mucosal vaccine composition to effectively induce mucosal immunity.

[0018]

[0019] To achieve the above object, the present invention provides a mucosal vaccine composition comprising at least one type of antigen, tannic acid, and at least one type of cell-penetrating peptide (CPP).

[0020]

[0021] According to one embodiment of the present invention, a mucosal vaccine composition is provided in which the mass ratio of the antigen, tannic acid, and CPP included in the present invention is 1:0.5 to 10:0.5 to 10.

[0022] According to one embodiment of the present invention, a mucosal vaccine composition is provided in which the CPP included in the present invention is a cationic CPP.

[0023] According to one embodiment of the present invention, a mucosal vaccine composition is provided in which the length of the cationic CPP included in the present invention is composed of 5 to 30 amino acid residues, and the content of arginine and lysine amino acid residues among the amino acid residues is 35% to 100%.

[0024] According to one embodiment of the present invention, the cationic CPP included in the present invention provides a mucosal vaccine composition derived from a randomly synthesized sequence that does not exist naturally or a portion of a sequence of a naturally existing protein.

[0025] According to one embodiment of the present invention, a mucosal vaccine composition is provided in which the antigen included in the present invention is a microbial-derived antigen.

[0026] According to one embodiment of the present invention, the microorganism included in the present invention comprises a mucosal vaccine composition, wherein at least one microorganism is selected from the group consisting of viruses, bacteria, protozoa, and fungi.

[0027] According to one embodiment of the present invention, a mucosal vaccine composition is provided, wherein the virus included in the present invention is at least one selected from the group consisting of an attenuated virus, an inactivated virus, a virus fragment, and an epitope derived from a virus fragment.

[0028] According to one embodiment of the present invention, a mucosa to which the mucosal vaccine composition of the present invention is applied is provided, wherein the mucosa is at least one mucosa selected from the group consisting of oral mucosa, ocular mucosa, ear mucosa, genital mucosa, pharyngeal mucosa, respiratory tract mucosa, nasal mucosa, bronchial mucosa, pulmonary mucosa, gastric mucosa, intestinal mucosa, and rectal mucosa of a human or animal.

[0029] According to one embodiment of the present invention, a mucosal vaccine composition of the present invention is provided, wherein the mucosal vaccine composition further comprises a pharmaceutically acceptable excipient.

[0030] According to one embodiment of the present invention, the mucosal vaccine composition of the present invention provides a mucosal vaccine composition that induces a humoral immune response.

[0031] According to one embodiment of the present invention, a method for producing a mucosal vaccine composition comprising an antigen, a CPP, and tannic acid is provided, comprising the steps of: (a) mixing at least one type of antigen with at least one type of cell-penetrating peptide (CPP) or tannic acid; and (b) mixing tannic acid or CPP into the mixture of step (a).

[0032] According to one embodiment of the present invention, a method of inducing an immune response by administering a mucosal vaccine composition of the present invention to a subject in need of treatment or prevention of a disease is provided.

[0033] According to one embodiment of the present invention, a method for treating or preventing an infectious disease caused by a microorganism is provided using the mucosal vaccine composition of the present invention.

[0034]

[0035] The mucosal vaccine composition of the present invention is in the form of a 2D patch that can effectively attach to the mucosa, particularly the nasal mucosa, and can be attached for a sufficient period of time to induce mucosal immunity, and has the effect of being removed from the body after a certain period of time.

[0036] Therefore, the mucosal vaccine composition of the present invention not only overcomes the limitations of traditional spherical nanoparticles, but can also be used in the development of vaccines for preventing respiratory diseases.

[0037] In addition, the mucosal vaccine composition of the present invention has high stability, and thus has great advantages in vaccine distribution.

[0038] Figure 1 is a UV-Vis spectral analysis graph for GFP, GFP / CPP, TA, and GFP / CPP / TA nanoparticles manufactured in the present invention.

[0039] Figure 2 is an AFM image of GFP, GFP / CPP, and GFP / CPP / TA nanoparticles manufactured in the present invention.

[0040] Figure 3 is a schematic diagram of the structure of GFP / CPP and GFP / CPP / TA nanoparticles manufactured in the present invention.

[0041] Figure 4 shows the results of mucin adhesiveness analysis of GFP, GFP / TA, and GFP / CPP / GFP / CPP / TA nanoparticles manufactured in the present invention. Figure 4A shows a schematic diagram of the experiment and fluorescence microscopic photographs of each group, and Figure 4B shows the GFP signal intensity of each group.

[0042] Figure 5 shows the results of cell (EpH4) adhesion analysis of GFP, GFP / TA, GFP / CPP, and GFP / CPP / TA nanoparticles manufactured in the present invention. Figure 5A shows a schematic diagram of the experiment and fluorescence microscopic photographs of each group, and Figure 5B shows the GFP signal intensity of each group.

[0043] Figure 6 shows the results of mucin adhesiveness analysis of GFP / CPP / TA nanoparticles containing various types of CPP, including GFP, GFP / TA, TFP / Tat / TA, TFP / Pen / TA, GFP / R9 / TA, and GFP / PEP1 / TA nanoparticles.

[0044] Figure 7 shows the distribution status when GFP, GFP / TA, GFP / CPP, and GFP / CPP / TA nanoparticles manufactured in the present invention were injected into the nasal cavity of a mouse. Figure 7A shows the observation site, Figure 7B shows the distribution degree of GFP nanoparticles in Transverse Section 3, and Figure 7C shows the distribution degree of GFP / CPP / TA nanoparticles and macrophages and dendritic cells.

[0045] Figure 8 shows the degree of distribution of GFP / CPP / TA nanoparticles and macrophages and dendritic cells in the nasal mucosa and submucosal layer 2 hours after intranasal injection of the GFP / CPP / TA nanoparticles manufactured in the present invention into a mouse.

[0046] Figure 9 shows the distribution of GFP / CPP / TA nanoparticles in the nasal cavity and lungs 24 hours after intranasal injection of GFP / CPP / TA nanoparticles into a mouse according to the present invention.

[0047] Figure 10 shows the results of measuring the immune response induced in the nasal mucosa 3 weeks after intranasal injection of the GFP / CPP / TA nanoparticles manufactured in the present invention into a mouse. Figure 10A is a schematic diagram of the humoral immune mechanism occurring in the nasal mucosa, and Figure 10B is the results of quantitative analysis of antibodies (IgA, IgG, IgM) against GFP produced in BALF.

[0048]

[0049] Hereinafter, the present invention will be described in more detail to help understand the present invention.

[0050] The term “pathogen” as used herein refers to an organism, including a microorganism, that causes disease in another organism by directly infecting the organism or by producing toxins within the organism after infection (e.g., bacteria that produce pathogenic toxins, etc.). The terms “pathogen” and “pathogen” are used interchangeably herein.

[0051] The terms “host” and “subject” as used herein refer to an organism to be treated by the composition and / or method of the present invention.

[0052] The term “pharmaceutically acceptable substance” as used herein refers to a substance that does not substantially cause an adverse allergic or immunological reaction when administered to a host (e.g., an animal or a human).

[0053] The terms “immunity” and “immune response” as used herein refer to protection from disease upon exposure to antigens such as pathogens. Immunity may be innate immunity (antigen-nonspecific immunity) and / or acquired immunity (antigen-specific immunity).

[0054] “Pathogen product” as described herein means all types of products derived from a pathogen, including pathogen fragments, peptides (epitopes), proteins, nucleic acids, membrane proteins and polysaccharides derived from the pathogen.

[0055] The term “antigen” as used herein refers to a substance capable of inducing an immune response in a subject. For example, it refers to all types of substances, including the pathogen itself, pathogen fragments derived from the pathogen, and epitopes derived from the pathogen and capable of binding to antigen-presenting cells (APCs) in the subject.

[0056] As used herein, the terms “increased immune capacity,” “immune activation,” and “increased immunogenicity” all have the same meaning and refer to an increase in the level of acquired immunity against an antigen in a group administered the vaccine of the present invention compared to a group that was not administered. Acquired immunity can be measured by an increased level of effector T cells / effector B cells / memory T cells, etc. stimulated in response to an antigen presented by antigen-presenting cells. However, in the present invention, for example, an increase in antibody production against an immunized antigen is considered to be an increase in immune capacity.

[0057] The terms “2D nanopatch,” “2D nanostructure,” “2D complex,” and “2D planar structure” described in this specification are all used interchangeably to refer to an antigen / CPP / TA complex having adhesive properties.

[0058]

[0059] The present invention provides a mucosal vaccine composition comprising at least one type of antigen, at least one type of cell-penetrating peptide (CPP) and tannic acid.

[0060] The antigen included in the mucosal vaccine composition of the present invention is a substance for inducing an immune response in a subject and is derived from an antigen derived from a foreign substance or microorganism.

[0061] The above microbial antigens are antigens that can cause infectious diseases in the subject. In the case of infectious antigens, since the purpose is to prevent infectious diseases and it is necessary to preemptively form antibodies through vaccine administration, it is preferable to use the mucosal vaccine composition of the present invention. The mucosal vaccine composition of the present invention is suitable for activating humoral immunity, which produces antigen-specific antibodies.

[0062] In addition, the antigen used in the present invention may be an antigen derived from a microorganism causing an infection, and the microorganism may be at least one selected from the group consisting of viruses, bacteria, protozoa, and fungi.

[0063] Additionally, the virus may be at least one selected from the group consisting of an attenuated virus, an inactivated virus, a virus fragment, and an epitope derived from a virus fragment.

[0064] The virus may be derived from a virus such as an influenza virus A, an influenza virus B, an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a varicella virus (e.g., orthopox virus such as smallpox or vaccinia, or molluscum contagiosum), a rhinovirus, an enterovirus, a paramyxovirus (e.g., parainfluenza virus, measles virus, respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS-COVID2), a parvovirus, a papillomavirus, a hepatitis virus (e.g., HAV, HBV, HCV), a dengue virus, a lentivirus, or a retrovirus (e.g., a lentivirus such as HIV).

[0065] The above bacteria may be derived from bacteria of the genus Escherichia, Enterobacter, Salmonella, Staphylococcus aureus, Diplococcus, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Streptococcus pneumoniae, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella.

[0066] In addition, the virus included in the mucosal vaccine composition of the present invention may include as an antigen at least one selected from the group consisting of an attenuated virus, an inactivated virus, a virus fragment, and an epitope derived from a virus fragment.

[0067] At this time, the antigen may include multiple different types of viruses, or multiple different strains of a single virus. Furthermore, an epitope capable of inducing humoral immunity can be designed by analyzing viral protein fragments and TCR binding capacity, and a peptide epitope composed of 10-20 amino acid residues, artificially synthesized, can be used as the antigen.

[0068]

[0069] The mucosal vaccine composition of the present invention comprises at least one type of cell-penetrating peptide (CPP).

[0070] The CPP used in the present invention is preferably a cationic CPP. The cationic CPP is composed of 5 to 30 amino acid residues, and among the amino acid residues, the content of cationic amino acid residues, namely arginine and lysine, is preferably 35% to 100%.

[0071] The cationic CPP may be derived from a randomly synthesized sequence that does not exist naturally or from a portion of a sequence of a naturally occurring protein.

[0072] The above cationic CPP can be selected from commonly known cationic CPPs.

[0073] The above commonly known cationic CPP may be at least one selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 1), RQIKIWFQNRRMKWKK (SEQ ID NO: 2), RRRRR (SEQ ID NO: 3), RRRRRRRR (SEQ ID NO: 4), RRRRRRRRR (SEQ ID NO: 5), YARVRRRGPRR (SEQ ID NO: 6), RRRRPRRRTTRRRR (SEQ ID NO: 7), VSRRRRRRGGRRRR (SEQ ID NO: 8), and RQIKIWFQNRRMKWKK (SEQ ID NO: 9).

[0074] The CPP used in the present invention is not intended for cell membrane penetration but for non-covalent binding with an antigen, and need not be limited to a CPP commonly known as such, and it is sufficient if the content of arginine and lysine residues, which are cationic amino acid residues, among the entire amino acid sequence is 35% to 100%.

[0075] The mucosal vaccine composition of the present invention includes tannic acid (TA), a type of polyphenol. Tannic acid is a molecule containing numerous phenolic hydroxyl groups commonly found in edible plants and is known to be capable of forming complexes with various biomolecules, including proteins, DNA, synthetic polymers, and mucosal proteins such as mucin. However, historically, mucoadhesives have been developed as 3D spherical nanoparticles, and a 2D patch-type delivery system for nasal adhesion has never been developed.

[0076] In the present invention, GFP was used as an antigen material to facilitate tracking. When GFP and CPP of YGRKKRRQRRR (SEQ ID NO: 1) were mixed (GFP / CPP), it was confirmed that spherical nanoparticles were formed. When tannic acid was additionally mixed here (GFP / CPP / TA), it was confirmed that the spherical nanoparticles changed into a 2D patch shape and adhered to the mucosa. In addition, the same results were confirmed for viruses. Specifically, when AAV was used as an antigen and AAV / CPP / TA was mixed, a 2D nanopatch was formed, and it was confirmed that this nanopatch had membrane adhesiveness.

[0077]

[0078] The content ratio of each component above may be 1: 0.5 to 10: 0.5 to 10 based on the weight of antigen: CPP: tannic acid. In addition, the content ratio of each component above may be 1: 0.5 to 10: 0.5 to 5 based on the weight of antigen: CPP: tannic acid. In the content ratio above, when the content of CPP is 0.5 to 5 based on the antigen, the 2D nanopatch formation ability is stable without a significant difference. In addition, when the content of tannic acid is in the range of 0.5 to 5 based on the antigen, a stable 2D nanopatch is formed, but when it is greater than 10, there is a problem that the height of the 2D patch increases.

[0079] In the production of the antigen / CPP / TA 2D nanopatch of the present invention, the production may include a step of mixing at least one type of antigen and at least one type of CPP or tannic acid; and a step of mixing tannic acid or CPP into the mixture.

[0080] Therefore, the 2D nanopatch of the present invention can be manufactured by mixing an antigen and CPP and then mixing tannic acid, or by mixing an antigen and tannic acid and then mixing CPP.

[0081] This phenomenon occurred when tannic acid, which has the property of binding to various proteins, was inserted into the nanoparticles between GFP and CPP, thereby changing the original spherical nanoparticle (GFP / CPP) into a planar patch-shaped nanoparticle (GFP / CPP / TA), and as a result, the planar patch-shaped structure (complex; 2D nanopatch) was able to bind to mucin on the mucosal surface.

[0082] In one embodiment of the present invention, GFP (Green Fluorescence Protein) was used as the antigen material, but even if various antigen materials such as viruses and antigenic peptides are used, the same spherical antigen / CPP is formed, and when tannic acid is added thereto, it is changed into a 2D patch shape and can bind to mucin.

[0083] The GFP / CPP / TA vaccine composition of the present invention was confirmed to successfully induce mucosal immunity (production of IgG, IgA, and IgM in BALF; induction of humoral immunity) when administered intranasally to the mouse. Above all, 2 days after intranasal administration, GFP was present not only on the mucosal surface and the lamina propria layer, which is the submucosal layer of the nasal cavity, but also coexisted with macrophages and / or dendritic cells, indicating that an active immune response was induced. However, GFP was not detected in the nasal cavity and lungs 24 hours after administration, suggesting that the injected vaccine composition is decomposed or excreted in vivo, and thus no further antigen exposure occurs.

[0084] This phenomenon enables rapid antigen release or removal after a short period of antigen exposure following the induction of an immune response, thereby demonstrating the usefulness of the vaccine composition of the present invention as a mucosal vaccine delivery platform.

[0085] The mucosal vaccine composition of the present invention can be administered to at least one mucosa selected from the group consisting of oral mucosa, ocular mucosa, ear mucosa, genital mucosa, pharyngeal mucosa, respiratory tract mucosa, nasal mucosa, bronchial mucosa, pulmonary mucosa, gastric mucosa, intestinal mucosa, and rectal mucosa of a human or animal.

[0086] The mucosal vaccine composition of the present invention may include a “pharmaceutically acceptable carrier.” The pharmaceutically acceptable carrier may include one or more solvents, dispersion media, coatings, wetting agents (e.g., sodium lauryl sulfate), isotonic agents, antioxidants, surfactants, and absorption delaying agents, disintegrating agents (e.g., potato starch or sodium starch glycolate), and the like.

[0087] The mucosal vaccine composition of the present invention may be in the form of a liquid, spray, semi-solid formulation, or solid formulation. The mucosal vaccine composition of the present invention can be suitably administered to the nasal mucosa of a human or animal using a known method in the above-described formulation.

[0088] Hereinafter, the present invention will be described in detail by examples.

[0089] However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. The scope of the invention is determined by the entire detailed description and claims.

[0090]

[0091]

[0092] <Example>

[0093] <Example 1> Fabrication of 2D nanopatch and analysis of morphological characteristics

[0094] GFP (Green-Fluorescence protein) was purchased from Abcam (Cambridge, UK), HIV-1 Tat peptide (YGRKKRRQRRR) was purchased from Sigma-Aldrich (St. Louis, USA), Penetratin peptide (RQIKIWFQNRRMKWKK), polyarginine (R9) peptide (RRRRRRRRR), and Pep-1 peptide (KETWWETWWTEWSQPKKKRKV) were all purchased from MedChemExpress (New Jersey, USA), and tannic acid and mucin were purchased from Sigma-Aldrich (St. Louis, USA).

[0095] To prepare 2D nanopatches, three main components (GFP, CPP, and TA) were used in equal amounts (1 μg each), and TA was pre-dissolved in distilled water (DDW). The CPP used was YGRKKRRQRRR, a TAT (Transactivator of transcription) CPP sequence derived from HIV-1.

[0096] To form 2D nanopatches, GFP and CPP were mixed and incubated at 37°C for 30 minutes. TA was then added to the mixture and mixed. The GFP-only group and the GFP / CPP mixture group served as controls. In the GFP-only group, 1 μg of GFP was used in the experiment without any additives, while the GFP / CPP mixture was prepared by mixing 1 μg of GFP and 1 μg of CPP in the same ratio as used for the 2D nanopatches, followed by incubation at 37°C for 30 minutes.

[0097] The characterization of the previously fabricated 2D nanopatch particles was performed using two main techniques: UV-Vis spectroscopy (Hewlett-Packard 8453, USA) and tapping-mode AFM (NX-10, Park Systems). UV-Vis spectroscopy was performed on GFP, GFP / CPP, and GFP / CPP / TA.

[0098]

[0099] GFP exhibited three distinct absorption peaks at 280 nm (black), which were attributed to aromatic amino acids (Trp and Tyr), 397 nm (neutral chromophore), and 475 nm (anionic form), respectively. Since the amino acid composition of CPP (Tat; YGRKKRRQRRR) does not contain aromatic residues, the absorption at 280 nm did not change significantly when mixed with GFP, resulting in almost identical spectra. TA exhibited two peaks at 279 nm and 320 nm, reflecting the protonated galloyl group and its quinone form, respectively, with an absorbance ratio of 1.56 (A279 / A320). However, when the GFP / CPP / TA complex was formed, the contribution of TA to the overall absorbance spectrum became dominant, and the A279 / A320 ratio decreased to 0.86, reflecting the basic pH environment within the complex. These changes are attributed to the basic environment provided by the interaction between the arginine-rich sequence of CPP and the galloyl hydroxyl of TA, which promotes the conversion of the phenolic group (A279) of TA to the quinone group (A320). Furthermore, the increase in scattering absorption at 400–600 nm indicates the successful formation of the GFP / CPP / TA complex.

[0100] Each group (GFP, GFP / CPP, GFP / CPP / TA) was further analyzed using atomic force microscopy (AFM). Imaging was performed using silicon SPM sensor (NCHR-50, Nanosensors) cantilevers tuned to a resonant frequency of 320 kHz. AFM scans covered an area of ​​10 × 10 μm² at a scan rate of 0.5 Hz, ensuring detailed visualization. Height, line profile, and overall morphological analysis were analyzed using XEI software.

[0101] AFM images revealed that GFP-only particles were barely detectable, measuring approximately 3–4 nm in height (Fig. 2A). GFP / CPP significantly increased particle heights to 466 nm and 267 nm (Fig. 2B), indicating the formation of spherical complexes.

[0102] In contrast, the GFP / CPP / TA complex exhibited a distinct morphology from the spherical GFP / CPP. AFM images revealed a planar 2D nanopatch structure, with a height of less than 50 nm (Fig. 2C). This transformation from a 3D spherical structure (GFP / CPP) to a 2D planar structure (GFP / CPP / TA) appears to have been driven by TA. TA possesses the ability to bind to various proteins, and its adhesive properties appear to have facilitated the insertion reaction between GFP and CPP, thereby changing the 3D spherical structure to a 2D planar patch shape (Fig. 3).

[0103]

[0104] In addition, when experimenting with AAV (Adeno-associated virus) particles as an antigen, AAV alone did not form any identifiable structures or 2D nanopatches, and when AAV was mixed with CPP, some structures were observed, but they had small surface areas and could not attach to mucin. However, when AAV was mixed with CPP and TA, it was confirmed that a 2D nanopatch-shaped structure that could be widely attached to the surface was formed, similar to GFP / CPP / TA.

[0105] The degree of 2D nanopatch formation according to the weight ratio of GFP / CPP / TA was confirmed through AFM images using the above-described manufacturing method.

[0106] As a result, when the relative weight of GFP was fixed at 1 and the relative weight of DPP was increased from 0.5 to 10, a 2D patch shape similar to that of 1:1 was observed. However, a larger 2D patch shape was not observed compared to the 1:1:1 condition. However, when the relative weight of TA was increased from 0.5 to 5, it was similar to the 1:1:1 condition, but when the relative weight of TA was increased from 0.5 to 5, a 3D patch shape with adhesiveness but high height was observed. Therefore, the optimal weight ratio of antigen / CPP / TA was determined to be 1:0.5 to 10:0.5 to 5 (Table 1).

[0107]

[0108] GroupGFP:CPP:TA ratio2D patch formation degree11:1:1+++21:0.5:1++31:5:1+++41:10:1+++51:1:0.5++61:1:5++71:1:10+

[0109] (Judgment criteria: Relative area and height of each 2D nanopatch based on AFM images)

[0110]

[0111] <Example 2> Analysis of mucin adhesiveness of 2D nanopatches

[0112] To analyze the mucin adhesiveness of the 2D nanopatches, mucin was first coated onto chamber slides as follows. Chamber slides (Sigma, C6682) were incubated with 200 ng / mL mucin protein dissolved in 20 mM HEPES for 1 hour at room temperature. Equal amounts of GFP, GFP / TA, GFP / CPP, and GFP / CPP / TA were then added, and the chamber slides were washed with distilled water. Cover glasses were then mounted on the chamber slides, and images were captured using a fluorescence microscope. Fluorescence intensity was measured using ImageJ software (NIH, MD, USA). As a result, only the GFP / CPP / TA group exhibited a strong and significantly higher fluorescence signal (Fig. 4A, 4B), indicating that the planar 2D nanopatches exhibited mucoadhesive properties.

[0113]

[0114] <Example 3> Analysis of cell adhesion of 2D nanopatches

[0115] To analyze the cell adhesion of the 2D nanopatches, the mouse epithelial cell line EpH4 was used. EpH4 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, MA, USA) supplemented with 1× antibiotic-antimycotic solution (Thermo Fisher Scientific) and 5% fetal bovine serum (Corning, NY, USA) at 37°C under 95% air and 5% CO2. The culture medium was changed every 2 days.

[0116] 1 × 10 5Cells / well were seeded in 24-well plates (Corning). 24 h after seeding, the cell culture medium was replaced, and cells were treated with GFP, GFP / TA, GFP / CPP, and GFP / CPP / TA, respectively. Each treatment was prepared to contain 1 μg of each agent in a total volume of 2.25 μL. After treatment, the plates were immediately washed with Dulbecco's phosphate-buffered saline (DPBS). Fluorescence imaging was performed using Cytation 5 (BioTeck, VT, USA) at absorption / emission wavelengths of 480 / 510 nm. GFP signal intensity was quantified using ImageJ software using automatic threshold settings. The nuclei of each cell were stained with DAPI.

[0117] The staining results showed that only the GFP / CPP / TA group (the fourth cell surface image in Fig. 5A) exhibited a strong fluorescent signal, indicating strong adhesion to EpH4 cells. The GFP signal intensity in the GFP / CPP / TA group was significantly increased compared to the other groups (Fig. 5B).

[0118]

[0119] In contrast, the GFP / CPP composition showed minimal fluorescence signal in the cell surface and Z-stack images, suggesting weak intracellular uptake, despite the formation of nanoparticles in the AFM image (Fig. 2B) (third image in Fig. 5A). Meanwhile, the GFP / CPP / TA complex was strongly attached to the outer cell membrane due to the adhesive properties of TA (Fig. 5B).

[0120] However, for the GFP / TA complex, almost no complex was detected that adhered to the cell surface or was taken up into the cell. This suggests that TA acts as a small molecular adhesive (∼Da) that can bind to large GFP molecules (∼kDa), but is not exposed on the surface of the GFP / TA particle.

[0121]

[0122] <Example 4> Analysis of mucin adhesiveness of 2D nanopatches containing various types of CPPs

[0123] To determine whether a specific category of cell-penetrating peptides (CPPs) among various types of CPPs is effective, 2D nanopatches were fabricated using cationic CPPs Tat (SEQ ID NO: 1), Pen (SEQ ID NO: 2), and R9 (SEQ ID NO: 4) peptides and an amphipathic CPP PEP1 peptide (SEQ ID NO: 10). GFP / CPP / TA complexes using each CPP were fabricated using the method of Example 1, and the UV-Vis profiles were analyzed. Then, the mucin adhesiveness of each complex was analyzed using the method of Example 2. As a result, among the four complexes, only cationic CPPs Tat, Pen, and R9 showed mucin adhesiveness in the form of a patch-type complex, whereas the GFP / PEP1 / TA complex containing the amphipathic CPP PEP1 did not show mucin adhesiveness (Fig. 6). Therefore, it can be seen that only protein / cationic CPP / TA can form the desired 2D-shaped nanopatch.

[0124]

[0125] SEQ ID NO: 1YGRKKRRQRRR2RQIKIWFQNRRMKWKK3RRRRR4RRRRRRRR5RRRRRRRRRR6YARVRRRGPRR7RRRRPRRRTTRRRR8VSRRRRRRGGRRRR9RQIKIWFQNRRMKWKK10KETWWETWWTEWSQPKKKRKV

[0126]

[0127] <Example 5> Analysis of the effectiveness of 2D nanopatches (in vivo)

[0128] <Example 5-1> Nasal immunization using GFP-2D nanopatch

[0129] Since GFP can induce an immune response and produce antibodies, the GFP 2D nanopatch of the present invention was administered into the nasal cavity of mice to analyze its actual in vivo effects. Eight-week-old ICR female mice were purchased from Koatech (Pyeongtaek, Gyeonggi, Korea). Mice were administered 20 μL of GFP, GFP / TA, GFP / CPP, or GFP / CPP / TA complex intranasally under anesthesia.

[0130] Each treatment substance was prepared to contain 10 μg of each substance in a total volume of 20 μl. For example, GFP was prepared with 10 μg of GFP in a volume of 20 μl, GFP / TA was prepared with 10 μg of GFP and TA each in a volume of 20 μl, and the remaining treatment substances were prepared in the same manner.

[0131] Mice were euthanized 2 hours after drug administration. The hard palate was then exposed through bilateral incisions at the labial angle of each mouse. The hard palate was carefully dissected with fine forceps and fixed overnight in 4% paraformaldehyde in PBS solution (Thermo Fisher Scientific) at 4°C. The tissues were then decalcified for 6 hours in Calci-Clear Rapid (National Diagnostics, GA, USA). After decalcification, frozen blocks were prepared using 30% sucrose, embedded in OCT compound (Leica Microsystems, Wetzlar, Germany), and stored at -80°C. The nasal cavity was sectioned transversely at 15 μm thickness from the second palatal ridge (T3) using a Leica cryosectioner (Leica Microsystems) and stored at -80°C. This study was approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-231218-4) and performed in accordance with the approved guidelines.

[0132] To confirm the distribution of the drug after injection into the nasal cavity, transverse section 3, which includes the NALT, was selected as shown in Fig. 7A, and the distribution of each GFP was confirmed to confirm the distribution of the injected drug. In particular, GFP was slightly detected in the GFP / TA complex (indicated by a white triangle), and GFP was hardly detected in the GFP alone or GFP / CPP group. In contrast, the GFP / CPP / TA complex exhibited strong adhesive properties to the nasal mucosa (Fig. 7B). In the GFP / CPP / TA complex-treated group, GFP was observed throughout the entire nasal cavity of the mouse, and it can be seen that it was simply attached to the mucosal layer (gray triangle with a white border) or moved to the lamina propria of the submucosal layer (white triangle with a black border). We confirmed that GFP in the mucosa could contact APCs (CD11c-positive dendritic cells or / and F4 / 80-positive macrophages, indicated by white border and black triangle) in the submucosal area (Fig. 7C).

[0133]

[0134] Microfold cells (M cells) within the nasal mucosal epithelium play a crucial role in delivering antigens to lymph nodes, so the GFP / CPP / TA complex may increase the probability that the antigen (GFP) encounters M cells. Since the CPP-conjugated peptide is cell-permeable, it could be predicted that it could enhance the immune response by promoting the accumulation of peptide antigens in submucosal lymph nodes. However, the GFP / CPP complex did not induce GFP penetration into the submucosal layer (Fig. 8). Therefore, the GFP observed in the nasal lamina propria layer is likely not due to CPP migration into the nasal mucosal epithelium, but rather due to the mucosal adhesive ability of the GFP / CPP / TA complex, which covers a wide mucosal area. Furthermore, the GFP observed in the mucosal and submucosal layers 2 hours after administration provides strong evidence that it can induce mucosal immune activation.

[0135]

[0136] <Example 5-2> Analysis of the intranasal persistence time of GFP-2D nanopatch

[0137] In the development of vaccine compositions that induce immune responses, it is crucial that the administered vaccine degrades after a certain period of time, ensuring optimal antigen exposure time and safety. Therefore, the amount of GFP / CPP / TA remaining in the nasal cavity and lungs 24 hours after intranasal injection of the GFP / CPP / TA complex was measured, confirming that the GFP / CPP / TA complex was excreted from the lungs and nasal cavity within 24 hours (Figure 9). These results suggest that the vaccine composition of the present invention has utility as a mucosal vaccine delivery platform, as it enables rapid antigen excretion following a short period of antigen exposure following the induction of an immune response.

[0138]

[0139] <Example 5-3> Analysis of intranasal immune function (anti-GFP immunoglobulin) of GFP-2D nanopatch

[0140] The intranasal immunogenicity of the GFP-2D nanopatch can be confirmed by analyzing the level of antibody production against the antigen, as shown in Figure 10A. To this end, bronchoalveolar lavage fluids (BALF) were collected from euthanized mice 3 weeks after intranasal drug administration. BALF was collected using a slightly modified method from a previously established method. A 30-gauge syringe was inserted into the trachea, with the needle tip pointed toward the lungs and secured with forceps. 200 μl of DPBS in the syringe was slowly injected and withdrawn. Anti-GFP antibodies of each isotype in the collected BALF were quantified using an indirect enzyme-linked immunosorbent assay (ELISA).

[0141] The GFP / CPP / TA 2D nanopatch treatment group showed increased concentrations of IgA, IgG, and IgM against GFP compared to the control group. In particular, the increase in IgA, an indicator of mucosal immunity, indicates that the GFP / CPP / TA 2D nanopatch of the present invention is more suitable for activating mucosal immunity than GFP / TA spherical nanoparticles (Fig. 10).

[0142]

[0143] <Example 6> Stability analysis of GFP / CPP / TA 2D patches under harsh conditions

[0144] In order to analyze the stability of the GFP / CPP / TA 2D patch, which is a vaccine composition of the present invention, the GFP / CPP / TA 2D patch manufactured by the method of Example 1 was incubated in buffers of pH 2, 4, 7, 10, and 12 for 1 hour, and then AFM was measured. As a result, the height of the 2D patch at pH 2, 4, 7, and 10 did not change significantly at the level of 50 nm, but under extremely alkaline conditions of pH 12, the height tended to increase up to 200 nm. Therefore, it can be seen that the 2D patch of the present invention exhibits an overall stable structure except under extremely alkaline conditions of pH 12.

[0145] To confirm the temperature stability of the GFP / CPP / TA 2D patch, the manufactured GFP / CPP / TA 2D patch was incubated at RT (25°C), 40°C, and -80°C for approximately 2 hours, and then AFM was measured. As a result, it was confirmed that the GFP / CPP / TA 2D patch of the present invention maintains a stable structure even at typical high temperatures and extremely low temperatures, as it showed a stable structure under all conditions.

[0146] In addition, it was confirmed that the GFP / CPP / TA 2D patch of the present invention exhibited a stable structure even after rehydration by adding distilled water after complete drying at room temperature for 16 hours.

[0147] Therefore, it can be seen that the vaccine composition of the present invention has a very stable structure that maintains a 2D patch even in a wide range of pH, high and very low temperatures, and rehydration after drying, and such a feature can serve as a great advantage in vaccine distribution.

Claims

1. A mucosal vaccine composition comprising at least one type of antigen, at least one type of cell-penetrating peptide (CPP) and tannic acid.

2. In paragraph 1, A mucosal vaccine composition wherein the mass ratio of the above antigen, CPP and tannic acid is 1:0.5 to 10:0.5 to 10.

3. In paragraph 2, A mucosal vaccine composition having a mass ratio of 1:0.5 to 10:0.5 to 5.

4. In paragraph 2, A mucosal vaccine composition wherein the above CPP is a cationic CPP.

5. In paragraph 4, A mucosal vaccine composition wherein the cationic CPP has a length of 5 to 30 amino acid residues, and the content of arginine and lysine amino acid residues among the amino acid residues is 35% to 100%.

6. In paragraph 5, A mucosal vaccine composition wherein the cationic CPP is derived from a randomly synthesized sequence that does not exist naturally or a portion of a sequence of a naturally existing protein.

7. In paragraph 1, A mucosal vaccine composition wherein the above antigen is a microbial-derived antigen.

8. In paragraph 7, A mucosal vaccine composition, wherein the microorganism is at least one selected from the group consisting of viruses, bacteria, protozoa, and fungi.

9. In paragraph 8, A mucosal vaccine composition, wherein the virus is at least one selected from the group consisting of an attenuated virus, an inactivated virus, a virus fragment, and an epitope derived from a virus fragment.

10. In paragraph 1, A mucosal vaccine composition, wherein the mucosa is at least one mucosa selected from the group consisting of oral mucosa, ocular mucosa, ear mucosa, genital mucosa, pharyngeal mucosa, respiratory tract mucosa, nasal mucosa, bronchial mucosa, pulmonary mucosa, gastric mucosa, intestinal mucosa, and rectal mucosa of a human or animal.

11. In paragraph 10, A mucosal vaccine composition wherein the above mucosa is a nasal mucosa.

12. In any one of paragraphs 1 to 11, A mucosal vaccine composition further comprising a pharmaceutically acceptable excipient.

13. In any one of paragraphs 1 to 11, The above vaccine composition is a mucosal vaccine composition that induces a humoral immune response.

14. A method for producing a mucosal vaccine composition according to any one of claims 1 to 11, (a) a step of mixing at least one type of antigen and at least one type of cell-penetrating peptide (CPP) or tannic acid; and (b) a step of mixing tannic acid or CPP into the mixture of step (a); A method for producing a mucosal vaccine composition comprising an antigen, CPP and tannic acid.

Images