Extracellular endoplasmic reticulum containing coronavirus-derived antigen protein or the gene encoding said protein, and its uses

Abstract

The present invention relates to an extracellular vesicle containing a coronavirus-derived antigen protein or a gene encoding the protein, and its use. In the present invention, an activated immune cell loaded with an antigen protein or mRNA was used to administer an extracellular vesicle derived therefrom to a mouse for immunization, and the excellent antigen-specific neutralizing antibody production and T cell response induction effect were confirmed, and excellent stability and antibody production effect were experimentally confirmed when the extracellular vesicle was stored at room temperature and refrigerated conditions after freeze-drying. Thus, it is expected that the extracellular vesicle according to the present invention or a vaccine composition containing the same can function as a platform applicable to various diseases with excellent antigen-specific immune response induction effect and stability, and can be usefully utilized in the field of vaccine development for preventing or treating various diseases, including infectious diseases, particularly coronavirus infections.

Description

Technical Field

[0001] The present invention relates to extracellular vesicles containing a coronavirus-derived antigen protein or a gene encoding the protein, and uses thereof.

Background Art

[0002] A vaccine is a pharmaceutical product that confers acquired immunity against a specific disease or pathogen to animals including humans. It mainly has a structure similar to the antigen recognition site of a microbial pathogen that causes disease, but unlike the pathogen, it has no pathogenicity. Currently, various types of vaccines against various pathogens and various diseases have been developed, but there are still many diseases that require the development of more effective and safer vaccines.

[0003] Extracellular vesicles are nanovesicles with a size of several tens of nm to several hundreds of nm composed of a double lipid membrane, and are composed of substances having biological activities such as proteins, lipids, and genes. Such extracellular vesicles typically include, in addition to exosomes, ectosomes, microvesicles, or apoptotic bodies. Conventionally, such extracellular vesicles have been regarded as waste secreted from cells, but recently, the clinical significance of extracellular vesicles has emerged and various studies are underway. In particular, exosomes, which are spherical vesicles excreted by cells, have various information such as proteins and DNA of the parent cell, and the development of cancer diagnostic markers and sensors that utilize this as a biomarker has been actively progressing.

[0004] Exosomes are small membrane-bound vesicles (approximately 30-100 nm in diameter) secreted by various cells. Electron microscopy studies have shown that they do not detach directly from the plasma membrane, but rather originate in specific intracellular compartments called multivesicular bodies (MVBs) before being released and secreted outside the cell. In other words, when the multivesicular bodies and plasma membrane fuse, the vesicles are released into the extracellular environment, and these are called exosomes. It is known that not only red blood cells, but also various types of immune cells including B lymphocytes, T lymphocytes, dendritic cells, platelets, and macrophages, as well as tumor cells and stem cells, produce and secrete exosomes while alive.

[0005] Mammalian cell-derived exosomes are known to be involved in various physiological functions such as hemostasis, tissue regeneration, stem cell maintenance, regulation of inflammation / immune responses, and embryonic development. In particular, exosomes derived from immune cells have been reported to be able to transmit inflammatory cytokines and to directly or indirectly present antigens to promote immune responses (Nat Rev Immunol. 2009 Aug;9(8):581-93). Based on these characteristics and in vivo functions of exosomes, they are being researched and developed for applications such as nano-sized substance delivery methods, mediators, and biomarkers in fields such as oncology, immunotherapy, and regenerative medicine.

[0006] While research is being conducted to use exosomes for vaccine applications, it is still limited to certain diseases, and research and development of vaccine platform technologies applicable to a wide range of diseases, including infectious diseases, remains insufficient. [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors of this invention have conducted research efforts to develop an extracellular endoplasmic reticulum-based vaccine platform that can be universally applied to various diseases. As a result, they have confirmed that the extracellular endoplasmic reticulum acts as a potent immunostimulant and antigen carrier, inducing the production of antigen-specific neutralizing antibodies and antigen-specific T cell responses, thereby completing the present invention.

[0008] Therefore, the present invention aims to provide an extracellular endoplasmic reticulum containing a coronavirus-derived antigen protein or a gene encoding said protein.

[0009] Another objective of the present invention is to provide a vaccine composition for the prevention or treatment of coronavirus infection, which includes the extracellular endoplasmic reticulum.

[0010] Furthermore, another objective of the present invention is to provide a health functional food for the prevention or improvement of coronavirus infection, which includes the extracellular endoplasmic reticulum.

[0011] Furthermore, another objective of the present invention is to provide a method for preventing or treating coronavirus infection, which includes the step of administering the extracellular vesicles to an individual in need.

[0012] Furthermore, another objective of the present invention is to provide the use of the extracellular endoplasmic reticulum for the manufacture of agents for the prevention or treatment of coronavirus infection.

[0013] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. [Means for solving the problem]

[0014] To achieve the above-described objectives of the present invention, the present invention provides an extracellular endoplasmic reticulum containing a coronavirus-derived antigen protein or a gene encoding said protein.

[0015] In one embodiment of the present invention, the coronavirus may be SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2).

[0016] In another embodiment of the present invention, the antigen protein may be a receptor binding domain (RBD) protein of a coronavirus-derived spike protein.

[0017] In yet another embodiment of the present invention, the antigen protein or the gene encoding the protein may be located within the extracellular endoplasmic reticulum.

[0018] In yet another embodiment of the present invention, the extracellular endoplasmic reticulum may be derived from one or more selected from the group consisting of animal cells, plant cells, or microorganisms.

[0019] In yet another embodiment of the present invention, the animal cells may be one or more cells selected from the group consisting of somatic cells, germ cells, immune cells, nerve cells, and tumor cells.

[0020] In yet another embodiment of the present invention, the extracellular endoplasmic reticulum may be derived from activated immune cells.

[0021] In yet another embodiment of the present invention, the extracellular endoplasmic reticulum may express one or more selected from the group consisting of CD63, CD81, and Flotillin 1.

[0022] Furthermore, in one embodiment of the present invention, a vaccine composition for the prevention or treatment of coronavirus infection, comprising the extracellular endoplasmic reticulum, is provided.

[0023] In addition, in one embodiment of the present invention, there is provided a health functional food for preventing or improving coronavirus infection, which contains the extracellular vesicles.

[0024] In addition, in one embodiment of the present invention, there is provided a method for preventing or treating coronavirus infection, which includes the step of administering the extracellular vesicles to an individual who needs them.

[0025] In addition, in one embodiment of the present invention, there is provided the use of the extracellular vesicles for the manufacture of a medicament for preventing or treating coronavirus infection.

Advantages of the Invention

[0026] In the present invention, as a result of immunizing mice by administering extracellular vesicles derived from activated immune cells loaded with an antigen protein or mRNA, excellent antigen-specific neutralizing antibody production and T cell response induction effects were confirmed. When stored at room temperature and refrigerated conditions after lyophilization, excellent stability and antibody production effects were experimentally confirmed. From this, the extracellular vesicles or vaccine compositions containing the same according to the present invention function as a platform applicable to various diseases having excellent antigen-specific immune response induction effects and stability, and are expected to be usefully utilized in the field of vaccine development for preventing or treating infectious diseases, particularly various diseases including coronavirus infection.

Brief Description of the Drawings

[0027] FIG. 1 shows the results of confirming that THP-1 cells activated by treatment with LPS exhibit the phenotype of M1 macrophages. FIG. 1a is a microscopic image showing the morphology of resting THP-1 cells and activated THP-1 cells, and FIG. 1b shows the results of measuring the mRNA levels of pro-inflammatory cytokines markers IL-1β, IL-6, IL-8, TNF-α and iNOS in each of the above THP-1 cells. Figure 2 shows the results of analyzing the characteristics of exosomes isolated from activated THP-1 cells. Figure 2a shows the diameter of the isolated exosomes measured via Nanosight Tracking Analysis (NTA). Figure 2b is a transmission electron microscope (TEM) image of the exosomes. Figure 2c shows the results of Western blotting using cell lysates and isolated exosomes to measure the expression levels of the exosome markers CD63, CD81, and Flotillin 1 proteins. Figure 3 shows the results of confirming antibody production in mice immunized by either activated THP-1 cell-derived exosomes (COVID 19-ImmunExo) carrying the SARS-CoV-2-derived spike protein RBD according to the present invention, or by administering both an adjuvant and the RBD protein. Figure 3a is a simplified diagram showing the experimental method and analysis schedule for mouse immunization using the exosomes. Figure 3b shows the results of analyzing the antibody production effect by administering the exosomes (ImmunExo) at different concentrations to mice or by administering both an adjuvant and the RBD protein together (Adj+RBD 5ug), and performing a SARS-CoV-2 sVNT test using serum samples diluted 50-fold after 7, 21, 30, and 50 days. Figure 3c shows the results of analyzing the antibody production effect by performing a SARS-CoV-2 sVNT test on serum isolated 10-fold or 50-fold after 50 days in the same experimental group as in Figure 3b. This is the result of analyzing the antibody production effect after conducting an sVNT test. Figure 4 shows the degree of binding of SARS-CoV-2 RBD-specific serum antibodies to exosomes derived from immune cells (THP-1 cells or KG-1 cells) carrying RBD protein or mRNA. Specifically, (1) Figure 4a shows the results of analyzing the degree of binding of SARS-CoV-2 RBD-specific serum antibodies by measuring absorbance after 50 days when serum was separated from each experimental group of mice, in the same manner as in Figure 3c, after administering different concentrations of THP-1 cell-derived exosomes or adjuvant together with RBD protein. (2) Figure 4b shows the degree of binding of SARS-CoV-2 RBD-specific serum antibodies by measuring absorbance after 7 days when serum was separated from KG-1 cell-derived exosomes or adjuvant together with RBD protein. Figure 4c shows the results of analyzing the degree of binding of SARS-CoV-2 RBD-specific serum antibodies by measuring absorbance after administering THP-1 cell-derived exosomes or an adjuvant together with RBD mRNA, and separating the serum after 7 and 14 days. Figure 4d shows the results of analyzing the degree of binding of SARS-CoV-2 RBD-specific serum antibodies by measuring absorbance after administering KG-1 cell-derived exosomes or an adjuvant together with RBD mRNA, and separating the serum after 7 and 14 days. Figure 5 shows the results of an IFN-γELISPOT (Enzyme-linked immunospot) assay performed on splenocytes isolated from activated THP-1 cell-derived exosomes (ImmunExo) loaded with SARS-CoV-2-derived spike protein RBD, or from mice immunized by administering both an adjuvant and RBD protein. Figure 5a shows the results of the IFN-γELISPOT assay, indicating the presence or absence of spots and a quantitative graph of the number of spots. Figure 5b shows the results of ELISA performed using splenocyte culture medium to measure IFN-γ protein levels. Figure 6 shows the results of confirming the storage stability of activated THP-1 cell-derived exosomes loaded with SARS-CoV-2-derived spike protein RBD according to the present invention. Figure 6a is a simplified diagram showing the experimental schedule for analyzing the effects of exosomes. Figure 6b shows the results of immunizing mice with exosomes stored at 4°C for 7 days after lyophilization at doses of 0.1, 0.5, or 1 ug, respectively. Serum was obtained on days 7 and 21 after administration, and the SARS-CoV-2 sVNT test was performed. The antibody production effect was compared with that of exosomes in their original state (fresh) that were not stored by lyophilization. Figure 6c shows the results of immunizing mice with exosomes stored at room temperature for 7 days after lyophilization at doses of 1 ug, respectively. Serum was obtained on days 7 and 21 after administration, respectively. The SARS-CoV-2 sVNT test was performed. The antibody production effect was compared with that of exosomes in their original state (fresh) that were not stored by lyophilization. [Best Mode for Carrying Out the Invention]

[0028] This invention relates to a vaccine platform technology that can be usefully utilized in the field of vaccine development for the prevention or treatment of various infectious diseases by utilizing extracellular vesicles that function as potent immunostimulants and antigen carriers.

[0029] The present invention will be described in detail below.

[0030] The present invention provides an extracellular endoplasmic reticulum containing a coronavirus-derived antigen protein or a gene encoding said protein.

[0031] In the present invention, the coronavirus may be SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2).

[0032] Furthermore, in the present invention, the antigen protein may be a receptor binding domain (RBD) protein of a coronavirus-derived spike protein, and the gene encoding the antigen protein may be a nucleotide encoding the receptor binding domain (RBD) protein of a coronavirus-derived spike protein. The nucleotide may also be DNA, RNA, or a combination thereof.

[0033] Furthermore, in the present invention, the antigen protein or the gene encoding the protein may be located within the extracellular endoplasmic reticulum.

[0034] The method for loading the antigen protein or the gene encoding the protein into the extracellular endoplasmic reticulum is not specifically limited, and a person skilled in the art can appropriately select and use a method that is generally known in the art.

[0035] As used in the present invention, the term "antigen" refers to a molecule that can induce an immune response to produce antibodies within a host organism. The antigen is the target of the antibody, and each antibody is produced antigen-specific in response to the antigen after immune system cells come into contact with it. In the present invention, the antigen includes, as described above, proteins, genes encoding such proteins, and mRNA forms of such genes, as long as they can induce an immune response. The origin of the antigen is not specifically limited and may include, in non-limiting examples, those derived from microorganisms, including viruses, bacteria, or fungi, and those derived from cancer cells.

[0036] The term "extracellular endoplasmic reticulum" as used in the present invention refers to small, membrane-bound spheres derived from cells, and such spheres are highly diverse in their names depending on the origin of the cell from which they are formed and the method by which they are formed. The present invention may include one or more selected from the group consisting of exosomes, ectosomes, microvesicles, and apoptotic bodies, which are named according to the method by which they are formed in cells, and may, but is not limited to, exosomes.

[0037] The extracellular endoplasmic reticulum may be derived from one or more natural systems, for example, from the group consisting of animals, plants, and microorganisms, or it may be an artificially produced extracellular endoplasmic reticulum. The cells may also be cells isolated from a natural organism. The cells may also be derived from any type of animal or plant, including humans and non-human mammals. The animal cells may be one or more cells selected from the group consisting of somatic cells, germ cells, immune cells, nerve cells, and tumor cells.

[0038] Furthermore, the extracellular endoplasmic reticulum may be derived from activated immune cells, specifically activated THP-1 cells or KG-1 cells. In this case, the activation may be carried out by lipopolysaccharide (LPS), and when the THP-1 cells are activated, the THP-1 cells undergo a change in cell morphology similar to that of macrophages, and at the same time, can express one or more proteins selected from the group consisting of IL-1β, IL-6, IL-8, TNF-α, and iNOS.

[0039] In this invention, as an example in a specific example, changes in the phenotype of immune cells activated by antigen stimulation were analyzed, and the characteristics of exosomes isolated therefrom were observed.

[0040] Specifically, in one embodiment of the present invention, when human mononuclear cell line THP-1 cells were treated with lipopolysaccharide (LPS), it was confirmed that the cell morphology changed and M1 macrophage markers were expressed compared to cells that were not treated with LPS (see Example 1).

[0041] The somatic cells are cells that make up the body of any type of animal, including mammals, including humans, and in the present invention, they may include all types of cells except germ cells, immune cells, nerve cells, and tumor cells.

[0042] The aforementioned immune cells are cells that determine the immune response in the body. They play a role in defending against and overcoming invading pathogens, foreign substances, and viruses, regulating immunity, and eliminating cancer cells that are constantly being produced in the body. Specifically, they may include, but are not limited to, NK cells (natural killer cells), dendritic cells, T cells, B cells, macrophages, and lymphocytes.

[0043] The aforementioned neuronal cells are cells that constitute the nervous system, express ion pathways such as sodium pathways and potassium pathways, and unlike other cells, can transmit signals electrically. They also exchange various types of information with adjacent neuronal cells by sending and receiving chemical signals through structures called synapses. In the present invention, the term "neuronal cell" is not limited to any specific type and may include all cells that secrete extracellular endoplasmic reticulum.

[0044] The term "tumor cells" is used interchangeably with "cancer cells" and refers to cells that grow rapidly and irregularly because, unlike normal cells, their cell cycle is not regulated. In this invention, the term "tumor cells" is not specifically limited to any tissue of origin and may include any tumor cells that secrete extracellular endoplasmic reticulum.

[0045] Furthermore, in one embodiment of the present invention, the extracellular endoplasmic reticulum may express one or more selected from the group consisting of CD63, CD81, and Flotillin 1, and more specifically, it may express all of CD63, CD81, and Flotillin 1.

[0046] In other embodiments of the present invention, it was confirmed that the activated immune cell-derived exosomes, preferably activated TH-1 cell-derived exosomes, have an average diameter of 154.0 nm and express the exosome marker proteins CD63, CD81, and Flotillin 1 (see Example 2).

[0047] Furthermore, the present invention provides a vaccine composition for the prevention or treatment of coronavirus infection, comprising the extracellular endoplasmic reticulum.

[0048] The terms "extracellular endoplasmic reticulum," "coronavirus," and "antigen" mentioned above may be within the scope described above.

[0049] As used in the present invention, the term "prevention" means all actions that suppress or delay the onset of a particular disease or disorder by administering the vaccine composition according to the present invention.

[0050] As used in the present invention, the term "treatment" means all actions by which the administration of the vaccine composition according to the present invention improves or beneficially alters the symptoms of a particular disease or disorder.

[0051] As used in the present invention, the term "vaccine" encompasses all of the following: induction of an immune response to the antigen from a host, including humans, thereby preventing infection or reinfection by the pathogen or antigen, reducing the severity of symptoms or eliminating symptoms, or substantially or completely eliminating a disease caused by the pathogen or antigen. Therefore, the vaccine composition of the present invention is preferably administered prophylactically to an individual before infection by the pathogen or before the onset of the disease, but it can also be administered therapeutically after infection by the pathogen or after the onset of the disease.

[0052] In one embodiment of the present invention, activated THP-1 cells or KG-1 cells were prepared by loading SARS-CoV-2-derived spike protein receptor binding domain (RBD) protein or mRNA onto exosomes. These exosomes were administered subcutaneously to mice for immunization, and the presence or absence of neutralizing antibody production was analyzed. The results showed that even at very small doses, similar levels of RBD-specific IgG and neutralizing antibody titers were observed compared to cases where an adjuvant and the RBD protein or mRNA were administered together (see Examples 3-5).

[0053] Furthermore, in the present invention, the vaccine composition may simultaneously induce antigen-specific antibody production and a T cell-mediated immune response.

[0054] In another embodiment of the present invention, splenocytes were isolated from mice immunized with the exosomes, and the RBD antigen-specific T cell response was analyzed. The results showed that while the T cell response was not induced in mice administered with both an adjuvant and the RBD, a strong T cell response was induced in mice immunized with the exosomes (see Example 6).

[0055] Furthermore, in the present invention, the vaccine composition may be freeze-dried.

[0056] The inventors confirmed the specific immune response induction and storage stability of the vaccine composition based on examples. Specifically, to evaluate the storage stability of the exosome according to the present invention, lyophilized exosomes (COVID19-ImmunExo) were stored under refrigerated conditions at 4°C for 7 days or at room temperature, and then subcutaneously immunized in mice. Analysis of the presence or absence of antibody production confirmed that excellent stability and antibody production effect were maintained even after storage under the respective conditions (see Example 7).

[0057] Based on the embodiments of the present invention described above, activated immune cell-derived exosomes showed directionality towards lymph nodes after subcutaneous injection and were mainly absorbed by local macrophages and dendritic cells. It was found that dendritic cells caused MHC-I / peptide complexes to appear in the exosomes, which more effectively generated neutralizing antibodies against SARS-CoV-2 and could induce RBD-specific T cell responses in immunized mouse splenocytes.

[0058] Based on the results confirmed in the examples, the present invention strongly suggests that nano-sized extracellular vesicles containing exosomes secreted from cells can act as potent immunostimulants and antigen carriers, and can serve as a platform for designing effective vaccines against targeted diseases.

[0059] The vaccine composition of the present invention can be manufactured in any suitable, pharmaceutically acceptable formulation. For example, it can be manufactured in the form of an immediate-administration solution or suspension, a concentrated stock solution suitable for dilution before administration, or in a reconstituteable form, such as lyophilized, cryopreserved, or cryopreserved formulation.

[0060] The vaccine composition of the present invention can be formulated to further include a pharmaceutically acceptable carrier. Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipids, liposomes, microspheres, or nanospherical particles. These may form complexes with or be associated with a transport means and may be transported in vivo using transport systems commonly known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0061] Furthermore, the pharmaceutically acceptable carriers typically include diluents, excipients, stabilizers, and preservatives. Suitable diluents may be non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or aqueous solvents such as brine (preferably 0.8% brine) or water containing a buffer medium (preferably 0.05 M phosphate buffer). Suitable excipients may be starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene glycol, water, and ethanol. Suitable stabilizers may be carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, or proteins such as animal, plant, or microbial proteins such as powdered milk, serum albumin, and casein. Suitable preservatives may include thimerosal, melthiolate, gentamicin, neomycin, nystatin, ampotericin B, tetracycline, penicillin, streptomycin, and polymyxin B.

[0062] The vaccine composition of the present invention may further contain an antigen adjuvant. The antigen adjuvant may consist of one or more substances that enhance the immune response to an antigen. The antigen adjuvant may be, for example, complete Freund's solution, incomplete Freund's solution, saponins, gel-like aluminum adjuvants, surfactants (e.g., lysolecithin, pluronic glycol, polyanions, peptides, oils or hydrocarbon emulsions, etc.), vegetable oils (cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, etc.

[0063] The vaccine composition of the present invention can be manufactured by methods commonly used in the art to which the present invention pertains. The vaccine composition can be manufactured in oral or parenteral formulations, the parenteral formulations of which may be administered via any one of the following routes of administration selected from the group consisting of transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, or epidural.

[0064] The term "administration" means introducing a specific substance into an individual in an appropriate manner, and "individual" refers to all living organisms, including humans, rats, mice, and livestock, that can carry coronavirus infection. Specifically, it may also refer to mammals, including humans.

[0065] The vaccine composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to exhibit a vaccine effect without causing side effects or severe or excessive immune responses. The level of the effective dose can vary depending on various factors, including the disorder to be treated, the severity of the disorder, the activity of the vaccine substance, the route of administration, the rate of protein removal, the duration of treatment, substances used in combination with or concurrently with the vaccine composition, the individual's age, weight, sex, dietary habits, general health status, and factors generally known in the medical field.

[0066] Furthermore, the present invention provides a health functional food for the prevention or improvement of coronavirus infection, which includes the extracellular endoplasmic reticulum.

[0067] The terms "extracellular endoplasmic reticulum," "coronavirus," and "prevention" mentioned above may be within the scope described above.

[0068] The term "improvement" means all actions that at least reduce the severity of symptoms, for example, parameters related to the condition being treated. In this context, the health functional food may be used for the prevention or improvement of coronavirus infection, either before or after the onset of the disease, simultaneously with or separately from therapeutic drugs.

[0069] In the aforementioned health functional foods, the active ingredient can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately by conventional methods. The amount of active ingredient mixed can be suitably determined according to its intended use (prevention or improvement). Generally, when manufacturing food or beverages, the health functional foods can be added to the raw materials in an amount of approximately 15% by weight or less, more specifically, approximately 10% by weight or less. However, in the case of long-term intake for health and hygiene purposes or for health regulation purposes, the amount may be less than the aforementioned range.

[0070] The aforementioned health functional food may further contain one or more of the carrier, diluent, excipient, and additive, and may be formulated into one of the following dosage forms: tablets, pills, powders, granules, capsules, and liquids. Foods to which the compound described in this example can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

[0071] Specific examples of the carrier, excipient, diluent, and additive may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0072] In addition to containing the active ingredient, the aforementioned health functional food may contain other ingredients as essential components without any special restrictions. For example, it may contain various flavorings or natural carbohydrates as additional ingredients, as is the case with ordinary beverages. Examples of natural carbohydrates mentioned above include monosaccharides, e.g., glucose, fructose; disaccharides, e.g., maltose, sucrose; and ordinary sugars such as polysaccharides, e.g., dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings other than those mentioned above, natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) may be advantageously used. The proportion of the natural carbohydrates can be appropriately determined by those skilled in the art.

[0073] In addition to the above, a functional food according to one embodiment may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (for cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Such components can be used independently or in combination, and the proportions of such additives can be appropriately selected by those skilled in the art.

[0074] Furthermore, the present invention provides a method for preventing or treating coronavirus infection, which includes the step of administering the extracellular vesicles to an individual in need.

[0075] The terms "extracellular endoplasmic reticulum," "individual," "administration," "coronavirus infection," "prevention," and "treatment" mentioned above may be within the scope described above.

[0076] Furthermore, the present invention provides the use of the extracellular vesicle for the production of drugs for the prevention or treatment of coronavirus infection.

[0077] The terms "extracellular endoplasmic reticulum," "coronavirus infection," "prevention," and "treatment" mentioned above may be within the scope described above. [Modes for carrying out the invention]

[0078] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided solely to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0079] [Examples] Example 1. Observation of the phenotype of activated immune cells To develop a vaccine utilizing the extracellular endoplasmic reticulum, the inventors first selected immune cells, stimulated them with an antigen, and attempted to manufacture the vaccine using exosomes isolated from the activated immune cells. For this purpose, as an example of the immune cells, they activated THP-1 cells, a human mononuclear cell line, and observed the phenotype of the activated cells.

[0080] Specifically, THP-1 cells (8 × 10 5 THP-1 cells (cells / mL) were treated with lipopolysaccharide (LPS) at a concentration of 100 ng / mL to activate them. The cell morphology of these activated THP-1 cells was then observed under a microscope, comparing them to unactivated THP-1 cells that had not been treated with LPS. As shown in Figure 1a, it was confirmed that, compared to resting THP-1 cells, activated THP-1 cells exhibited morphological changes similar to those of macrophages.

[0081] Furthermore, based on the above results, in order to confirm whether THP-1 cells activated by LPS treatment differentiated into M1 macrophages, as reported in the art, we measured the mRNA levels of IL-1β, IL-6, IL-8, TNF-α, and iNOS, which are pro-inflammatory cytokine markers secreted by M1 macrophages. As a result, as shown in Figure 1b, it was found that the markers were hardly expressed in quiescent THP-1 cells, whereas in activated THP-1 cells, the expression of the markers increased, and in particular, the mRNA levels of IL-1β, IL-6, and IL-8 increased significantly.

[0082] The results described above show that THP-1 cells activated by LPS treatment exhibit the phenotype of M1 macrophages.

[0083] Example 2. Confirmation of the characteristics of activated immune cell-derived exosomes. To confirm the characteristics of the activated THP-1 cell-derived exosomes, the inventors isolated the exosomes from the cells through the following process. Specifically, to remove cells and cell debris, the cell culture medium was centrifuged at 1200 rpm for 5 minutes and the supernatant was collected. Then, to remove microparticles larger than the exosomes, the supernatant was passed through a 0.2 μm filter, and the exosomes were concentrated and washed using a tangential flow filtration (TFF) system.

[0084] Next, we performed Nanosight Tracking Analysis (NTA) and transmission electron microscopy to analyze the characteristics of the isolated exosomes. As a result, as shown in Figures 2a and 2b, we confirmed that the average diameter of the isolated exosomes was 154.0 nm and that they had a circular morphology.

[0085] Furthermore, the concentration of the separated exosomes was 2.6 × 10⁻⁶. 11The particle / ml concentration was quantitatively analyzed via a BCA assay and confirmed to be 3.5 mg / ml. Furthermore, to investigate whether the isolated exosomes expressed exosome markers, Western blotting was performed using cell lysates and isolated exosomes to measure the expression levels of the exosome markers CD63, CD81, and Flotillin 1. As is clear from Figure 2c, in the cell lysates, only CD63 was expressed at a weak level, while in the isolated exosomes, all of the marker proteins were expressed at high levels.

[0086] From the above results, it was clearly established that the exosomes isolated from the activated THP-1 cells possess the characteristics described above.

[0087] Example 3. Production of exosomes loaded with recombinant SARS-CoV-2-derived protein. The inventors attempted to verify the preventive effect of loading the receptor binding domain (RBD) protein of the SARS-CoV-2-derived spike protein into the activated THP-1 cell or KG-1 cell-derived exosomes isolated in Example 2, as a vaccine against SARS-CoV-2 infection.

[0088] To this end, we first prepared a protein construct (Cat.No.Ab-P0018,AbClon) in which the DNA sequence encoding the SARS-CoV-2-derived spike protein RBD (YP_009724390.1) (Ser325-Lys529) was fused at the C-terminus with the Fc region of mouse IgG2a. Subsequently, to load the RBD protein expressed from the construct into the activated THP-1 or KG-1 cell-derived exosomes, we performed 3 × 10⁶ 11(9 mg) of exosomes and 300 ug of the RBD protein were prepared, and after treatment with extracorporeal shockwave (ESW), the exosomes loaded with RBD protein were washed and concentrated using an ultracentrifuge (32,000 rpm, 2 hours).

[0089] Subsequently, quantitative analysis of the amount of RBD protein loaded into the THP-1 cell-derived exosomes using ELISA confirmed that a total of 40.754 ug of RBD protein was loaded into the exosomes. The inventors attempted to evaluate the effect of activated THP-1 cell or KG-1 cell-derived exosomes (ImmunExo RBD or COVID 19-ImmunExo) loaded with SARS-CoV-2-derived spike protein RBD, prepared as described above, against coronavirus (SARS-CoV-2) infection-19 (COVID-19) through the following experiments.

[0090] Example 4. Production of exosomes loaded with recombinant SARS-CoV-2 mRNA. The inventors attempted to verify the preventive effect of loading the receptor binding domain (RBD) mRNA of the SARS-CoV-2-derived spike protein into activated THP-1 cells or KG-1-derived exosomes isolated in Example 2, as a vaccine against SARS-CoV-2 infection.

[0091] Specifically, RBD mRNA was produced from a DNA template via in vitro transcription (IVT). The produced RBD mRNA was then prepared by preparing 9 mg of THP-1 or KG-1 cell-derived exosomes with 150 ug of RBD mRNA. After treatment with extracorporeal shockwave (ESW), the RBD mRNA-loaded exosomes were washed and concentrated using an ultracentrifuge (32000 rpm, 2 hours).

[0092] Subsequently, quantitative analysis of the amount of RBD mRNA loaded into the THP-1 cell or KG-1 cell-derived exosomes using qRT-PCR confirmed that a total of 81 ug was loaded into the THP-1 cell-derived exosomes and 64.5 ug into the KG-1 cell-derived exosomes. The inventors attempted to evaluate the effect of the activated THP-1 cell or KG-1 cell-derived exosomes (Exo+RBD mRNA) loaded with SARS-CoV-2-derived spike protein RBD mRNA, produced as described above, against coronavirus (SARS-CoV-2) infection-19 (COVID-19) through the following experiments.

[0093] Example 5. Confirmation of the vaccine efficacy of activated immune cell-derived exosomes against COVID-19. The inventors attempted to evaluate the efficacy of activated THP-1 cells or KG-1 cell-derived exosomes loaded with SARS-CoV-2-derived spike protein RBD protein or mRNA, as a vaccine against COVID-19, as described in Example 3.

[0094] For this purpose, in vivo immunization experiments were conducted using mice according to the schedule shown in Figure 3a. Specifically, activated THP-1 cell-derived exosomes (COVID-19-ImmunExo, or ImmunExo) loaded with the spike protein RBD protein were subcutaneously injected into the mice at various concentrations (0.005, 0.01, 0.05, 0.1, 0.2, 0.5, or 1 ug), with the same number of exosome particles injected. As a control group, mice were administered both an adjuvant and RBD protein (Adj+RBD), with the adjuvant being 500 ug of Al(OH)3 and 5 ug of RBD protein per mouse. Subsequently, the effects of the exosomes were evaluated from multiple perspectives through the following experiments.

[0095] Furthermore, activated KG-1 cell-derived exosomes loaded with the spike protein RBD protein (COVID-19-ImmunExo, or ImmunExo) were subcutaneously injected at a concentration of 1 ug, and activated THP-1 cell-derived exosomes loaded with the spike protein RBD mRNA (Exo+RBD mRNA) were subcutaneously injected at a concentration of 10 ug. In all cases, the number of exosome particles was the same, and as a control group, mice were administered an adjuvant along with RBD protein or mRNA (Adj+RBD or Adj+RBD mRNA). As an adjuvant, 500 ug of Al(OH)3 was administered per mouse, along with 5 ug of RBD protein and 10 ug of RBD mRNA. Subsequently, the effects of the exosomes were evaluated from multiple perspectives through the experiments described below.

[0096] 5-1.SARS-CoV-2 surrogate virus neutralization test (sVNT) The SARS-CoV-2 surrogate virus neutralization test is a blocking ELISA detection method that mimics the viral neutralization process. It is based on the inhibitory effect of neutralizing antibodies on the interaction between SARS-CoV-2 RBD and the human ACE2 (hACE2) receptor protein. The detection sensitivity of the test is 93.80%, and the inhibition efficiency (inhibition%) for the interaction was derived using the following formula 1. The cutoff value for the result was 20%. A value of 20% or higher was considered positive, indicating the detection of neutralizing antibodies against SARS-CoV-2. A value below 20% was considered not to have detected neutralizing antibodies.

[0097]

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[0098] Subsequently, mice were administered activated THP-1 cell-derived exosomes (COVID-19-ImmunExo) loaded with SARS-CoV-2-derived spike protein RBD protein at different concentrations as described above, or administered with an adjuvant and RBD protein together (Adj+RBD 5ug). Blood samples were collected at 7, 21, 30, and 50 days, and the serum was diluted 50-fold (X50 dilution). The suppression efficiency was calculated using the aforementioned formula.

[0099] As a result, as shown in Figure 3b, it was revealed that when the exosome (ImmunExo) was administered, the inhibitory efficiency increased in proportion to the administered concentration. When 1 ug of exosome was administered, a level of inhibitory efficiency very similar to that observed when both the adjuvant and RBD protein were administered was observed.

[0100] Based on the above results, serum was separated from blood collected 50 days later, and the inhibitory efficiency of the samples diluted 10-fold and 50-fold was analyzed. As is clear from Figure 3c, it was confirmed that administering 1 ug of the exosome (ImmunExo) resulted in an inhibitory efficiency level very similar to that obtained when 5 ug of both the adjuvant and RBD protein were administered together.

[0101] 5-2. Analysis of serum antibody response to SARS-CoV-2 (1) In the case of THP-1 cell-derived exosomes loaded with SARS-CoV-2-derived spike protein RBD protein In Example 5-1, the inventors administered activated THP-1 cell-derived exosomes (ImmunExo) loaded with SARS-CoV-2-derived spike protein RBD protein to mice at different concentrations, or administered both an adjuvant and RBD protein to each mouse. After 50 days, serum samples were obtained from each mouse, and the samples were diluted in PBS with 1% BSA at a ratio of 1:100 to 1:1000. The degree of antibody binding to SARS-CoV-2 RBD-specific serum antibodies was analyzed by measuring absorbance.

[0102] As a result, as shown in Figure 4a, the degree of antibody binding decreased in a similar manner as the mouse-derived serum was diluted in all groups, and was found to be approximately proportional to the administered concentration of the exosome. Furthermore, it was confirmed that overall antibody binding was highest when 1 ug of the exosome was administered, and when 0.5 ug was administered, it was similar to the group administered with both an adjuvant and RBD protein (Adj+RBD 5 ug).

[0103] (2) In the case of KG-1 cell-derived exosomes loaded with SARS-CoV-2-derived spike protein RBD protein The inventors administered activated KG-1 cell-derived exosomes (ImmunExo) loaded with SARS-CoV-2-derived spike protein RBD protein to mice produced in Example 3 at a concentration of 1 ug, or administered an adjuvant and 1 ug of RBD protein together to each mouse. After 7 days, serum samples were obtained from each mouse, and the samples were diluted in PBS with 1% BSA at a ratio of 1:100 to 1:1000. The degree of antibody binding of serum antibodies specific to SARS-CoV-2 RBD was analyzed by measuring absorbance.

[0104] As a result, as shown in Figure 4b, we were able to confirm that the degree of serum antibody binding specific to SARS-CoV-2 RBD was higher in the group compared to the control group.

[0105] (3) In the case of THP-1 cell-derived exosomes loaded with SARS-CoV-2-derived spike protein RBD mRNA The inventors administered activated THP-1 cell-derived exosomes (Exo+RBD mRNA) loaded with SARS-CoV-2 spike protein RBD mRNA to mice produced in Example 4 at a concentration of 10 ug, or administered both an adjuvant and 10 ug of RBD mRNA to each mouse. Serum samples were obtained from each mouse at 7 and 14 days, and then diluted in PBS supplemented with 1% BSA at a ratio of 1:100 to 1:1000. The degree of antibody binding of serum antibodies specific to SARS-CoV-2 RBD was analyzed by measuring absorbance.

[0106] As a result, as shown in Figure 4c, after 7 days, the degree of serum antibody binding specific to SARS-CoV-2 RBD could not be measured in any of the groups. However, after 14 days, it was confirmed that a significantly higher degree of serum antibody binding specific to SARS-CoV-2 RBD appeared compared to the group administered both the adjuvant and RBD mRNA, or the control group.

[0107] (4) In the case of KG-1 cell-derived exosomes loaded with SARS-CoV-2-derived spike protein RBD mRNA The inventors administered activated KG-1 cell-derived exosomes (Exo+RBD mRNA) loaded with SARS-CoV-2 spike protein RBD mRNA to mice produced in Example 4 at a concentration of 10 ug, or administered an adjuvant and 10 ug of RBD mRNA together to mice. Serum samples were obtained from each mouse at 7 and 14 days, and then diluted in PBS supplemented with 1% BSA at a ratio of 1:100 to 1:1000. The degree of antibody binding of serum antibodies specific to SARS-CoV-2 RBD was analyzed by measuring absorbance.

[0108] As a result, as shown in Figure 4d, after 7 days, unlike the other groups, only the group with activated KG-1 cell-derived exosomes loaded with SARS-CoV-2 spike protein RBD mRNA (Exo+RBD mRNA) was able to measure the degree of SARS-CoV-2 RBD-specific serum antibody binding. After 14 days, it was confirmed that this group exhibited a significantly higher degree of SARS-CoV-2 RBD-specific serum antibody binding compared to the groups administered both the adjuvant and RBD mRNA, or the control group.

[0109] 5-3.SARS-CoV-2 RBD-specific T cell response analysis In Example 5-1, the inventors isolated splenocytes from mice immunized by administering activated THP-1 cell-derived exosomes (ImmunExo) loaded with SARS-CoV-2-derived spike protein RBD protein, and performed an IFN-γELISPOT (Enzyme-linked immunospot) assay on these splenocytes to analyze the SARS-CoV-2 RBD-specific T cell response. The IFN-γELISPOT assay is a sensitive technique that detects antigen-specific T cells secreting cytokines at the single-cell level, can directly measure Th1 cell-mediated immune responses, is useful for monitoring the effectiveness of vaccines that induce cell-mediated immunity, and can directly measure the frequency of cytokine-producing T cells by confirming the number of spots generated through a colorimetric reaction.

[0110] To carry out the assay, 5 × 10⁶ mice-derived splenocytes from each of the mice in Example 5-1 5 Each well of the plate was dispensed with / well, treated with SARS-CoV-2-derived spike protein RBD protein at a concentration of 2 ug / mL, and then incubated for 24 hours. As a result, as shown in Figure 5a, no spots appeared in the group administered both the adjuvant and the RBD protein (Adj+RBD 5ug), whereas in the group administered the exosome according to the present invention (COVID19-ImmunExo), although the number of spots varied depending on the administration concentration, spots were generated in all groups. In particular, the group administered 0.5ug of the exosome had the largest number of spots.

[0111] Furthermore, mouse-derived splenocytes (1 × 10) administered together with the exosome or adjuvant according to the present invention and RBD protein using the same method as described above. 7 After treating the spleen cells (1 / well) with SARS-CoV-2-derived spike protein RBD protein at 2 ug / mL for 48 hours, ELISA was performed using the culture medium to measure the IFN-γ protein levels secreted from Th1 cells.

[0112] As a result, as can be seen from Figure 5b, and consistent with the results in Figure 5a, it was revealed that IFN-γ was completely absent in the culture medium of mouse-derived splenocytes administered with both the adjuvant and RBD protein, whereas IFN-γ was confirmed to be present in the culture medium of mouse-derived splenocytes administered with the exosome (ImmunExo) according to the present invention.

[0113] The results described above suggest that while administering the adjuvant and RBD protein together did not induce a Th1 cell-mediated immune response, exosome administration according to the present invention induced a strong Th1 cell-mediated immune response.

[0114] Example 6. Verification of storage stability and efficacy of freeze-dried COVID19-ImmunExo. To confirm the clinical applicability of activated THP-1 cell-derived exosomes (COVID19-ImmunExo) loaded with the SARS-CoV-2-derived spike protein RBD protein according to the present invention, the inventors verified the storage stability of said exosomes.

[0115] More specifically, the exosomes of the present invention were freeze-dried according to the schedule shown in Figure 6a, prepared in single-dose quantities at different concentrations, divided into tubes, frozen at -70°C for one day, and then freeze-dried. The freeze-dried exosomes were stored at 4°C (Figure 6b) or room temperature (Figure 6c) for 7 days, after which the exosomes prepared in the dosage form were administered to each mouse at the indicated concentrations. Subsequently, the SARS-CoV-2 sVNT test was performed on days 7 and 21 using the same method as in Example 5-1.

[0116] As a result, as shown in Figure 6b, when lyophilized exosomes stored at 4°C for 7 days were administered at concentrations of 0.1 ug, 0.5 ug, and 1 ug, an inhibitory effect was observed from the 7th day when 1 ug of exosomes was administered. When exosomes were administered at a concentration of 0.5 ug or higher on the 21st day, an inhibitory efficiency close to 50% was confirmed on the 21st day. Furthermore, as shown in Figure 6c, when lyophilized exosomes stored at room temperature for 7 days were administered at a concentration of 1 ug, an inhibitory effect was observed from the 7th day, and an inhibitory efficiency close to 40% was confirmed on the 21st day. These results indicate that the antibody production effect is similar to that of exosomes that are not lyophilized and stored for 7 days (fresh), as the inhibitory efficiency is similar under the same conditions. Therefore, it was found that the effect of the exosomes according to the present invention is stably maintained even when stored under the aforementioned conditions.

[0117] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified in other specific forms without altering the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects.

Claims

1. An extracellular endoplasmic reticulum containing a coronavirus-derived antigen protein or a gene encoding said protein, The antigen protein is selected from the group consisting of spike proteins or their fragments, nucleocapsid proteins or their fragments, membrane proteins or their fragments, and envelope proteins or their fragments. The extracellular endoplasmic reticulum is one in which either the antigen protein or the gene encoding the protein is carried, and The extracellular endoplasmic reticulum is an extracellular endoplasmic reticulum that does not contain polycationic lipids within it.

2. The extracellular endoplasmic reticulum according to claim 1, wherein the coronavirus is SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2).

3. The extracellular endoplasmic reticulum according to claim 1, wherein the antigen protein is a receptor binding domain (RBD) protein of a coronavirus-derived spike protein.

4. The extracellular endoplasmic reticulum according to claim 1, wherein the extracellular endoplasmic reticulum is derived from one or more selected from the group consisting of animal cells, plant cells, or microorganisms.

5. The extracellular endoplasmic reticulum according to claim 4, wherein the animal cell is one or more cells selected from the group consisting of somatic cells, germ cells, immune cells, nerve cells, and tumor cells.

6. The extracellular vesicle according to claim 1, wherein the extracellular vesicle is derived from activated immune cells.

7. The extracellular endoplasmic reticulum according to claim 1, wherein the extracellular endoplasmic reticulum expresses one or more selected from the group consisting of CD63, CD81, and Flotillin 1.

8. A vaccine composition for the prevention or treatment of coronavirus infection, comprising the extracellular endoplasmic reticulum as described in claim 1.

9. The vaccine composition according to claim 8, wherein the vaccine composition simultaneously induces antigen-specific antibody production and a T cell-mediated immune response.

10. The vaccine composition according to claim 8, wherein the vaccine composition is freeze-dried.

11. A health functional food for the prevention or improvement of coronavirus infection, comprising the extracellular endoplasmic reticulum described in claim 1.

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