Extracellular vesicles containing antigen proteins or genes encoding said proteins and uses thereof

Abstract

The present invention relates to an extracellular vesicle containing an antigen protein or a gene encoding the protein, and a use thereof, and more specifically, to an extracellular vesicle containing an antigen protein derived from a virus, a microorganism or a cancer cell or a gene encoding the protein, or a vaccine composition for preventing or treating viral infections, microbial infections or cancers comprising the same. The extracellular vesicle or the vaccine composition comprising the same according to the present invention is a platform applicable to various diseases, has excellent antigen-specific immune response induction effect and stability, and is expected to be effectively used in the field of vaccine development. The vaccines are useful for the prevention or treatment of various diseases including viral infections, microbial infections or cancers.

Description

Technical Field

[0001] The present invention relates to an extracellular vesicle containing an antigen protein or a gene encoding the protein and use thereof. Background Art

[0002] Vaccines are drugs that provide acquired immunity to specific diseases or pathogens for animals such as humans. Their structures are similar to the antigen recognition sites of the main disease-causing microbial pathogens, but unlike pathogens, they are not pathogenic. Currently, many types of vaccines have been developed for various pathogens and various diseases, 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 tens to hundreds of nanometers composed of a double lipid membrane, and are composed of bioactive substances such as proteins, lipids, and genes. As representative examples of these extracellular vesicles, in addition to exosomes, they also include extranuclear granules (ectosomes), microvesicles, bacterial outer membrane vesicles (Bacterial Outer membrane vesicles, Bacterial OMV), microbial extracellular vesicles or apoptotic bodies (apoptotic bodies) including yeast-derived extracellular vesicles, etc. In the past, these extracellular vesicles were considered to be waste secreted by cells, but recently, with the clinical significance of extracellular vesicles attracting attention, various studies are being conducted. In particular, exosomes are spherical vesicles released by cells, with various information such as proteins and DNA of mother cells, so cancer diagnostic markers and sensors using exosomes as biomarkers are being actively developed.

[0004] On the other hand, exosomes are tiny membrane-structured vesicles (about 30 to 100 nm in diameter) secreted by various cells. Studies using electron microscopes have shown that they do not fall directly from the plasma membrane, but originate from specific intracellular compartments called multivesicular bodies (MVBs), and are released and secreted outside the cell. That is, when multivesicular bodies fuse with the plasma membrane, the vesicles are released into the extracellular environment, which are called exosomes. For these exosomes, it is known that in addition to red blood cells, various types of immune cells including B lymphocytes, T lymphocytes, dendritic cells, platelets, macrophages, etc., as well as tumor cells, stem cells, etc., also produce and secrete exosomes when they are alive.

[0005] It is reported that mammalian cell-derived exosomes participate in a variety of physiological functions, such as hemostasis, tissue regeneration, stem cell maintenance, inflammation / immune response regulation and embryonic development. In particular, immune cell-derived exosomes can promote immune response by delivering inflammatory cytokines or directly or indirectly presenting antigens (Nat Rev Immunol. 2009 Aug; 9(8): 581-93). Based on these characteristics and in vivo functions of exosomes, exosomes are being studied and developed as nanoscale delivery carriers, mediators and biomarkers in the fields of oncology, immunotherapy and regenerative medicine.

[0006] Thus, research is underway on the use of extracellular vesicles, including exosomes or extracellular vesicles of microbial origin, for vaccines, but this is limited to certain diseases, and thus the development of vaccine platform technologies applicable to a variety of diseases, including viral or microbial infectious diseases or cancers, is still insufficient. Summary of the invention

[0007] Technical issues

[0008] The present inventors conducted research to develop an extracellular vesicle-based vaccine platform that is generally applicable to various diseases, and the results confirmed that extracellular vesicles act as powerful immunostimulants and antigen carriers to induce antigen-specific antibody production and antigen-specific T cell responses, thereby completing the present invention.

[0009] Therefore, an object of the present invention is to provide an extracellular vesicle containing an antigenic protein or a gene encoding the protein.

[0010] In addition, another object of the present invention is to provide a vaccine composition for preventing or treating viral infections, comprising: extracellular vesicles containing virus-derived antigenic proteins or genes encoding the proteins.

[0011] In addition, another object of the present invention is to provide a vaccine composition for preventing or treating microbial infections, which comprises: extracellular vesicles containing microbial-derived antigenic proteins or genes encoding the proteins.

[0012] In addition, another object of the present invention is to provide a vaccine composition for preventing or treating cancer, which comprises: extracellular vesicles containing cancer cell-derived antigen proteins or genes encoding the proteins.

[0013] In addition, another object of the present invention is to provide a health functional food for preventing or improving viral infections, which comprises: extracellular vesicles containing virus-derived antigenic proteins or genes encoding the proteins.

[0014] In addition, another object of the present invention is to provide a health functional food for preventing or improving microbial infections, which comprises: extracellular vesicles containing microbial-derived antigenic proteins or genes encoding the proteins.

[0015] In addition, another object of the present invention is to provide a health functional food for preventing or improving cancer, which comprises: extracellular vesicles containing cancer cell-derived antigen proteins or genes encoding the proteins.

[0016] In addition, another object of the present invention is to provide a method for preventing or treating viral infections, comprising: administering extracellular vesicles containing viral-derived antigenic proteins or genes encoding the proteins to an individual in need.

[0017] In addition, another object of the present invention is to provide a method for preventing or treating microbial infections, comprising: administering extracellular vesicles containing microbial-derived antigenic proteins or genes encoding the proteins to an individual in need.

[0018] In addition, another object of the present invention is to provide a method for preventing or treating cancer, which comprises: administering extracellular vesicles containing cancer cell-derived antigen proteins or genes encoding the proteins to an individual in need.

[0019] In addition, another object of the present invention is to provide a use of an extracellular vesicle in the preparation of a drug for preventing or treating a viral infection, wherein the extracellular vesicle contains a viral-derived antigen protein or a gene encoding the protein.

[0020] In addition, another object of the present invention is to provide a use of an extracellular vesicle in the preparation of a drug for preventing or treating a microbial infection, wherein the extracellular vesicle contains a microbial-derived antigen protein or a gene encoding the protein.

[0021] In addition, another object of the present invention is to provide a use of an extracellular vesicle in the preparation of a drug for preventing or treating cancer, wherein the extracellular vesicle contains a cancer cell-derived antigen protein or a gene encoding the protein.

[0022] However, the technical problems to be solved by the present invention are not limited to the above problems, and those skilled in the art can clearly understand other problems not mentioned from the following description.

[0023] Technical Solution

[0024] In order to achieve the above-mentioned object of the present invention, the present invention provides an extracellular vesicle containing an antigen protein or a gene encoding the protein.

[0025] In one embodiment of the present invention, the antigen protein may be derived from one or more selected from viruses, microorganisms and cancer cells.

[0026] In another embodiment of the present invention, the virus can be selected from one or more of adenovirus, smallpox virus, polio virus, measles virus, hepatitis C virus (Hepatitis C virus), human immunodeficiency virus-1 (Human Immunodeficiency Virus-1, HIV-1), hepatitis B virus (Hepatitis B virus, HBV), influenza virus (Influenza virus), respiratory syncytial virus (Respiratory Syncytial Virus), Herpes Simplex virus (Herpes Simplex virus), Human Papilloma virus (Human Papilloma virus), Zika virus (Zikavirus), Varicella-Zoster virus (Varicella-Zoster virus) and Severe Fever with Thrombocytopenia Syndrome virus (Severe Fever with Thrombocytopenia Syndrome virus).

[0027] In another embodiment of the present invention, the antigen protein can be selected from one or more of the p24 protein derived from human immunodeficiency virus-1, the s protein derived from hepatitis B virus, the H1N1 protein derived from influenza virus, the RSV-F protein derived from respiratory syncytial virus, and the glycoprotein E (Glycoprotein E) (VZE) derived from varicella-zoster virus.

[0028] In another embodiment of the present invention, the microorganism can be selected from the group consisting of Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida, andida, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and one or more microorganisms of the order Mucorales.

[0029] In another embodiment of the present invention, the cancer cells may be derived from one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer and glioblastoma.

[0030] In another embodiment of the present invention, the antigen protein can be selected from one or more of MAGE1 / 2 / 3, WT1, CDK4, MUC-1, HER2, PSA, E6 / E7 of HPV16 and HCV.

[0031] In another embodiment of the present invention, the antigen protein or the gene encoding the protein can be loaded into the extracellular vesicles.

[0032] In another embodiment of the present invention, the extracellular vesicles may be derived from one or more selected from animal cells, plant cells or microorganisms.

[0033] In another embodiment of the present invention, the animal cell may be one or more cells selected from somatic cells, germ cells, immune cells, neural cells and tumor cells.

[0034] In another embodiment of the present invention, the extracellular vesicles may be derived from activated immune cells or bacterial outer membranes.

[0035] In another embodiment of the present invention, when the extracellular vesicles are derived from animal cells, the extracellular vesicles may express one or more proteins selected from CD9, CD63, CD81, Alix, TSG101, Syntenin 1 and Flotillin 1.

[0036] In another embodiment of the present invention, when the extracellular vesicles are derived from plant cells, the extracellular vesicles may express one or more proteins selected from syntaxin (PEN1), tetraspanin 8 (Tet8) and heat shock protein (HSP).

[0037] In another embodiment of the present invention, when the extracellular vesicles are derived from a microorganism, the extracellular vesicles may express one or more proteins selected from OmpA, flagellin, HSP70 and beta-glucan.

[0038] In addition, in one embodiment of the present invention, a vaccine composition for preventing or treating viral infections is provided, which comprises: an extracellular vesicle containing a virus-derived antigen protein or a gene encoding the protein.

[0039] In another embodiment of the present invention, the viral infection can be selected from one or more of adenovirus infection, smallpox virus infection, poliovirus infection, measles virus infection, hepatitis C virus infection, human immunodeficiency virus-1 (HIV-1) infection, hepatitis B virus (HBV) infection, influenza virus infection, respiratory syncytial virus infection, herpes simplex virus infection, human papillomavirus infection, Zika virus infection, varicella-zoster virus infection and severe fever with thrombocytopenia syndrome virus infection.

[0040] In one embodiment of the present invention, a vaccine composition for preventing or treating microbial infection is provided, comprising: an extracellular vesicle containing a microbial-derived antigen protein or a gene encoding the protein.

[0041] In another embodiment of the present invention, the microbial infection can be selected from the group consisting of Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida, andida, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and one or more microbial species of the order Mucorales.

[0042] In addition, in one embodiment of the present invention, a vaccine composition for preventing or treating cancer is provided, which comprises: an extracellular vesicle containing a cancer cell-derived antigen protein or a gene encoding the protein.

[0043] In another embodiment of the present invention, the cancer may be selected from one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer and glioblastoma.

[0044] In another embodiment of the present invention, the vaccine composition can simultaneously induce antigen-specific antibody production and T cell-mediated immune response.

[0045] In another embodiment of the present invention, the vaccine composition may be freeze-dried.

[0046] In addition, in one embodiment of the present invention, a health functional food for preventing or improving viral infections is provided, which comprises: an extracellular vesicle containing a virus-derived antigen protein or a gene encoding the protein.

[0047] In addition, in one embodiment of the present invention, a health functional food for preventing or improving microbial infection is provided, which comprises: an extracellular vesicle containing a microbial-derived antigen protein or a gene encoding the protein.

[0048] In addition, in one embodiment of the present invention, a health functional food for preventing or improving cancer is provided, which comprises: an extracellular vesicle containing a cancer cell-derived antigen protein or a gene encoding the protein.

[0049] In addition, in one embodiment of the present invention, a method for preventing or treating viral infection is provided, comprising: administering extracellular vesicles containing a virus-derived antigen protein or a gene encoding the protein to an individual in need thereof.

[0050] In addition, in one embodiment of the present invention, a method for preventing or treating a microbial infection is provided, comprising: administering an extracellular vesicle containing a microorganism-derived antigen protein or a gene encoding the protein to an individual in need thereof.

[0051] In addition, in one embodiment of the present invention, a method for preventing or treating cancer is provided, comprising: administering extracellular vesicles containing a cancer cell-derived antigen protein or a gene encoding the protein to an individual in need thereof.

[0052] In addition, in one embodiment of the present invention, there is provided a use of an extracellular vesicle in the preparation of a drug for preventing or treating a viral infection, wherein the extracellular vesicle contains a virus-derived antigen protein or a gene encoding the protein.

[0053] In addition, in one embodiment of the present invention, there is provided a use of an extracellular vesicle in the preparation of a drug for preventing or treating a microbial infection, wherein the extracellular vesicle contains a microbial-derived antigen protein or a gene encoding the protein.

[0054] In addition, in one embodiment of the present invention, there is provided a use of an extracellular vesicle in the preparation of a drug for preventing or treating cancer, wherein the extracellular vesicle contains a cancer cell-derived antigen protein or a gene encoding the protein.

[0055] Beneficial Effects

[0056] In the present invention, mice are immunized by administering activated immune cells or extracellular vesicles derived from bacterial outer membranes carrying antigenic proteins or mRNA, and as a result, the experiment confirms excellent antigen-specific antibody production and T cell response induction effects. Thus, the extracellular vesicle or vaccine composition containing the same according to the present invention is a platform applicable to various diseases, which has excellent antigen-specific immune response induction effect and stability, and is expected to be effectively used in the field of vaccine development for the prevention or treatment of various diseases, such as viral infections, microbial infections or cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 shows the results that THP-1 cells activated by lipopolysaccharide (LPS) treatment exhibit M1 macrophage phenotype. Figure 1a The microscopic images are of the morphology of unactivated THP-1 cells (Resting THP-1) and activated THP-1 cells (Activated THP-1). Figure 1b The results are as follows: the mRNA levels of pro-inflammatory cytokines markers IL-1b, IL-6, IL-8, TNF-α and iNOS were measured in each of the THP-1 cells.

[0058] Figure 2 shows the results of characterization of exosomes isolated from activated THP-1 cells. Figure 2a The diameter of the isolated exosomes was measured by Nanosight Tracking Analysis (NTA). Figure 2b To show the cryogenic transmission electron microscopy (cryo-EM) image of the exosomes, Figure 2c The results are as follows: Western blotting was performed using cell lysates and isolated exosomes to measure the expression levels of exosomal markers CD63, CD81, Syntenin 1, and Flotillin 1 proteins.

[0059] Figure 3 The figure shows the results of characteristic analysis of outer membrane vesicles isolated from attenuated bacteria, and the results of measuring the diameter of the isolated bacterial outer membrane vesicles by NTA (Nanosight Tracking Analysis).

[0060] FIG4 shows the degree of binding of antibodies specific to H1N1 protein or RSV-F protein in the serum of exosomes derived from immune cells (THP-1 cells) loaded with H1N1 protein or RSV-F protein. Specifically, (1) Figure 4aThe results show that after administering THP-1 cell-derived exosomes or adjuvants together with H1N1 proteins, the serum was separated 14 days later and then the binding degree of antibodies specific to influenza virus-derived H1N1 proteins in the serum was analyzed by measuring absorbance, 2) Figure 4b The results show that after administering THP-1 cell-derived exosomes or adjuvants together with RSV-F protein, the serum was separated 14 days later and then the binding degree (antibody binding) of antibodies specific to RSV-F protein derived from respiratory syncytial virus in the serum was analyzed by measuring absorbance.

[0061] Figure 5 Experimental schedule for analysis of serum antibody responses and specific T cell responses to extracellular vesicles loaded with mRNA of viral-derived proteins.

[0062] Figure 6 Results of analysis of serum antibody responses in mice administered extracellular vesicles loaded with influenza virus-derived H1N1 mRNA.

[0063] Figure 7 Results of serum antibody response analysis of mice administered extracellular vesicles loaded with varicella-zoster virus-derived glycoprotein E mRNA.

[0064] Figure 8 Results of serum antibody response analysis of mice administered extracellular vesicles loaded with respiratory syncytial virus-derived RSV-F mRNA.

[0065] Fig. 9 Results of analysis of specific T cell responses in mice administered extracellular vesicles loaded with influenza virus-derived H1N1 mRNA.

[0066] Fig.10 Results of analysis of specific T cell responses in mice administered extracellular vesicles loaded with varicella-zoster virus-derived glycoprotein E mRNA. DETAILED DESCRIPTION

[0067] The present invention relates to a vaccine platform technology that can be effectively used in the field of vaccine development for preventing or treating various infectious diseases or cancers using extracellular vesicles as potent immunostimulants and antigen carriers.

[0068] Hereinafter, the present invention will be described in detail.

[0069] The present invention provides an extracellular vesicle containing an antigen protein or a gene encoding the protein.

[0070] As used herein, the term "antigen" refers to a molecule that can induce an immune response in a host individual to produce antibodies. As the target of the antibody, each antibody is specific to the antigen so that the cells of the immune system respond to it after contact with the antigen. In the present invention, as long as the antigen is in a form that can induce an immune response as described above, it can include proteins, genes encoding the proteins, mRNA forms of the genes, and fragments of the proteins or mRNAs. The source of the antigen is not specifically limited. As non-limiting examples, it can include: antigens of viral origin; antigens of microbial origin including bacteria, archaea and fungi; and antigens of cancer cell origin.

[0071] That is, in the present invention, the antigen protein may be derived from one or more selected from viruses, microorganisms, and cancer cells.

[0072] The virus can be selected from one or more of adenovirus, smallpox virus, polio virus, measles virus, hepatitis C virus, human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), influenza virus, respiratory syncytial virus, herpes simplex virus, human papilloma virus, Zika virus, varicella-zoster virus and severe fever with thrombocytopenia syndrome virus. Specifically, it can be selected from influenza virus, respiratory syncytial virus, herpes simplex virus, human papilloma virus, virus), Zika virus, Varicella-Zoster virus and Severe Fever with Thrombocytopenia Syndrome virus, more specifically, it can be selected from one or more of influenza virus, Varicella-Zoster virus and respiratory syncytial virus.

[0073] In the present invention, when the antigen protein is derived from human immunodeficiency virus, the antigen protein may be p24 protein derived from human immunodeficiency virus-1.

[0074] In addition, in the present invention, when the antigen protein is derived from hepatitis B virus, the antigen protein may be S protein derived from hepatitis B virus.

[0075] In addition, in the present invention, when the antigen protein is derived from influenza virus, the antigen protein can be selected from one or more of H1N1 protein, H2N2 protein, H3N2 protein, H5N1 protein, H7N7 protein, H7N9 protein, H9N2 protein and H10N7 protein derived from influenza virus, specifically, it can be H1N1 protein, more specifically, it can be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0076] The polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 may include a polypeptide having a sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 1, and the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 may contain the amino acid sequence of SEQ ID NO: 1 and its functional variants. Functional variants refer to all similar sequences with some amino acid substitutions at the amino acid positions without affecting the biological properties of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0077] In addition, in the present invention, when the antigen protein is derived from respiratory syncytial virus, the antigen protein may be RSV-F protein derived from respiratory syncytial virus, specifically, may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0078] In addition, the polypeptide composed of the amino acid sequence of SEQ ID NO: 2 may contain a polypeptide having a sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 2, respectively. The polypeptide composed of the amino acid sequence of SEQ ID NO: 2 may contain the amino acid sequence of SEQ ID NO: 2 and its functional variants.

[0079] In addition, in the present invention, when the antigen protein is derived from varicella-zoster virus, the antigen protein may be glycoprotein E (Glycoprotein E) derived from varicella-zoster virus, specifically, may be a polypeptide consisting of the amino acid sequence of SEQ ID NO:3.

[0080] In addition, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 may contain a polypeptide having a sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the amino acid sequence of SEQ ID NO: 3, respectively. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 may contain the amino acid sequence of SEQ ID NO: 3 and its functional variants.

[0081] The term "homology" is intended to indicate the degree of similarity to a wild-type amino acid sequence. Such homology comparison can be performed using comparison programs well known in the art, and the homology between two or more sequences can be calculated as a percentage (%).

[0082] In the present invention, the microorganism includes prokaryotic microorganisms and eukaryotic microorganisms, the prokaryotic microorganism includes true bacteria (bacteria) and archaea, and the eukaryotic microorganism includes fungi, algae and protozoa. In addition, the true bacteria (bacteria) includes gram-positive bacteria and gram-negative bacteria, and the fungi includes molds and yeasts. Specifically, the microorganism can be selected from the group consisting of Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida, The invention further comprises one or more microorganisms of the genera da, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium and the order Mucorales.

[0083] In addition, in the present invention, the cancer cells may be derived from one or more of the following cancers: acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, mammary cancer, medullary carcinoma); brain cancer; cancer (e.g., meningioma, glioblastoma, glioblastoma (e.g., astrocytoma, oligodendroglioma, medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma;Endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophilia; gall bladder cancer; gastric cancer cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma); throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer); heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis amyloidosis);kidney cancer (e.g., nephroblastoma, also known as Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disease (mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM), also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis);neuroendocrine cancer (e.g., gastroenteropancreaticneuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); disease); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndrome; intraepithelial neoplasm; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer);soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma, synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva), specifically, may be derived from one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, glioblastoma, gastric cancer, pancreatic cancer, liver cancer, cervical cancer, carcinoid tumor, endometrial cancer, kidney cancer, testicular cancer, glioma, thyroid cancer, skin cancer, and lymphoma, more specifically, may be derived from one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma. ;

[0084] In the present invention, when the antigen protein is derived from cancer cells, the antigen protein may be one or more proteins selected from MAGE1 / 2 / 3, WT1, CDK4, MUC-1, HER2, PSA, E6 / E7 of HPV16 and HCV.

[0085] In the present invention, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., broad-spectrum biological activity) and reduced or enhanced antigenicity, a protecting group can be attached to the N- or C-terminus of the antigen protein. The protecting group can be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or a polyethylene glycol (PEG), but can include but is not limited to any modification that can improve the antigen protein, especially the stability of the antigen protein.

[0086] The term "stability" may refer not only to in vivo stability for protecting the antigen protein from attacks by protein-cleaving enzymes in vivo, but also to storage stability (eg, storage stability at room temperature).

[0087] In the present invention, the antigen protein can be derived from nature, or can be obtained by various polypeptide synthesis methods known in the art. As an example, it can be prepared using gene recombination and protein expression systems, or can be prepared by chemical synthesis such as protein synthesis (for example, liquid phase or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemical method) and cell-free protein synthesis methods. In addition, as an example, the antigen protein can be a product obtained by culturing peptides, plant-derived tissues or cells or microorganisms (for example, bacteria or fungi, particularly yeast).

[0088] In the present invention, when the antigen protein is derived from human immunodeficiency virus, the gene encoding the antigen protein may be a gene encoding p24 protein derived from human immunodeficiency virus-1.

[0089] In addition, in the present invention, when the antigen protein is derived from hepatitis B virus, the gene encoding the antigen protein may be a gene encoding the S protein derived from hepatitis B virus.

[0090] In addition, in the present invention, when the antigen protein is derived from influenza virus, the gene encoding the antigen protein may be a gene encoding one or more proteins selected from H1N1 protein, H2N2 protein, H3N2 protein, H5N1 protein, H7N7 protein, H7N9 protein, H9N2 protein and H10N7 protein derived from influenza virus, specifically, it may be a gene encoding H1N1 protein, more specifically, it may be a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0091] In addition, when the antigen protein is derived from respiratory syncytial virus, the gene encoding the antigen protein may be a gene encoding RSV-F protein derived from respiratory syncytial virus, specifically, a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0092] In addition, in the present invention, when the antigen protein is derived from varicella-zoster virus, the gene encoding the antigen protein can be a gene encoding glycoprotein E (Glycoprotein E) derived from varicella-zoster virus, specifically, it can be a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

[0093] In addition, in the present invention, the gene encoding the cancer cell-derived antigen protein may be a gene encoding one or more proteins selected from the group consisting of MAGE1 / 2 / 3, WT1, CDK4, MUC-1, HER2, PSA, E6 / E7 of HPV16 and HCV.

[0094] In the present invention, the gene encoding the antigen protein can be, for example, DNA, RNA or a combination or modification thereof, and can be double-stranded or single-stranded. In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, titer, efficacy, etc.), changed specificity (e.g., broad-spectrum biological activity) and reduced or enhanced antigenicity, the gene encoding the antigen protein can encode an amino acid sequence incorporating a protective group at the N- or C-terminus of the antigen protein, and can include the protective group without limitation, as long as the protective group is a component that can particularly improve stability.

[0095] Furthermore, in the present invention, the antigen protein or the gene encoding the protein can be loaded into the extracellular vesicles.

[0096] The method for loading the antigen protein or the gene encoding the protein into extracellular vesicles is not specifically limited, and those skilled in the art can appropriately select and use methods known in the relevant technical field. For example, the antigen protein can be loaded into extracellular vesicles after the extracellular vesicles are treated with extracorporeal shock waves.

[0097] As used herein, the term "extracellular vesicle" refers to a small sphere surrounded by a cell-derived membrane, which varies greatly depending on the source of the cell or the method of production that forms its name. In the present invention, it can include one or more selected from exosomes, ectosomes, microvesicles, bacterial outer membrane vesicles (Bacterial OMV), microbial-derived extracellular vesicles including yeast-derived extracellular vesicles, and apoptotic bodies, specifically, exosomes or bacterial-derived outer membrane vesicles, but not limited thereto.

[0098] The extracellular vesicle may be derived from nature, for example, one or more extracellular vesicles selected from animals, plants and microorganisms, or may be artificially produced extracellular vesicles. In addition, the cell may be a cell separated from a natural organism. In addition, the cell may be derived from any type of animal or plant, including humans and non-human mammals. The animal cell may be one or more cells selected from somatic cells, germ cells, immune cells, neural cells and tumor cells, specifically, immune cells, more specifically, activated immune cells.

[0099] In addition, in the present invention, when the extracellular vesicle is derived from a microorganism, the microorganism may be a bacterium, and the extracellular vesicle may be an outer membrane vesicle derived from a bacterium.

[0100] In the present invention, the animal cell may be one or more cells selected from Vero, MDCK, MRC-5, serum-free Hi-5, Hi-5, WI-38, PER.C6, HEK-293 and CEF.

[0101] The somatic cells are cells constituting the body of any type of animal including mammals such as humans, and in the present invention, they may include all types of cells except germ cells, immune cells, neural cells, and tumor cells.

[0102] The immune cells are cells that determine the immune response in the body. They play a role in defense, control immunity, and eliminate cancer cells that are constantly generated in the body in order to resist and overcome external invading pathogens, foreign bodies, viruses, etc. Specifically, they may include but are not limited to NK cells (natural killer cells), dendritic cells, T cells, B cells, macrophages, and lymphocytes.

[0103] The neuronal cells, as a cell constituting the nervous system, express ion channels such as sodium channels and potassium channels. Unlike other cells, they can transmit signals electrically and exchange chemical signals through a structure called a synapse to exchange various information with other adjacent neuronal cells. In the present invention, the specific type of the neuronal cells is not limited, and any cell that secretes extracellular vesicles can be included.

[0104] The tumor cells have the same meaning as cancer cells. Unlike normal cells, tumor cells refer to cells that grow rapidly and irregularly due to unregulated cell cycles. In the present invention, the source tissue of the tumor cells is not specifically limited, as long as the tumor cells secrete extracellular vesicles.

[0105] In the present invention, when the extracellular vesicles are derived from animal cells, the extracellular vesicles may express one or more proteins selected from CD9, CD63, CD81, Alix, TSG101, Syntenin 1 and Flotillin 1.

[0106] In addition, in the present invention, when the extracellular vesicles are derived from plant cells, the extracellular vesicles may express one or more proteins selected from syntaxin (PEN1), tetraspanin 8 (Tet8) and heat shock protein (HSP).

[0107] In the present invention, when the extracellular vesicles are derived from a microorganism, the extracellular vesicles may express one or more proteins selected from OmpA, flagellin, HSP70 and β-glucan. Specifically, when the microorganism is a bacterium, the extracellular vesicles may express one or more proteins selected from OmpA and flagellin. When the microorganism is a fungus, the extracellular vesicles may express one or more proteins selected from HSP70 and β-glucan.

[0108] In addition, the extracellular vesicles can be derived from activated immune cells, specifically, activated THP-1 cells or KG-1 cells. At this time, the activation can be completed by lipopolysaccharide (LPS) or α-galactosylceramide (α-GC). When THP-1 cells are activated, THP-1 cells are similar to macrophages, and their cell shape can change. At the same time, they can express one or more proteins selected from IL-1b, IL-6, IL-8, TNF-α and iNOS. Specifically, IL-1b, IL-6, IL-8, TNF-α and iNOS can all be expressed.

[0109] In the present invention, as an example of a specific embodiment, the phenotypic changes of immune cells activated by antigen stimulation are analyzed, the properties of exosomes isolated therefrom are observed, and outer membrane vesicles are isolated from attenuated bacteria and their properties are observed.

[0110] Specifically, in one embodiment of the present invention, as a result of treating THP-1 cells, a human monocytic cell line, with lipopolysaccharide (LPS), it was confirmed that the cells changed in shape and expressed markers of M1 macrophages compared to cells not treated with LPS (see Example 1).

[0111] In another embodiment of the present invention, it is confirmed that the activated immune cell-derived exosomes, preferably activated THP-1 cell-derived exosomes, have an average diameter of 128.0 nm and express exosomal marker proteins CD63, CD81 and raftin 1 (see Example 2).

[0112] In another embodiment of the present invention, the culture fluid of the attenuated bacterial strain BL21 was centrifuged, the supernatant was collected, and the supernatant was filtered, concentrated, and washed to prepare outer membrane vesicles (OMV). The results of NTA analysis of the characteristics of the outer membrane vesicles showed that the average diameter of the outer membrane vesicles was 119.2 nm, and the concentration of the isolated outer membrane vesicles was 1.5×10 11 The protein concentration was 2.2 mg / ml as confirmed by quantitative analysis using BCA assay (see Example 3).

[0113] In another embodiment of the present invention, activated THP-1 cell-derived exosomes loaded with influenza virus-derived H1N1 protein or respiratory syncytial virus-derived RSV-F protein were prepared, and then subcutaneously administered to mice for immunization, and analyzed for the production of antibodies. The results confirmed that, compared with the case where an adjuvant and the H1N1 protein or RSV-F protein were used in combination, even with a much smaller amount, a similar or higher level of H1N1 or RSV-F-specific IgG formation could be achieved (see Examples 4 and 6).

[0114] In another embodiment of the present invention, activated THP-1 cell-derived exosomes carrying influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA are prepared, or cell-derived outer membrane vesicles carrying influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA are prepared (see Example 5).

[0115] In another embodiment of the present invention, the activated THP-1 cell-derived exosomes or bacteria-derived outer membrane vesicles loaded with influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 5 were subcutaneously administered to mice for immunization, and the production of antibodies was analyzed. The results confirmed that compared with the control group, the activated THP-1 cell-derived exosomes or bacteria-derived outer membrane vesicles showed a better degree of formation of H1N1, RSV-F or VZE-specific IgG (see Example 7-1).

[0116] In another embodiment of the present invention, the activated THP-1 cell-derived exosomes or cell-derived outer membrane vesicles loaded with influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 4 were subcutaneously administered to mice for immunization, and then spleen cells were isolated from the immunized mice and the H1N1 or VZE antigen-specific T cell response was analyzed by the amount of IFN-γ. The results confirmed that compared with the control group, mice immunized with activated THP-1 cell-derived exosomes or bacteria-derived outer membrane vesicles induced stronger T cell responses (see Example 7-2).

[0117] The extracellular vesicles according to the present invention were administered to mice for immunization, and the results confirmed that the antigen-specific antibody production and T cell response induction effects were excellent, so that the extracellular vesicles according to the present invention can be effectively used in the field of vaccine development, which is used to prevent or treat various diseases including viral infections, microbial infections or cancer.

[0118] In the present invention, the viral infection can be selected from one or more of adenovirus infection, smallpox virus infection, polio virus infection, measles virus infection, hepatitis C virus infection, human immunodeficiency virus-1 infection, hepatitis B virus infection, influenza virus infection, respiratory syncytial virus infection, herpes simplex virus infection, human papillomavirus infection, Zika virus infection, varicella-zoster virus infection and fever with thrombocytopenia syndrome virus infection, specifically, it can be selected from one or more of human immunodeficiency virus-1 infection, hepatitis B virus infection, influenza virus infection, respiratory syncytial virus infection and varicella-zoster virus infection, more specifically, it can be selected from one or more of influenza virus infection, varicella-zoster virus infection and respiratory syncytial virus infection.

[0119] In addition, in the present invention, the microbial infection can be selected from the group consisting of Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida Infection with one or more microorganisms of the genera , Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and the order Mucorales.

[0120] In addition, in the present invention, the cancer may be one or more of the following cancers: acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, mammary cancer, medullary carcinoma); brain cancer; cancer (e.g., meningioma, glioblastoma, glioblastoma (e.g., astrocytoma, oligodendroglioma, medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma;Endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophilia; gall bladder cancer; gastric cancer cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma); throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer); heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharyngeal cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis amyloidosis);Kidney cancer (e.g., nephroblastoma, also known as Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disease (mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM), also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis);neuroendocrine cancer (e.g., gastroenteropancreaticneuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); disease); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndrome; intraepithelial neoplasm; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer);soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma, synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva), specifically, it may be one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, glioblastoma, gastric cancer, pancreatic cancer, liver cancer, cervical cancer, carcinoid tumor, endometrial cancer, kidney cancer, testicular cancer, glioma, thyroid cancer, skin cancer, and lymphoma, more specifically, it may be one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma. ;

[0121] In addition, the present invention provides a vaccine composition for preventing or treating viral infections, comprising: an extracellular vesicle containing a virus-derived antigen protein or a gene encoding the protein.

[0122] The "virus-derived antigen protein", "gene encoding the protein", "extracellular vesicle", "virus", "viral infection" and the like can be within the above scope.

[0123] As used herein, the term "prevention" refers to any action to inhibit a particular disease or disorder, or to delay the onset of the disease, by administering a vaccine composition according to the present invention.

[0124] As used herein, the term "treatment" refers to any action to ameliorate or beneficially alter the symptoms of a particular disease or disorder by administering a vaccine composition according to the present invention.

[0125] As used herein, the term "vaccine" refers to a vaccine that prevents infection or reinfection caused by a pathogen or antigen, reduces the severity of symptoms or eliminates symptoms, or actually or completely eliminates a disease caused by the pathogen or antigen by inducing an immune response to the antigen in a host, including humans. Therefore, the vaccine composition of the present invention is preferably administered to an individual prophylactically before infection by the pathogen or before the onset of the disease, but can also be administered therapeutically after infection by the pathogen or after the onset of the disease.

[0126] In addition, in the present invention, the vaccine composition can simultaneously induce antigen-specific antibody production and T cell-mediated immune response.

[0127] In an embodiment of the present invention, activated THP-1 cell-derived exosomes loaded with H1N1 protein derived from influenza virus or RSV-F protein derived from respiratory syncytial virus were prepared, and then subcutaneously administered to mice for immunization, and analyzed for the production of antibodies. The results confirmed that, compared with the case where an adjuvant and the H1N1 protein or RSV-F protein were used in combination, even with a much smaller amount, a similar or higher level of H1N1 or RSV-F-specific IgG formation could be achieved.

[0128] In another embodiment of the present invention, the activated THP-1 cell-derived exosomes or bacteria-derived outer membrane vesicles loaded with influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 5 were subcutaneously administered to mice for immunization, and the production of antibodies was analyzed. The results confirmed that compared with the control group, the activated THP-1 cell-derived exosomes and bacteria-derived outer membrane vesicles showed a better degree of formation of H1N1, RSV-F or VZE-specific IgG.

[0129] In another embodiment of the present invention, the activated THP-1 cell-derived exosomes or cell-derived outer membrane vesicles loaded with influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 5 were subcutaneously administered to mice for immunization, and then spleen cells were isolated from the immunized mice and the H1N1 or VZE antigen-specific T cell response was analyzed by the amount of IFN-γ. The results confirmed that compared with the control group, mice immunized with activated THP-1 cell-derived exosomes or bacteria-derived outer membrane vesicles induced stronger T cell responses.

[0130] In addition, in the present invention, the vaccine composition may be freeze-dried.

[0131] The inventors confirmed the specific immune response induction and storage stability of the vaccine composition. Specifically, in order to evaluate the storage stability of the extracellular vesicles according to the present invention, the freeze-dried exosomes were stored under 4°C refrigeration for 7 days or stored at room temperature, and then subcutaneously administered to mice for immunization and analyzed for antibody production. The results confirmed that even after storage under the conditions, the excellent stability and antibody production effect were maintained.

[0132] The immune cell-derived exosomes activated by the embodiment of the present invention showed lymph node tropism after subcutaneous injection, and were mainly absorbed by local macrophages and dendritic cells. MHC-I / peptide complexes appeared in exosomes by dendritic cells. It can be seen that in addition to more effectively producing neutralizing antibodies against viruses, this can also induce H1N1, VZE or RSV-F specific T cell responses in the spleen cells of immunized mice.

[0133] Thus, the present invention strongly suggests, based on the results demonstrated in the Examples, that nanoscale extracellular vesicles including exosomes secreted by cells or outer membrane vesicles derived from bacteria can be used as powerful immunostimulants and antigen carriers, and can serve as a platform for designing effective vaccines against target diseases.

[0134] The vaccine composition of the present invention can be prepared in the form of any suitable pharmaceutically acceptable formulation. For example, it can be prepared in the form of a ready-to-use solution or suspension, or a concentrated stock solution suitable for dilution before administration, or a reconstituted form, such as a freeze-dried, lyophilized or frozen formulation.

[0135] The vaccine composition of the present invention can be formulated to further include a pharmaceutically acceptable carrier. Specifically, the carrier can be, for example, a colloidal suspension, a powder, a saline solution, a lipid, a liposome, a microsphere (microspheres) or a nano-spherical particle. They can form a complex and a delivery vehicle or be associated with a complex and a delivery vehicle, and can be transported in vivo using a delivery system known in the art, such as a lipid, a liposome, a microparticle, gold, a nanoparticle, a polymer, a condensing agent, a polysaccharide, a polyamino acid, a dendrimer, a saponin, an adsorption enhancing substance or a fatty acid.

[0136] In addition, the pharmaceutically acceptable carrier generally includes diluents, excipients, stabilizers, preservatives, etc. Suitable diluents may be non-aqueous solvents or aqueous solvents, etc., the non-aqueous solvents may be, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, etc., the aqueous solvents may be, for example, saline (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer), etc., suitable excipients may be starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, ethylene glycol, water, ethanol, etc., suitable stabilizers may be carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamic acid, glucose, etc., or animal, vegetable or microbial proteins such as milk powder, serum albumin, casein, etc. Suitable preservatives may be thimerosal, thimerosal sodium, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, and the like.

[0137] The vaccine composition of the present invention may also include an antigen adjuvant. The antigen adjuvant may be composed of one or more substances that enhance the immune response to the antigen. The antigen adjuvant may be, for example, selected from complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, gel-like aluminum adjuvant, surfactant (e.g., lysolecithin, polyether glycol, polyanion, peptide, oil or hydrocarbon emulsion, etc.), vegetable oil (cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, squalene, GLA-SE, muramyl dipeptide, lipopolysaccharide (LPS), Quil A, alum (aluminum salts, Aluminum salts), Al(OH)3 and α-galactosylceramide (α-Galactosylceramide, α-GC), specifically Al(OH)3.

[0138] The vaccine composition of the present invention can be prepared by a method commonly used in the technical field to which the present invention belongs. The vaccine composition can be prepared as an oral or parenteral preparation, and the parenteral preparation can preferably be administered by any route selected from transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal or epidural.

[0139] The term "administer" means introducing a predetermined substance into an individual by an appropriate method, and "individual" means any organism that can carry a viral infection, such as humans, rats, mice, and livestock, etc. As a specific example, it may be a mammal, including humans.

[0140] The vaccine composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to an amount sufficient to show the effect of the vaccine without causing side effects or severe or excessive immune responses, and the level of the effective dose may vary according to a variety of factors, such as the disorder to be treated, the severity of the disorder, the activity of the vaccine substance, the route of administration, the rate of protein elimination, the duration of treatment, the substances combined with or used simultaneously with the vaccine composition, the age, weight, sex, dietary habits, general health status of the individual, and factors well known in the medical field.

[0141] In addition, the present invention provides a vaccine composition for preventing or treating microbial infections, comprising: an extracellular vesicle containing a microbial-derived antigen protein or a gene encoding the protein.

[0142] The "antigen protein derived from microorganisms", "gene encoding the protein", "extracellular vesicles", "microorganisms", "microbial infections", "prevention", "treatment", "vaccine compositions" and the like can be within the above scope.

[0143] In addition, the present invention provides a vaccine composition for preventing or treating cancer, comprising: an extracellular vesicle containing a cancer cell-derived antigen protein or a gene encoding the protein.

[0144] The "cancer cell-derived antigen protein", "gene encoding the protein", "extracellular vesicles", "cancer cells", "cancer", "prevention", "treatment", "vaccine composition" and the like can be within the above scope.

[0145] In addition, a health functional food for preventing or improving viral infections is provided, which comprises: an extracellular vesicle containing a virus-derived antigen protein or a gene encoding the protein.

[0146] The "virus-derived antigen protein", "gene encoding the protein", "extracellular vesicle", "virus", "viral infection", "prevention" and the like can be within the above scope.

[0147] The term "improvement" may refer to any action that at least alleviates a parameter associated with the state being treated, such as the degree of a symptom. In this case, the health functional food may be used simultaneously with or alone with a drug for treatment before or after the onset of the relevant disease to prevent or improve viral infections.

[0148] The health functional food of the present invention can also include an antigen adjuvant. The antigen adjuvant can be, for example, selected from complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, gel-like aluminum adjuvant, surfactant (for example, lysolecithin, polyether glycol, polyanion, peptide, oil or hydrocarbon emulsion, etc.), vegetable oil (cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, squalene, GLA-SE, muramyl dipeptide, lipopolysaccharide (LPS), Quil A, alum (aluminum salts, Aluminum salts), Al(OH)3 and α-galactosylceramide (α-Galactosylceramide, α-GC) one or more, specifically Al(OH)3.

[0149] In the health functional food, the active ingredient can be directly added to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). Usually, when preparing food or beverages, based on raw materials, the addition amount of the health functional food can be about 15% by weight or less, more specifically, about 10% by weight or less. However, if it is taken in for a long time for health and hygiene purposes or health control purposes, the amount can be less than or equal to the range.

[0150] The health functional food may include one or more of a carrier, a diluent, an excipient and an additive, and may be configured as one selected from tablets, pills, powders, granules, powders, capsules and liquid preparations. The food to which the compound according to one embodiment may be added includes various foods, powders, granules, tablets, capsules, syrups, beverages, chewing gum, tea, vitamin complexes, health functional foods, etc.

[0151] Specific examples of the carrier, excipient, diluent and additive can be selected from at least one of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, polyvinyl pyrrolidone, methylcellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0152] In addition to containing the active ingredients, the health functional food may also contain other ingredients as essential ingredients without any particular restrictions. For example, like ordinary beverages, various flavoring agents or natural carbohydrates may be contained as additional ingredients. As the above-mentioned natural carbohydrates, for example, monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than the above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizic acid, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The ratio of the natural carbohydrates may be appropriately determined by the selection of those skilled in the art.

[0153] In addition to the above, the health functional food according to one aspect may include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used alone or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.

[0154] In addition, a health functional food for preventing or improving microbial infections is provided, which includes: an extracellular vesicle containing a microbial-derived antigen protein or a gene encoding the protein.

[0155] The "antigen protein derived from microorganisms", "gene encoding the protein", "extracellular vesicles", "microorganisms", "microbial infections", "prevention", "improvement", "health functional foods" and the like can be within the above scope.

[0156] In addition, a health functional food for preventing or improving viral infections is provided, which includes: an extracellular vesicle containing a cancer cell-derived antigen protein or a gene encoding the protein.

[0157] The "cancer cell-derived antigen protein", "gene encoding the protein", "extracellular vesicles", "cancer cells", "cancer", "prevention", "improvement", "health functional food" and the like can be within the above scope.

[0158] In addition, the present invention provides a method for preventing or treating viral infection, comprising: administering an extracellular vesicle containing a virus-derived antigen protein or a gene encoding the protein to an individual in need thereof.

[0159] The “virus-derived antigen protein”, “gene encoding the protein”, “extracellular vesicle”, “individual”, “administration”, “viral infection”, “prevention”, “treatment” and the like can be within the above scope.

[0160] In addition, the present invention provides a method for preventing or treating a microbial infection, comprising: administering an extracellular vesicle containing a microorganism-derived antigenic protein or a gene encoding the protein to an individual in need thereof.

[0161] The “antigenic protein derived from microorganisms”, “gene encoding the protein”, “extracellular vesicles”, “individual”, “administration”, “microbial infection”, “prevention”, “treatment” and the like can be within the above scope.

[0162] In addition, the present invention provides a method for preventing or treating cancer, comprising: administering an extracellular vesicle containing a cancer cell-derived antigen protein or a gene encoding the protein to an individual in need thereof.

[0163] The “cancer cell-derived antigen protein”, “gene encoding the protein”, “extracellular vesicle”, “individual”, “administration”, “cancer”, “prevention”, “treatment” and the like can be within the above scope.

[0164] In addition, the present invention provides a use of an extracellular vesicle in preparing a drug for preventing or treating a viral infection, wherein the extracellular vesicle contains a virus-derived antigen protein or a gene encoding the protein.

[0165] The "virus-derived antigen protein", "gene encoding the protein", "extracellular vesicles", "viral infection", "prevention", "treatment" and the like can be within the above scope.

[0166] In addition, the present invention provides a use of an extracellular vesicle in preparing a drug for preventing or treating a microbial infection, wherein the extracellular vesicle contains a microbial-derived antigen protein or a gene encoding the protein.

[0167] The "antigenic protein derived from microorganisms", "gene encoding the protein", "extracellular vesicles", "microbial infection", "prevention", "treatment" and the like can be within the above scope.

[0168] In addition, the present invention provides a use of an extracellular vesicle in preparing a drug for preventing or treating cancer, wherein the extracellular vesicle contains a cancer cell-derived antigen protein or a gene encoding the protein.

[0169] The "cancer cell-derived antigen protein", "gene encoding the protein", "extracellular vesicles", "cancer", "prevention", "treatment" and the like can be within the above scope.

[0170] Implementation

[0171] Hereinafter, reference is made to preferred embodiments to facilitate understanding of the present invention. However, the following embodiments are provided only to make the present invention easier to understand, and the content of the present invention is not limited to the following embodiments.

[0172] [Example]

[0173] Example 1. Phenotypic observation of activated immune cells

[0174] In order to develop a vaccine using extracellular vesicles, the inventors first selected immune cells, stimulated the immune cells with antigens, and attempted to prepare a vaccine using exosomes isolated from activated immune cells. To this end, human monocytic cell line THP-1 cells were activated as an example of the immune cells, and the phenotype of the activated cells was observed.

[0175] Specifically, THP-1 cells (8×10 5 The cells were activated by adding 100 cells / mL of LPS and then observing the cell morphology under a microscope together with the THP-1 cells that were not activated because they were not treated with LPS. Figure 1a As shown, compared with resting THP-1 cells, the cell morphology of activated THP-1 cells became similar to that of macrophages.

[0176] Further, based on the results, as reported in the art, in order to confirm whether THP-1 cells activated by LPS treatment differentiate into M1 macrophages, the mRNA levels of pro-inflammatory cytokines markers IL-1b, IL-6, IL-8, TNF-α and iNOS secreted by M1 macrophages were measured. The results confirmed that Figure 1b As shown, in non-activated THP-1 cells, the markers were hardly expressed, while in activated THP-1 cells, the expression of the markers increased, especially the mRNA levels of IL-1b, IL-6 and IL-8 increased significantly.

[0177] The results indicate that THP-1 cells activated by LPS treatment exhibit the phenotype of M1 macrophages.

[0178] Example 2: Confirmation of the characteristics of activated immune cell-derived exosomes

[0179] In order to confirm the characteristics of the activated THP-1 cell-derived exosomes, the present inventors isolated exosomes from cells by the following process. Specifically, in order to remove cells and cell debris, the cell culture medium was centrifuged at 1200 rpm for 5 minutes, the supernatant was collected, and then the supernatant was filtered through a 0.2 um filter to remove microparticles larger than exosomes, and then the exosomes were concentrated and washed using a tangential flow filtration (TFF) system.

[0180] Next, the characteristics of the isolated exosomes were analyzed by NTA (Nanosight Tracking Analysis) and cryo-electron microscopy. Figure 2a and 2b As shown, the isolated exosomes had an average diameter of 128.0 nm and had a round shape.

[0181] In addition, the concentration of isolated exosomes was 2.6×10 11 The results of quantitative analysis by BCA assay confirmed that the exosome protein concentration was 3.5 mg / ml. Further, in order to investigate whether the isolated exosomes expressed exosome markers, Western blotting was performed using cell lysate and isolated exosomes to measure the expression levels of exosome markers CD63, CD81 and raft protein 1. The results confirmed that Figure 2c As shown, only CD63 was weakly expressed in cell lysates, while the above marker proteins were all highly expressed in the isolated exosomes.

[0182] It can be clearly seen from the above results that the exosomes isolated from the activated THP-1 cells are exosomes having the above-mentioned properties.

[0183] Example 3: Confirmation of the characteristics of outer membrane vesicles derived from attenuated bacteria

[0184] In order to confirm the characteristics of the outer membrane vesicles (OMV) derived from the attenuated bacteria (BL21 strain), the inventors isolated outer membrane vesicles (OMV) from the bacteria by the following process. Specifically, in order to remove cells and cell debris, the culture solution was centrifuged at 3000 rpm for 15 minutes, the supernatant was collected, and then the supernatant was filtered through a 0.2um filter to remove large-sized microparticles, and then the outer membrane vesicles (OMV) were concentrated and washed using a tangential flow filtration (TFF) system.

[0185] Next, the properties of the isolated outer membrane vesicles were analyzed by NTA (Nanosight Tracking Analysis). Figure 3 As shown in Figure 2, the average diameter of the isolated outer membrane vesicles was 119.2 nm, and the concentration of the isolated outer membrane vesicles was 1.5×10 11 The protein concentration was 2.2 mg / ml as confirmed by quantitative analysis using BCA assay.

[0186] Example 4: Preparation of exosomes carrying recombinant virus-derived proteins

[0187] The present inventors attempted to verify the preventive effect of the activated THP-1 cell-derived exosomes isolated in Example 2 as a vaccine against viral infections by loading influenza virus-derived H1N1 protein or respiratory syncytial virus-derived RSV-F protein onto the activated THP-1 cell-derived exosomes isolated in Example 2.

[0188] To this end, constructs of influenza virus-derived H1N1 protein (A / Puerto Rico / 8 / 1934; accession number #ABD77675.1) and respiratory syncytial virus-derived RSV-F (fusion glycoprotein; accession number #P03420.1) were first prepared. Then, in order to load the H1N1 protein or RSV-F protein expressed from the construct into the activated THP-1 cell-derived exosomes, 1.4×10 11 The exosomes (3 mg) and 50 μg of the H1N1 protein or RSV-F protein were treated with extracorporeal shock wave (ESW), and then the exosomes carrying the H1N1 protein or RSV-F protein were washed and concentrated using an ultracentrifuge (32000 rpm, 16 hours).

[0189] Then, the amount of H1N1 protein or RSV-F protein loaded into the THP-1 cell-derived exosomes was quantitatively analyzed using ELISA, and the results confirmed that a total of 41.1 μg of H1N1 protein was loaded into the exosomes, and a total of 36.43 μg of RSV-F protein was loaded into the exosomes. The inventors attempted to evaluate the effect of the activated THP-1 cell-derived exosomes (ImmunExo H1N1) loaded with influenza virus-derived H1N1 protein or the activated THP-1 cell-derived exosomes (ImmunExo RSV-F) loaded with respiratory syncytial virus-derived RSV-F protein prepared above on viral infections through the following experiments.

[0190] Example 5: Preparation of extracellular vesicles containing recombinant virus-derived mRNA

[0191] The inventors attempted to verify the preventive effect of the vaccine against viral infections by loading influenza virus-derived H1N1 (Influenza A H1N1Hawaii / 70 / 2019 hemagglutinin) mRNA, respiratory syncytial virus-derived RSV-F (Human respiratory syncytialvirus (RSV) (A2) fusion glycoprotein) mRNA, and varicella-zoster virus-derived glycoprotein E (Varicella-Zoster virus glycoprotein E, VZE) mRNA into the activated THP-1 cell-derived exosomes isolated in Example 2 or the bacteria-derived outer membrane vesicles (Bacteria Outer membrane vesicles, Bacteria OMV) isolated in Example 3.

[0192] Specifically, the mRNA of each protein was prepared from a DNA template by an in vitro transcription (IVT) process by rnagene. For the prepared mRNA, THP-1 cell-derived exosomes or bacterial-derived OMVs (6 mg) and 100 ug of each mRNA were prepared and subjected to extracorporeal shockwave (ESW) treatment, and then the exosomes carrying the mRNA of each viral-derived protein were washed and concentrated using an ultracentrifuge (32000 rpm, 2 hours).

[0193] Then, qRT-PCR was used to quantitatively analyze the amount of each mRNA loaded into the THP-1 cell-derived exosomes or bacteria-derived OMVs, and the results confirmed that a total of 67.08ug, 60.3ug, and 66.3ug (H1N1, RSV-F, and VZE) were loaded into the THP-1 cell-derived exosomes, and 64.36ug, 66.5ug, and 65ug (H1N1, RSV-F, and VZE) were loaded into the bacteria-derived OMVs. The inventors attempted to evaluate the effects of the activated THP-1 cell-derived exosomes or bacteria-derived OMVs prepared above, which were loaded with mRNAs of various virus-derived proteins, on various viral infections through the following experiments.

[0194] Example 6: Confirmation of the vaccine efficacy of activated immune cell-derived exosomes carrying virus-derived proteins against viruses

[0195] The present inventors attempted to evaluate the efficacy of activated THP-1 cell-derived exosomes loaded with influenza virus-derived H1N1 protein or respiratory syncytial virus-derived RSV-F protein prepared in Example 4 as a vaccine against the virus.

[0196] (1) Analysis of serum antibody responses to influenza virus in THP-1 cell-derived exosomes loaded with influenza virus-derived H1N1 protein

[0197] The present inventors obtained serum samples from mice prepared in Example 4 to which activated THP-1 cell-derived exosomes (ImmunExo) carrying influenza virus-derived H1N1 protein were administered at a concentration of 5 μg, or from mice to which adjuvant Al(OH)3 500 μg and H1N1 protein 5 μg were administered together 14 days later, and then diluted the samples into PBS containing 1% BSA at a ratio of 1:100 to 1:1000, and analyzed the binding degree of specific antibodies against influenza virus-derived H1N1 protein in the serum by measuring the absorbance.

[0198] The results confirmed that Figure 4a As shown, although the amount of exosomes in the group administered with H1N1 protein was much smaller, the degree of binding of specific antibodies against influenza virus H1N1 in the serum was similar to that in the group administered with adjuvant and H1N1 protein at the same time.

[0199] (2) Analysis of serum antibody responses to the virus in THP-1 cell-derived exosomes carrying RSV-F protein

[0200] The inventors obtained serum samples 14 days later from mice prepared in Example 4 to which activated THP-1 cell-derived exosomes (ImmunExo) carrying RSV-F protein from respiratory syncytial virus were administered at a concentration of 8 μg, or from mice administered with adjuvant Al(OH)3 500 μg and RSV-F protein 8 μg. The samples were diluted in PBS containing 1% BSA at a ratio of 1:100 to 1:1000, and the binding degree of specific antibodies against RSV-F protein from respiratory syncytial virus in the serum was analyzed by measuring the absorbance.

[0201] The results confirmed that Figure 4b As shown, although the amount of exosomes in the group administered with RSV-F protein was much smaller, the degree of binding of specific antibodies against respiratory syncytial virus RSV-F in the serum was better than that in the group administered with adjuvant and RSV-F protein at the same time.

[0202] Example 7: Confirmation of the vaccine efficacy of extracellular vesicles carrying mRNA of virus-derived proteins against viruses

[0203] The present inventors attempted to evaluate the efficacy of activated THP-1 cell-derived exosomes or bacteria-derived outer membrane vesicles (OMVs) loaded with influenza virus-derived H1N1 mRNA, respiratory syncytial virus-derived RSV-F mRNA, or varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 5 as vaccines against viruses.

[0204] 7-1. Analysis of serum antibody responses against extracellular vesicles carrying mRNA of viral-derived proteins

[0205] (1) Extracellular vesicles carrying influenza virus-derived H1N1 mRNA

[0206] The present inventors administered 10ug of activated THP-1 cell-derived exosomes carrying the influenza virus-derived H1N1 mRNA prepared in Example 5 or 10ug of bacterial-derived outer membrane vesicles (Bacteria OMV) carrying influenza virus-derived H1N1 mRNA to B6C3F1 / Slc mice (female) by subcutaneous injection. Three weeks later, an equal amount of extracellular vesicles was subcutaneously injected. Five weeks after the first administration, that is, 35 days later, the mice were killed to obtain serum samples. Then, the serum samples were diluted in PBS containing 1% BSA at a ratio of 1:100 to 1:1000, and the binding degree of specific antibodies against influenza virus-derived H1N1 protein in the serum was analyzed by measuring the absorbance ( Figure 5 ).

[0207] The results confirmed that Figure 6 As shown, mice administered with activated THP-1 cell-derived exosomes and bacteria-derived OMVs loaded with influenza virus-derived H1N1 mRNA both showed increased levels of antibody binding (IgG) against influenza virus H1N1 protein in the serum compared with the control group.

[0208] (2) Extracellular vesicles carrying varicella-zoster virus-derived glycoprotein E mRNA

[0209] The present inventors administered 10ug of activated THP-1 cell-derived exosomes carrying varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 5 or 10ug of bacterial-derived outer membrane vesicles (Bacteria OMVs) carrying varicella-zoster virus-derived glycoprotein E mRNA to B6C3F1 / Slc mice (female) by subcutaneous injection. Three weeks later, an equal amount of extracellular vesicles was subcutaneously injected. Five weeks after the first administration, that is, 35 days later, the mice were killed to obtain serum samples. Then, the serum samples were diluted in PBS containing 1% BSA at a ratio of 1:100 to 1:1000, and the binding degree (antibody binding) of specific antibodies against varicella-zoster virus-derived glycoprotein E in the serum was analyzed by measuring the absorbance ( Figure 5 ).

[0210] The results confirmed that Figure 7 As shown, compared with the control group, mice administered with activated THP-1 cell-derived exosomes and bacteria-derived OMVs loaded with varicella-zoster virus-derived glycoprotein E mRNA showed an increased degree of binding of specific antibodies (IgG) against varicella-zoster virus-derived glycoprotein E in the serum.

[0211] (3) Extracellular vesicles carrying RSV-F mRNA from respiratory syncytial virus

[0212] The inventors administered 10ug of activated THP-1 cell-derived exosomes carrying RSV-F mRNA from respiratory syncytial virus prepared in Example 5 or 10ug of bacterial-derived outer membrane vesicles (Bacteria OMV) carrying RSV-F mRNA from respiratory syncytial virus to B6C3F1 / Slc mice (female) by subcutaneous injection, and obtained serum samples 2 weeks after the first administration, i.e. 14 days later. Then, the serum samples were diluted in PBS containing 1% BSA at a ratio of 1:100 to 1:1000, and the binding degree of specific antibodies to RSV-F protein from respiratory syncytial virus in serum was analyzed by measuring the absorbance.

[0213] The results confirmed that Figure 8 As shown, compared with the control group, mice administered with activated THP-1 cell-derived exosomes and bacteria-derived OMVs loaded with RSV-F mRNA from respiratory syncytial virus both showed an increased degree of binding of specific antibodies (IgG) against RSV-F protein from respiratory syncytial virus in the serum.

[0214] 7-2. Analysis of specific T cell responses to extracellular vesicles carrying mRNA of viral-derived proteins

[0215] (1) Extracellular vesicles carrying influenza virus-derived H1N1 mRNA

[0216] The present inventors administered 10ug of activated THP-1 cell-derived exosomes carrying the influenza virus-derived H1N1 mRNA prepared in Example 5 or 10ug of bacterial-derived outer membrane vesicles (Bacteria OMVs) carrying influenza virus-derived H1N1 mRNA to B6C3F1 / Slc mice (female) by subcutaneous injection. Three weeks later, the same amount of extracellular vesicles were subcutaneously injected. Five weeks after the first administration, i.e., 35 days later, the mice were killed and spleen cells were isolated. Then, the spleen cells (1×10 7 / ml) for 48 hours, and then ELISA was performed using the culture fluid of the spleen cells to measure the level of IFN-γ protein secreted from Th1 cells.

[0217] The results confirmed that Fig. 9 As shown, IFN-γ protein was not detected in the culture medium of the control group, while IFN-γ was present in the culture medium of spleen cells of mice administered with activated THP-1 cell-derived exosomes and bacteria-derived OMVs loaded with influenza virus-derived H1N1 mRNA.

[0218] The above results indicate that, while the Th1 cell-mediated immune response was not induced in the control group, a stronger Th1 cell-mediated immune response was induced by the administration of the extracellular vesicles according to the present invention.

[0219] (2) Extracellular vesicles carrying varicella-zoster virus-derived glycoprotein E mRNA

[0220] The present inventors administered 10ug of activated THP-1 cell-derived exosomes carrying varicella-zoster virus-derived glycoprotein E mRNA prepared in Example 5 or 10ug of bacterial-derived outer membrane vesicles (Bacteria OMVs) carrying varicella-zoster virus-derived glycoprotein E mRNA to B6C3F1 / Slc mice (female) by subcutaneous injection. Three weeks later, an equal amount of extracellular vesicles was subcutaneously injected. Five weeks after the first administration, i.e., 35 days later, the mice were killed and spleen cells (splenocytes) were isolated. Then, the spleen cells (1×10 7 / ml) for 48 hours, and then ELISA was performed using the culture fluid of the spleen cells to measure the level of IFN-γ protein secreted from Th1 cells.

[0221] The results confirmed that Fig.10 As shown, IFN-γ protein was not detected in the culture medium of the control group, while IFN-γ was present in the culture medium of spleen cells of mice administered with activated THP-1 cell-derived exosomes and bacteria-derived OMVs loaded with varicella-zoster virus-derived glycoprotein E mRNA.

[0222] The above results indicate that, while the Th1 cell-mediated immune response was not induced in the control group, a stronger Th1 cell-mediated immune response was induced by the administration of the extracellular vesicles according to the present invention.

[0223] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will appreciate that the present invention can be easily modified into other specific forms without changing the technical ideas or essential features of the present invention. Therefore, the above embodiments should be understood in all aspects as illustrative rather than restrictive.

Claims

An extracellular vesicle comprising: an antigen protein or a gene encoding the protein.

2. The extracellular vesicle according to claim 1, wherein The antigen protein is derived from one or more selected from viruses, microorganisms and cancer cells.

3. The extracellular vesicle according to claim 2, wherein The virus is selected from one or more of adenovirus, smallpox virus, polio virus, measles virus, hepatitis C virus, human immunodeficiency virus-1, hepatitis B virus, influenza virus, respiratory syncytial virus, herpes simplex virus, human papillomavirus, Zika virus, varicella-zoster virus and fever with thrombocytopenia syndrome virus.

4. The extracellular vesicle according to claim 1, wherein The antigen protein is selected from one or more of p24 protein derived from human immunodeficiency virus-1, s protein derived from hepatitis B virus, H1N1 protein derived from influenza virus, RSV-F protein derived from respiratory syncytial virus, and glycoprotein E derived from varicella-zoster virus.

5. The extracellular vesicle according to claim 2, wherein The microorganism is selected from one or more microorganisms of the genera Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ayerococcus, Candida, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Lepidoptera, Absidia, Bottle Mould, Entomophthora, Otoderma, Frog Fecal Mould, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Trichosporon and Mucorales.

6. The extracellular vesicle according to claim 2, wherein The cancer cells are derived from one or more of the following cancers: head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma.

7. The extracellular vesicle according to claim 1, wherein The antigen protein is selected from one or more of MAGE1 / 2 / 3, WT1, CDK4, MUC-1, HER2, PSA, E6 / E7 of HPV16 and HCV.

8. The extracellular vesicle according to claim 1, wherein The antigen protein or the gene encoding the protein is loaded into the extracellular vesicles.

9. The extracellular vesicle according to claim 1, wherein The extracellular vesicles are derived from one or more selected from animal cells, plant cells or microorganisms.

10. The extracellular vesicle according to claim 9, wherein The animal cells are one or more cells selected from somatic cells, germ cells, immune cells, neural cells and tumor cells.

11. The extracellular vesicle according to claim 9, wherein When the extracellular vesicles are derived from animal cells, the extracellular vesicles express one or more proteins selected from CD9, CD63, CD81, Alix, TSG101, endocytin 1 and raftin 1.

12. The extracellular vesicle according to claim 9, wherein When the extracellular vesicles are derived from plant cells, the extracellular vesicles express one or more proteins selected from syntaxin (PEN1), tetraspanin 8 (Tet8) and heat shock protein (HSP).

13. The extracellular vesicle according to claim 9, wherein When the extracellular vesicles are derived from a microorganism, the extracellular vesicles express one or more proteins selected from OmpA, flagellin, HSP70 and β-glucan.

14. A vaccine composition for preventing or treating viral infections, comprising: extracellular vesicles containing virus-derived antigenic proteins or genes encoding the proteins.

15. A vaccine composition for preventing or treating microbial infections, comprising: extracellular vesicles containing microbial-derived antigenic proteins or genes encoding the proteins.

16. A vaccine composition for preventing or treating cancer, comprising: extracellular vesicles containing cancer cell-derived antigenic proteins or genes encoding said proteins.

17. The vaccine composition according to any one of claims 14 to 16, wherein The vaccine composition simultaneously induces antigen-specific antibody production and T cell-mediated immune responses.

18. A health functional food for preventing or improving viral infections, comprising: extracellular vesicles containing virus-derived antigenic proteins or genes encoding the proteins.

19. A health functional food for preventing or improving microbial infections, comprising: extracellular vesicles containing microbial-derived antigenic proteins or genes encoding the proteins.

20. A health functional food for preventing or improving cancer, comprising: extracellular vesicles containing cancer cell-derived antigen proteins or genes encoding the proteins.

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