Extracellular vesicles having an antigen protein or a gene encoding the protein and uses thereof

Abstract

The present invention relates to extracellular vesicles having an antigen protein or a gene encoding the protein and uses thereof, and more particularly, to extracellular vesicles having an antigen protein derived from a virus, microorganism, cancer cell or a gene encoding the protein, or a vaccine composition for preventing or treating a viral infection, a microbial infection or cancer containing the same. The extracellular vesicles according to the present invention or the vaccine composition containing the same have an excellent effect of inducing an antigen-specific immune response and stability, and as a platform applicable to various diseases, it is expected to be usefully utilized in the field of vaccine development for preventing or treating various diseases including viral infections, microbial infections or cancer.

Description

Technical Field

[0001] The present invention relates to extracellular vesicles having an antigen protein or a gene encoding the protein and uses thereof.

Background Art

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

[0003] Extracellular vesicles are nanovesicles with a size of several tens to several hundreds of nm composed of a double lipid membrane, and are composed of substances having bioactive substances such as proteins, lipids, and genes. Representative examples of these extracellular vesicles include exosomes. In addition, extracellular vesicles derived from microorganisms such as ectosomes, microvesicles, bacterial outer membrane vesicles (Bacterial OMV), extracellular vesicles derived from yeast, or apoptotic bodies are included. Conventionally, these extracellular vesicles were regarded as residues secreted from cells, but in recent years, as the clinical significance of extracellular vesicles has emerged, various studies have been conducted. In particular, exosomes, which are spherical vesicles excreted by cells, have various information such as proteins and DNA of the mother cell, and the development of cancer diagnostic markers and sensors using this as a biomarker has been actively carried out.

[0004] On the one hand, exosomes are small vesicles with a membrane structure (approximately 30 - 100 nm in diameter) secreted from various cells. In studies using electron microscopy, they are not directly isolated from the plasma membrane but are observed to be derived from specific intracellular compartments called multivesicular bodies (MVBs) and released and secreted outside the cell. That is, when the fusion of multivesicular bodies and the plasma membrane occurs, the vesicles are released into the extracellular environment, and these are called exosomes. Such exosomes are known to be produced and secreted by various immune cells including red blood cells, B lymphocytes, T lymphocytes, dendritic cells, platelets, macrophages, as well as tumor cells and stem cells while they are alive.

[0005] Exosomes derived from mammalian cells are known to be involved in various physiological functions such as hemostasis, tissue regeneration, maintenance of stem cells, regulation of inflammatory and immune responses, and embryonic development. In particular, exosomes derived from immune cells have been reported to promote immune responses by directly or indirectly transmitting inflammatory cytokines and presenting antigens (Nat Rev Immunol. 2009 Aug;9(8):581 - 93). Based on such characteristics and functions in vivo of exosomes, exosomes are being researched and developed for uses such as nano - sized substance transport means, mediators, and biomarkers in the fields of oncology, immunotherapy, and regenerative medicine.

[0006] As described above, research is underway to use extracellular vesicles including exosomes or extracellular vesicles derived from microorganisms for vaccines, but it is still limited to some diseases. The research and development of technologies for vaccine platforms applicable to various diseases, including viral and microbial infections and cancer, is insufficient. - form is the actual situation.

Summary of the Invention

Problems to be Solved by the Invention

[0007] As a result of intensive research to develop an extracellular vesicle-based vaccine platform that can be widely applied to various diseases, the present inventors have found that extracellular vesicles act as powerful immunostimulants and carriers of antigens, inducing the production of antigen-specific antibodies and antigen-specific T cell responses, and thus have completed the present invention.

[0008] Therefore, an object of the present invention is to provide extracellular vesicles having an antigen protein or a gene encoding the protein.

[0009] Another object of the present invention is to provide a vaccine composition for preventing or treating viral infections, which contains extracellular vesicles having a virus-derived antigen protein or a gene encoding the protein.

[0010] Another object of the present invention is to provide a vaccine composition for preventing or treating microbial infections, which contains extracellular vesicles having a microbe-derived antigen protein or a gene encoding the protein.

[0011] Another object of the present invention is to provide a vaccine composition for preventing or treating cancer, which contains extracellular vesicles having a cancer cell-derived antigen protein or a gene encoding the protein.

[0012] Another object of the present invention is to provide a health functional food for preventing or improving viral infections, which contains extracellular vesicles having a virus-derived antigen protein or a gene encoding the protein.

[0013] Another object of the present invention is to provide a health functional food for preventing or improving microbial infections, which contains extracellular vesicles having a microbe-derived antigen protein or a gene encoding the protein.

[0014] Furthermore, another object of the present invention is to provide a health functional food for preventing or improving cancer, which contains extracellular vesicles having an antigen protein derived from cancer cells or a gene encoding the protein.

[0015] Furthermore, a further object of the present invention is to provide a method for preventing or treating a viral infection disease, which includes a step of administering extracellular vesicles having a viral antigen protein or a gene encoding the protein to an individual in need thereof.

[0016] Furthermore, another object of the present invention is to provide a method for preventing or treating a microbial infectious disease, which includes a step of administering extracellular vesicles having a microbial antigen protein or a gene encoding the protein to an individual in need thereof.

[0017] In addition, another object of the present invention is to provide a method for preventing or treating cancer, which includes a step of administering extracellular vesicles having a cancer cell-derived antigen protein or a gene encoding the protein to an individual in need thereof.

[0018] Furthermore, another object of the present invention is to provide the use of extracellular vesicles having a viral antigen protein or a gene encoding the protein for the manufacture of a preventive or therapeutic agent for viral infectious diseases. Another object of the present invention is to provide the use of extracellular vesicles having a microbial antigen protein or a gene encoding the protein for the manufacture of a preventive or therapeutic agent for microbial infectious diseases.

[0019] Furthermore, another object of the present invention is to provide the use of extracellular vesicles having a cancer cell-derived antigen protein or a gene encoding the protein for the manufacture of a preventive or therapeutic agent for cancer.

[0020] Furthermore, another object of the present invention is to provide the use of extracellular vesicles having a cancer cell-derived antigen protein or a gene encoding the protein for the manufacture of a preventive or therapeutic agent for cancer.

[0021] However, the technical problems to be solved by the present invention are not limited to the problems described above, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

Means for Solving the Problems

[0022] In order to achieve the object of the present invention as described above, the present invention provides extracellular vesicles having an antigen protein or a gene encoding the protein.

[0023] In one embodiment of the present invention, the antigen protein can be derived from one or more selected from the group consisting of viruses, microorganisms, and cancer cells.

[0024] In another embodiment of the present invention, the virus can be one or more selected from the group consisting of adenovirus, smallpox virus, poliovirus, measles virus, hepatitis C virus, human immunodeficiency virus type 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.

[0025] In another embodiment of the present invention, the antigen protein may be one or more selected from the group consisting of p24 protein derived from human immunodeficiency virus type 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 (VZE) derived from varicella-zoster virus.

[0026] In another embodiment of the present invention, the microorganism is Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida, Coccidioides, Cryptococc One or more microorganisms selected from the group consisting of Aspergillus, Candida, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and Mucorales.

[0027] In another embodiment of the present invention, the cancer cells may be derived from one or more cancers selected from the group consisting of head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma.

[0028] In another embodiment of the present invention, the antigen protein may be one or more selected from the group consisting of MAGE1 / 2 / 3, WT1, CDK4, MUC-1, HER2, PSA, HPV16, and E6 / E7 of HCV.

[0029] In yet another embodiment of the present invention, the antigen protein or the gene encoding the protein may be carried within the extracellular vesicles.

[0030] In another embodiment of the present invention, the extracellular vesicles can be derived from one or more cells selected from the group consisting of animal cells, plant cells, or microorganisms.

[0031] In another embodiment of the present invention, the animal cells may be one or more cells selected from the group consisting of somatic cells, germ cells, immune cells, nerve cells, and tumor cells.

[0032] In still another embodiment of the present invention, the extracellular vesicles may be derived from activated immune cells or the outer membrane of bacteria.

[0033] In another embodiment of the present invention, when the extracellular vesicles are derived from animal cells, the extracellular vesicles can express one or more proteins selected from the group consisting of CD9, CD63, CD81, Alix, TSG101, syntenin 1, and flotillin 1.

[0034] In another embodiment of the present invention, when the extracellular vesicles are derived from plant cells, the extracellular vesicles can express one or more proteins selected from the group consisting of syntaxin (PEN1), tetraspanin 8 (Tet8), and heat shock protein (HSP).

[0035] In another embodiment of the present invention, when the extracellular vesicles are derived from microorganisms, the extracellular vesicles may express one or more proteins selected from the group consisting of OmpA, flagellin, HSP70, and β-glucan.

[0036] Also, in one embodiment of the present invention, there is provided a vaccine composition for preventing or treating viral infections, which contains extracellular vesicles having a viral antigen protein or a gene encoding the protein.

[0037] In other embodiments of the present invention, the viral infection is an adenovirus infection, natural poxvirus infection, poliovirus infection, measles virus infection, hepatitis C virus infection, human immunodeficiency virus type 1 (Human It can be one or more selected from the group consisting of infections with 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.

[0038] In an embodiment of the present invention, there is provided a vaccine composition for preventing or treating microbial infections, which contains an antigen protein derived from a microorganism or extracellular vesicles having a gene encoding the protein.

[0039] In another embodiment of the present invention, the microbial infection is caused by one or more microorganisms selected from the group consisting of Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylocococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and Mucorales.

[0040] In one embodiment of the present invention, there is also provided a vaccine composition for preventing or treating cancer, which contains an extracellular vesicle having an antigen protein derived from cancer cells or a gene encoding the protein.

[0041] In other embodiments of the present invention, the cancer may be one or more selected from the group consisting of head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma.

[0042] In still other embodiments of the present invention, the vaccine composition may simultaneously induce the production of antigen-specific antibodies and a T cell-mediated immune response. In another embodiment of the present invention, the vaccine composition may be lyophilized and may be.

[0043] Also, in one embodiment of the present invention, there is provided a health functional food for preventing or improving a viral infectious disease, which contains an antigen protein derived from a virus or an extracellular vesicle having a gene encoding the protein.

[0044] Also, in one embodiment of the present invention, there is provided a health functional food for preventing or improving a microbial infectious disease, which contains an antigen protein derived from a microorganism or an extracellular vesicle having a gene encoding the protein.

[0045] Also, in one embodiment of the present invention, there is provided a health functional food for preventing or improving cancer, which contains an antigen protein derived from cancer cells or an extracellular vesicle having a gene encoding the protein.

[0046] Furthermore, in one embodiment of the present invention, there is provided a method for preventing or treating a viral infectious disease, which includes the step of administering an antigen protein derived from a virus or an extracellular vesicle having a gene encoding the protein to an individual in need thereof.

[0047] Also, in one embodiment of the present invention, there is provided a method for preventing or treating a microbial infectious disease, which includes the step of administering an antigen protein derived from a microorganism or an extracellular vesicle having a gene encoding the protein to an individual in need thereof.

[0048] In addition, in one embodiment of the present invention, there is provided a method for preventing or treating cancer, which includes administering extracellular vesicles having an antigen protein derived from cancer cells or a gene encoding the protein to an individual in need thereof.

[0049] In addition, in one embodiment of the present invention, there is provided the use of extracellular vesicles having an antigen protein derived from a virus or a gene encoding the protein for the manufacture of a prophylactic or therapeutic agent for viral infectious diseases.

[0050] In addition, in one embodiment of the present invention, there is provided the use of extracellular vesicles having an antigen protein derived from a microorganism or a gene encoding the protein for the manufacture of a prophylactic or therapeutic agent for microbial infectious diseases.

[0051] In addition, in one embodiment of the present invention, there is provided the use of extracellular vesicles having an antigen protein derived from cancer cells or a gene encoding the protein for the manufacture of a prophylactic or therapeutic agent for cancer.

Advantages of the Invention

[0052] In the present invention, as a result of immunizing mice by administering activated immune cells loaded with antigen proteins or mRNA or extracellular vesicles derived from the outer membrane of bacteria, it was experimentally confirmed that excellent antigen-specific antibodies were produced and a T cell response was induced. Thereby, the extracellular vesicles according to the present invention or a vaccine composition containing the same have an excellent effect of inducing an antigen-specific immune response and stability, and as a platform applicable to various diseases, it is expected to be usefully utilized in the field of vaccine development for the prevention or treatment of various diseases including viral infections, microbial infections or cancer.

Brief Description of the Drawings

[0053]

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BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] (BEST MODE FOR CARRYING OUT THE INVENTION) The present invention relates to a vaccine platform technology that can be usefully applied in the field of vaccine development for the prevention and treatment of various infectious diseases and cancers using extracellular vesicles that act as powerful immunostimulants and carriers of antigens.

[0055] Hereinafter, the present invention will be described in detail.

[0056] The present invention provides extracellular vesicles having an antigen protein or a gene encoding the protein.

[0057] As used herein, the term "antigen" means a molecule capable of inducing an immune response for producing an antibody within a host individual. An antigen is a target of an antibody, and each antibody is produced antigen-specifically in response thereto after cells of the immune system come into contact with the antigen. In the present invention, the antigen may include any of a protein, a gene encoding the protein, and an mRNA of the gene, as long as it can induce the immune response as described above, and may include a fragment of the protein or mRNA. The origin of the antigen is not particularly defined, and non-limiting examples include those derived from a virus, a microorganism such as bacteria (bacteria), archaea, fungi, and those derived from cancer cells.

[0058] That is, in the present invention, the antigen protein may be derived from one or more selected from the group consisting of a virus, a microorganism, and a cancer cell.

[0059] The virus is an adenovirus, a smallpox virus, a poliovirus, a measles virus, a hepatitis C virus, a human immunodeficiency virus type 1 It can be one or more selected from the group consisting of type (Human Immunodeficiency Virus-1: HIV-1), hepatitis B virus (Hepatitis B virus: HBV), influenza virus, respiratory syncytial virus, herpes simplex virus, human papillomavirus, Zika virus, varicella-zoster virus, and severe fever with thrombocytopenia syndrome virus. Specifically, it can be one or more selected from the group consisting of influenza virus, respiratory syncytial virus, herpes simplex virus, human papillomavirus, Zika virus, varicella-zoster virus, and severe fever with thrombocytopenia syndrome virus. More specifically, it can be one or more selected from the group consisting of influenza virus, varicella-zoster virus, and respiratory syncytial virus.

[0060] In the present invention, when the antigen protein is derived from human immunodeficiency virus, the antigen protein may be the p24 protein derived from human immunodeficiency virus-1.

[0061] In the present invention, when the antigen protein is derived from hepatitis B virus, the antigen protein may be the s protein derived from hepatitis B virus.

[0062] In the present invention, when the antigen protein is derived from an influenza virus, the antigen protein may be one or more selected from the group consisting of 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 H1N1 protein, and more specifically, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0063] The polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 may include polypeptides having 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, respectively. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 may include the amino acid sequence of SEQ ID NO: 1 and its functional variants. The functional variant means all similar sequences in which several amino acid substitutions occur at the amino acid positions without affecting the biological properties of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0064] In the present invention, when the antigen protein is derived from a respiratory syncytial virus, the antigen protein may be RSV-F protein derived from a respiratory syncytial virus, and specifically, it may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0065] Also, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 may include polypeptides having 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 consisting of the amino acid sequence of SEQ ID NO: 2 may include the amino acid sequence of SEQ ID NO: 2 and its functional variants. The polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 may include the amino acid sequence of SEQ ID NO: 2 and its functional variants.

[0066] In the present invention, when the antigen protein is derived from varicella-zoster virus, the antigen protein may be glycoprotein E derived from varicella-zoster virus, and specifically, it may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

[0067] In addition, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 can include polypeptides having 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 include the amino acid sequence of SEQ ID NO: 3 and its functional variants.

[0068] The term "homology" is for indicating the similarity with the 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 (%).

[0069] In the present invention, the microorganisms include both prokaryotic microorganisms and eukaryotic microorganisms. The prokaryotic microorganisms include eubacteria (bacteria) and archaebacteria, and the eukaryotic microorganisms include fungi, algae, and protozoa. Further, the eubacteria (bacteria) include both gram-positive bacteria and gram-negative bacteria, and the fungi include both molds and yeasts. Specifically, the microorganisms can be one or more genera of microorganisms 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, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and Mucorales.

[0070] In addition, in the present invention, the cancer cells are acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymph angioendotheliosarcoma, hemangiosarcoma); appendix cancer, monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma); bladder cancer, breast cancer (e.g., thymoma, papillary carcinoma, mammary cancer, medulla ry carcinoma), brain cancer (e.g., meningioma, glioblastoma, neuroglioma (e.g., astrocytoma, o ligodendroglioma)), 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 sarco ma), multiple idiopathic hemorrhagic sarcoma; endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer, uterine sarcoma, esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma), familial hypereosinophilia, gallbladder cancer; gastric 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 (head and neck squamouscarcarcinoma)), 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; ain disease);

[0071] Hypopharynx cancer; Inflammatory myofibroblastic tumors; Immunocytic 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, lung adenocarcinoma); Leiomyosarcoma: LMS; Mastocytosis (e.g., systemic mastocytosis); Myosarcoma; Myelodysplastic syndrome: MDS ; Mesothelioma; Myeloproliferative disorder: MPD (e.g., polycythemia Vera: PV, essential thrombocytosis: ET, myelofibrosis: MF, also known as agnogenic myeloid metaplasia: AMM, chronic idiopathic myelofibrosis, chronic myelogenous leukemia ); 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., gastroenteropanc reatic neuroendoctrine tumor: GEP-NET), carcinoid tumor)); osteosarcoma (e.g., bone cancer)); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma)); 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)); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia;

[0072] 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 rous 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 cancer; urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva). It may be derived from one or more cancers selected from the group consisting of, specifically, 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, endometrial cancer, kidney cancer, testicular cancer, glioblastoma, thyroid cancer, skin cancer, and lymphoma. More specifically, it may be derived from one or more cancers selected from the group consisting of head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma.

[0073] In the present invention, when the antigen protein is derived from cancer cells, the antigen protein may be one or more selected from the group consisting of MAGE1 / 2 / 3, WT1, CDK4, MUC-1, HER2, PSA, HPV16, and E6 / E7 of HCV.

[0074] In the present invention, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, titer, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activities), and decreased or enhanced antigenicity, it may be bound to the N-terminus or C-terminus of the antigen protein. The protecting group may be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristoyl group, a stearyl group, or polyethylene glycol (PEG), but it can be included without limitation as long as it is a component that can promote the modification of the antigen protein, particularly the stability of the antigen protein.

[0075] The term "stability" may mean not only in vivo stability that protects the antigen protein from the attack of proteolytic enzymes in the living body, but also storage stability (for example, room temperature storage stability).

[0076] In the present invention, the antigen protein may be of natural origin, and can also be obtained by various polypeptide synthesis methods widely known in the art. For example, it can be prepared using a gene recombination and protein expression system, or synthesized in vitro by chemical synthesis such as protein synthesis (for example, liquid phase or solid phase synthesis, condensation of fragments, F-MOC or T-BOC chemistry) and cell-free protein synthesis methods. Also, as an example, the antigen protein may be a product obtained by culturing peptides, extracts of plant-derived tissues or cells, or microorganisms (for example, bacteria or fungi, especially yeast).

[0077] In the present invention, when the antigen protein is derived from the human immunodeficiency virus the gene encoding the antigen protein may be a gene encoding the p24 protein derived from human immunodeficiency virus type 1.

[0078] In the present invention, when the antigen protein is derived from the hepatitis B virus, the gene encoding the antigen protein may be a gene encoding the s protein derived from the hepatitis B virus.

[0079] 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 the group consisting of 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.

[0080] 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, it may be a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0081] In the present invention, when the antigen protein is derived from varicella-zoster virus, the gene encoding the antigen protein may be a gene encoding glycoprotein E derived from varicella-zoster virus. Specifically, it may be a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

[0082] In the present invention, the gene encoding the antigen protein derived from cancer cells 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.

[0083] In the present invention, the gene encoding the antigen protein may be, for example, DNA, RNA, or a combination or modification thereof, and may be double-stranded or single-stranded. Further, the gene encoding the antigen protein may encode an amino acid sequence linked to the N-terminus or C-terminus of the antigen protein in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, titer, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activities), and decreased or enhanced antigenicity, and the protecting group can be included without limitation as long as it is a component that can promote stability in particular.

[0084] In the present invention, the antigen protein or the gene encoding the protein may also be carried on the extracellular vesicles.

[0085] The method of loading the antigen protein or the gene encoding the protein onto the extracellular vesicles is not particularly limited, and those skilled in the art can appropriately select and use known methods in the art. For example, the antigen protein can be loaded onto the extracellular vesicles after treating the extracellular vesicles with extracorporeal shock waves.

[0086] The term "extracellular vesicles" used in the present invention means small spheres surrounded by a membrane derived from cells, and such spheres are very diverse depending on the origin and production method of the cells from which they are named. In the present invention, it may include one or more selected from the group consisting of extracellular vesicles derived from microorganisms including exosomes, ectosomes, microvesicles, bacterial outer membrane vesicles (Bacterial OMV), extracellular vesicles derived from yeast, etc. named according to the production method of cells, and apoptotic bodies. Specifically, it may be an exosome or an outer membrane vesicle derived from bacteria, but is not limited thereto.

[0087] The extracellular vesicles may be extracellular vesicles derived from one or more selected from nature, for example, animals, plants, and microorganisms, or artificially produced extracellular vesicles. Further, the cells may be cells isolated from living organisms in nature. Furthermore, the cells may be derived from any animal or plant, including humans and non-human mammals. The animal cells may be one or more cells selected from the group consisting of somatic cells, germ cells, immune cells, nerve cells, and tumor cells. Specifically, they may be immune cells, and more specifically, activated immune cells.

[0088] In the present invention, when the extracellular vesicles are derived from microorganisms, the microorganisms may be bacteria, and the extracellular vesicles may be extracellular vesicles.

[0089] In the present invention, the animal cells may be one or more cells selected from the group consisting of Vero, MDCK, MRC-5, serum-free Hi-5, Hi-5, WI-38, PER.C6, HEK-293, and CEF. The somatic cells are cells that constitute the body of any animal, including mammals including humans. In the present invention, they may include all remaining types of cells excluding germ cells, immune cells, nerve cells, and tumor cells.

[0090] The immune cells are cells that determine the immune response in the body, defend against pathogens, foreign substances, viruses, etc. that invade from the outside so as to be able to resist and overcome them, regulate immunity, and play a role in eliminating cancer cells that are constantly produced in the body. Specifically, they can include, but are not limited to, natural killer cells (NK cells), dendritic cells, T cells, B cells, macrophages, and lymphocytes.

[0091] The above-mentioned neuronal cells are cells that constitute the nervous system. By expressing ion channels such as sodium channels and potassium channels, they can transmit signals electrically, which is different from other cells. Through a structure called synapses, they exchange chemical signals with adjacent other neuronal cells to exchange various information. In the present invention, the neuronal cells can include any cells as long as their types are not specified and they secrete extracellular vesicles.

[0092] The above-mentioned tumor cells are used in the same sense as cancer cells and refer to cells that, unlike normal cells, have an unregulated cell cycle and grow rapidly and irregularly. In the present invention, the origin tissues of the above-mentioned tumor cells are not specifically limited, and any tumor cells that secrete extracellular vesicles can be included.

[0093] In the present invention, when the above-mentioned extracellular vesicles are derived from animal cells, the extracellular vesicles can express one or more proteins selected from the group consisting of CD9, CD63, CD81, Alix, TSG101, syntenin 1, and flotillin 1 can.

[0094] Also, in the present invention, when the above-mentioned extracellular vesicles are derived from plant cells, the extracellular vesicles may express one or more proteins selected from the group consisting of syntaxin (PEN1), tetraspanin 8 (Tet8), and heat shock protein (HSP).

[0095] In the present invention, when the extracellular vesicles are derived from microorganisms, the extracellular vesicles may express one or more proteins selected from the group consisting of OmpA, flagellin, HSP70, and β-glucan. Specifically, when the microorganism is a bacterium, the extracellular vesicles may express one or more proteins selected from the group consisting of OmpA and flagellin. When the microorganism is a fungus, the extracellular vesicles may express one or more proteins selected from the group consisting of HSP70 and β-glucan.

[0096] Furthermore, the extracellular vesicles may be derived from activated immune cells. Specifically, they may be derived from activated THP-1 cells or KG-1 cells. At this time, the activation can be carried out by lipopolysaccharide (LPS) or α-galactosylceramide (α-GC). When the THP-1 cells are activated, the cell morphology of the THP-1 cells changes to be similar to that of macrophages, and at the same time, they may express one or more proteins selected from the group consisting of IL-1b, IL-6, IL-8, TNF-α, and iNOS. Specifically, they can express all of IL-1b, IL-6, IL-8, TNF-α, and iNOS.

[0097] In the present invention, in a specific embodiment, as an example, the change in the phenotype of immune cells activated by antigen stimulation was analyzed, the characteristics of exosomes isolated therefrom were observed, outer membrane vesicles isolated from attenuated bacteria were separated, and their characteristics were observed.

[0098] Specifically, in one embodiment of the present invention, as a result of treating THP-1 cells, a human mononuclear cell line, with lipopolysaccharides (LPS), it was confirmed that the cell morphology changed and markers of M1 macrophages were expressed when compared with cells not treated with LPS (see Example 1).

[0099] In another embodiment of the present invention, the exosomes derived from the activated immune cells, preferably the exosomes derived from activated THP-1 cells, have an average diameter of 128.0 nm and are confirmed to express exosomal marker proteins of CD63, CD81, and flotillin 1 (see Example 2).

[0100] In still another embodiment of the present invention, the supernatant of the culture of the attenuated BL21 strain was collected by centrifugation, passed through a filter, and then concentrated and washed to prepare outer membrane vesicles (OMVs). As a result of analyzing the characteristics of the outer membrane vesicles by NTA, 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 particles / ml. When the protein concentration was quantitatively analyzed by the BCA assay, it was confirmed to be 2.2 mg / ml (see Example 3).

[0101] In another embodiment of the present invention, exosomes derived from activated THP-1 cells loaded with H1N1 protein derived from influenza virus or RSV-F protein derived from respiratory syncytial virus were prepared, subcutaneously administered to mice for immunization, and then analyzed for whether antibodies were produced. As a result, compared with the case where an adjuvant and the H1N1 protein or RSV-F protein were administered together, it was confirmed that a much smaller amount showed an equivalent or higher level of formation of H1N1 or RSV-F-specific IgG (see Examples 4 and 6).

[0102] In a further embodiment of the present invention, exosomes derived from activated THP-1 cells loaded with H1N1 mRNA derived from influenza virus, RSV-F mRNA derived from respiratory syncytial virus, or glycoprotein E mRNA derived from varicella-zoster virus, or outer membrane vesicles derived from bacteria loaded with H1N1 mRNA derived from influenza virus, RSV-F mRNA derived from respiratory syncytial virus, or glycoprotein E mRNA derived from varicella-zoster virus were prepared (see Example 5).

[0103] Another embodiment of the present invention is H1N1 derived from influenza virus prepared in Example 5 After immunizing mice by subcutaneous administration of exosomes derived from activated THP-1 cells or outer membrane vesicles derived from bacteria loaded with mRNA of RSV-F derived from respiratory syncytial virus, mRNA of H1N1 derived from influenza virus prepared in Example 4, or glycoprotein E mRNA derived from varicella-zoster virus, and analyzing whether antibodies were produced, it was confirmed that both exosomes derived from activated THP-1 cells and outer membrane vesicles derived from bacteria showed a superior degree of RSV-F or VZE-specific IgG formation compared to the control group (see Example 7-1).

[0104] Another embodiment of the present invention is H1N1 derived from influenza virus prepared in Example 4 After immunizing mice by subcutaneous administration of exosomes derived from activated THP-1 cells or outer membrane vesicles derived from bacteria loaded with mRNA of RSV-F derived from respiratory syncytial virus, mRNA of H1N1 derived from influenza virus prepared in Example 4, or glycoprotein E mRNA derived from varicella-zoster virus, 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. As a result, it was confirmed that mice immunized with exosomes derived from activated THP-1 cells or outer membrane vesicles derived from bacteria all induced a strong T cell response compared to the control group (see Example 7-2).

[0105] As a result of immunizing mice by administering extracellular vesicles according to the present invention, it was confirmed that there were excellent effects of producing antigen-specific antibodies and inducing T cell responses. Therefore, the extracellular vesicles according to the present invention can be usefully utilized in the field of vaccine development for the prevention and treatment of various diseases including viral infections, microbial infections, and cancer.

[0106] In the present invention, the viral infectious disease is one or more selected from the group consisting of adenovirus infectious disease, smallpox virus infectious disease, poliovirus infectious disease, measles virus infectious disease, hepatitis C virus (Hepatitis C virus) infectious disease, human immunodeficiency virus type 1 (Human Immunodeficiency Virus-1: HIV-1) infectious disease, hepatitis B virus (Hepatitis B virus: HBV) infectious disease, influenza virus (Influenza virus) infectious disease, respiratory syncytial virus (Respiratory Syncytial Virus) infectious disease, herpes simplex virus (Herpes Simplex virus) infectious disease, human papillomavirus (Human Papilloma virus) infectious disease, Zika virus (Zika virus) infectious disease, varicella-zoster virus (Varicella-Zoster virus) infectious disease, and severe fever with thrombocytopenia syndrome virus (Severe Fever with Thrombocytopenia Syndromevirus) infectious disease, and specifically, it may be one or more selected from the group consisting of human immunodeficiency virus type 1 infectious disease, hepatitis B virus infectious disease, influenza virus infectious disease, respiratory syncytial virus infectious disease, and varicella-zoster virus infectious disease, and more specifically, it may be one or more selected from the group consisting of influenza virus infectious disease, varicella-zoster virus infectious disease, and respiratory syncytial virus infectious disease.

[0107] In another embodiment of the present invention, the microbial infection can be a microbial infection of one or more genera selected from the group consisting of Salmonella, Yersinia, Escherichia, Chlamydia, Xanthomonas, Erwinia, Pseudomonas, Ralstonia, Vibrio, Neisseria, Mycobacterium, Streptococcus, Staphylocococcus, Enterococcus, Lactobacillus, Aspergillus, Blastomyces, Ajellomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and Mucorales.

[0108] In addition, in the present invention, the cancer is 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., thymoma, papillary carcinoma, mammary cancer, medullary carcinoma), brain cancer (for example, meningioma, glioblastoma, neuroglioma (e.g., astrocytoma, oligodendroglioma)); medulloblastoma); bronchus cancer, carcinoid tumor 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 r (e.g., uterine cancer, uterine sarcoma sarcoma)); esophageal cancer, uterine sarcoma, esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer; gastric 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 (head and neck squamouscarcarcinoma)), throat cancer (e.g., laryngeal cancer), pharyngeal cancer,

[0109] nasopharyngeal cancer, oropharyngeal cancer)), heavy chain dise ase) (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic 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, lung adenocarcinoma); leiomyosarcoma: LMS; mastocytosis (e.g., systemic mastocytosis); Myosarcoma; myelodysplastic syndrome: MDS; mesothelioma;Myeloproliferative disorders (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), myelofibrosis (MF), also known as agnogenic myeloid metaplasia (AMM), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma)); s)); neuroendocrine cancer (e.g., gastroenteropancreatic neuroen docrine tumor: GEP-NET), carcinoid tumor)); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma));

[0110] 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); 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;It may be derived from one or more cancers selected from the group consisting of 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 cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva), specifically, it may be derived from one or more cancers selected from the group consisting of 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, endometrial cancer, kidney cancer, testicular cancer, glioblastoma, thyroid cancer, skin cancer, and lymphoma, more specifically, it may be one or more selected from the group consisting of head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, glioblastoma.;

[0111] Furthermore, the present invention provides a vaccine composition for preventing or treating viral infections, which contains an extracellular vesicle having a virus-derived antigen protein or a gene encoding the protein.

[0112] The “virus-derived antigen protein”, “gene encoding the protein”, “extracellular vesicle”, “virus”, “viral infection”, etc. can be within the ranges described above.

[0113] As used herein, the term “prevention” means all acts of suppressing a specific disease or disorder or delaying its onset by administration of the vaccine composition according to the present invention.

[0114] As used herein, the term "treatment" means any act by which the symptoms of a particular disease or disorder are alleviated or favorably changed by administration of the vaccine composition according to the present invention.

[0115] As used herein, the term "vaccine" means to prevent infection or reinfection by the pathogen or antigen, reduce the severity of symptoms or eliminate the symptoms, or actually or completely eliminate the disease caused by the pathogen or antigen by inducing an immune response against the antigen in a host including humans. Therefore, although it is desirable to prophylactically administer the vaccine composition of the present invention to an individual before infection with the pathogen or onset of the disease, it can be therapeutically administered after infection with the pathogen or onset of the disease.

[0116] In the present invention, the vaccine composition may also simultaneously induce the production of antigen-specific antibodies and a T cell-mediated immune response.

[0117] In an embodiment of the present invention, exosomes derived from activated THP-1 cells loaded with H1N1 protein derived from influenza virus or RSV-F protein derived from respiratory syncytial virus were prepared and subcutaneously administered to mice for immunization, and then whether antibodies were produced was analyzed. As a result, it was confirmed that even in a much smaller amount compared to the case where an adjuvant and the H1N1 protein or RSV-F protein were administered together, it showed a degree of formation of H1N1 or RSV-F-specific IgG equal to or higher than that level.

[0118] In another embodiment of the present invention, after exosomes derived from activated THP-1 cells or outer membrane vesicles derived from bacteria loaded with H1N1 mRNA derived from influenza virus, RSV-F mRNA derived from respiratory syncytial virus, or glycoprotein E mRNA derived from varicella-zoster virus prepared in Example 5 were subcutaneously administered to mice for immunization, the results of analyzing whether antibodies were produced showed that both exosomes derived from activated THP-1 cells and outer membrane vesicles derived from bacteria exhibited a superior degree of formation of H1N1, RSV-F, or VZE-specific IgG compared to the control group.

[0119] Another embodiment of the present invention is H1N1 derived from influenza virus prepared in Example 5 mRNA, RSV-F mRNA derived from respiratory syncytial virus, or exosomes derived from activated THP-1 cells or outer membrane vesicles derived from bacteria loaded with glycoprotein E mRNA derived from varicella-zoster virus were subcutaneously administered to mice for immunization. After that, 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. As a result, it was confirmed that mice immunized with exosomes derived from activated THP-1 cells or outer membrane vesicles derived from bacteria all induced a stronger T cell response compared to the control group.

[0120] In the present invention, the vaccine composition may be freeze-dried.

[0121] The present inventors confirmed the induction of specific immune responses and storage stability of the vaccine composition. Specifically, in order to evaluate the storage stability of the extracellular vesicles according to the present invention, freeze-dried exosomes were stored under refrigerated conditions at 4°C for 7 days, stored at room temperature, and then subcutaneously administered to mice for immunization to analyze whether antibodies were produced. As a result, it was confirmed that excellent stability and antibody production effects were maintained after each storage under the above conditions.

[0122] According to the above-described embodiments of the present invention, exosomes derived from activated immune cells show lymph node tropism after subcutaneous injection, are mainly absorbed by local macrophages and dendritic cells, and MHC-I / peptide complexes appear in the exosomes by dendritic cells. From this, it has been found that neutralizing antibodies against viruses are produced more effectively, and H1N1, VZE, or RSV-F specific T cell responses are induced in the spleen cells of immunized mice.

[0123] Therefore, based on the results confirmed in the examples, the present invention strongly suggests that nano-sized extracellular vesicles containing exosomes secreted from cells or outer membrane vesicles derived from bacteria can serve as a powerful immunostimulant and antigen carrier and can be a platform capable of designing an effective vaccine against a target disease.

[0124] The vaccine composition of the present invention can be prepared with any suitable pharmaceutically acceptable formulation. For example, it may be in the form of an immediate administration solution or suspension, a concentrated stock solution suitable for dilution before administration, or a form capable of reconstitution, such as a freeze-dried, lyophilized, or frozen formulation.

[0125] The vaccine composition of the present invention can be formulated by further including a pharmacologically acceptable carrier. Specifically, the carrier may be, for example, a colloidal suspension, powder, physiological saline, lipid, liposome, microspheres, or nanospherical particles. These may form or be associated with transport means and complexes, and may be transported in vivo using transport systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reactants, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0126] Furthermore, the pharmacologically acceptable carrier usually includes diluents, excipients, stabilizers, preservatives, etc. Suitable diluents may be non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or aqueous solvents such as saline (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer). Suitable excipients may be starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc. Suitable stabilizers can be carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamic acid, glucose, or proteins derived from animals, plants or microorganisms such as dried milk, serum albumin, casein. Suitable preservatives include thimerosal, methyl oleate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, etc.

[0127] The vaccine composition of the present invention may further contain an adjuvant for the antigen. The adjuvant for the antigen can consist of one or more substances that enhance the immune response to the antigen. The adjuvant for the antigen is, for example, complete Freund, incomplete Freund, saponin, gel-like aluminum adjuvant, surfactant (For example, it may be one or more selected from the group consisting of lysophosphatidylcholine, pluronic (registered trademark) glycol, multi-anion, peptide, oil, or hydrocarbon emulsion, etc.), vegetable oil (such as cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, squalene, GLA-SE, muramyl dipeptide, lipopolysaccharide (LPS), Quil A, Alum (aluminum salts), Al(OH)3, and α-galactosylceramide (α-GC). Specifically, it may be Al(OH)3.)

[0128] 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 may be prepared as an oral formulation or a parenteral formulation. The parenteral formulation can preferably be administered through any of the administration routes selected from the group consisting of transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural.

[0129] The term "administration" refers to introducing a predetermined substance into an individual in an appropriate manner. The term "individual" refers to all organisms that may carry a viral infection, such as humans, mice, livestock, etc. As a specific example, it may be a mammal including humans. The vaccine composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in the present invention means a sufficient amount to exhibit a vaccine effect and does not cause side effects or serious or excessive immune reactions or excessive immune reactions. The level of the effective dose can vary depending on various factors, including the disorder to be treated, the severity of the disease, the activity of the vaccine substance, the administration route, the rate of protein removal, the duration of treatment, the substances combined with or used simultaneously with the vaccine composition, the age, weight, gender, eating habits, general health status of the individual, and factors known in the medical field.

[0130] Furthermore, the present invention provides a vaccine composition for preventing or treating microbial infections, which comprises an antigen protein derived from a microorganism or extracellular vesicles having a gene encoding the protein.

[0131] The "antigen protein derived from a microorganism", "gene encoding the protein", "extracellular vesicles", "microorganism", "microbial infection", "prevention", "treatment", "vaccine composition", etc. may be those within the aforementioned ranges.

[0132] Furthermore, the present invention provides a vaccine composition for preventing or treating cancer, which comprises an antigen protein derived from cancer cells or extracellular vesicles having a gene encoding the protein.

[0133] The "antigen protein derived from cancer cells", "gene encoding the protein", "extracellular vesicles", "cancer cells", "cancer", "prevention", "treatment", "vaccine composition", etc. may be those within the aforementioned ranges.

[0134] In addition, the present invention provides a health functional food for preventing or improving viral infections, which comprises an antigen protein derived from a virus or extracellular vesicles having a gene encoding the protein.

[0135] The "antigen protein derived from a virus", "gene encoding the protein", "extracellular vesicles", "virus", "viral infection", "prevention", etc. may be those within the aforementioned ranges.

[0136] The term "improvement" can be meant to include all actions related to the parameters associated with the condition being treated, for example, at least reducing the severity. At this time, the health functional food may be used in combination with a therapeutic agent before and after the onset stage of the disease, or may be used alone for preventing or improving viral infections.

[0137] The health functional food of the present invention may further contain an adjuvant of an antigen. The adjuvant of the antigen may be, for example, complete Freund, incomplete Freund, saponin, gel-like aluminum adjuvant, a surfactant (such as lysophosphatidylcholine, poloxamer glycol, multi-anion, peptide, oil, or hydrocarbon emulsion, etc.), vegetable oil (such as cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, squalene, GLA-SE, muramyl dipeptide, lipopolysaccharide (LPS), Quil A, Alum (aluminum salts), Al(OH)3, and α-galactosylceramide (α-GC), and may be one or more selected from the group consisting of them. Specifically, it may be Al(OH)3.

[0138] In the health functional food, the active ingredient may be added to the food as it is, or used together with other foods or food ingredients, and may be appropriately used according to the normal method. The blending amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverage, the health functional food can be added to the raw materials in an amount of specifically about 15% by weight or less, more specifically about 10% by weight. However, when taken for a long time for health or health management purposes, the amount may be below the above range.

[0139] The health functional food comprises one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated in one dosage form selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Examples of foods that can be added to the compound according to one embodiment include various foods, powders, granules, tablets, capsules, syrups, beverages, chewing gums, teas, vitamin complexes, health functional foods, and the like.

[0140] Specific examples of the carrier, excipient, diluent, and additive may include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginic acid, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, molasses, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0141] Except for containing the active ingredient, the health functional food may contain other ingredients as essential ingredients without special restrictions. For example, similar to ordinary beverages, various flavoring agents and natural carbohydrates can be contained as additional ingredients. Examples of the natural carbohydrates described above include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and general saccharides, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavors (e.g., thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrates can be appropriately determined by those skilled in the art.

[0142] In addition to the above, the health functional food according to one aspect may contain various supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavors, coloring agents and enhancers (such as cheese and chocolate), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners adhesives, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination, and the ratio of these additives can also be appropriately selected by those skilled in the art.

[0143] Also provided is a health functional food for preventing or improving a microbial infectious disease, which contains an antigen protein derived from a microorganism or an extracellular vesicle having a gene encoding the protein.

[0144] The "antigen protein derived from a microorganism", "gene encoding the protein", "extracellular vesicle", "microorganism", "microbial infectious disease", "prevention", "improvement", "health functional food", etc. may be within the ranges described above.

[0145] Also provided is a health functional food for preventing or improving a viral infectious disease, which contains an antigen protein derived from a cancer cell or an extracellular vesicle having a gene encoding the protein.

[0146] The "antigen protein derived from a cancer cell", "gene encoding the protein", "extracellular vesicle", "cancer cell", "cancer", "prevention", "improvement", "health functional food", etc. may be within the ranges described above.

[0147] Furthermore, the present invention provides a method for preventing or treating a viral infectious disease, which includes administering to an individual in need thereof an extracellular vesicle having an antigen protein derived from a virus or a gene encoding the protein.

[0148] The "antigen protein derived from a virus", "gene encoding the protein", "extracellular vesicle", "individual", "administration", "viral infectious disease", "prevention", "treatment", etc. may be within the ranges described above. Furthermore, the present invention provides a method for preventing or treating a microbial infectious disease, which includes administering to an individual in need thereof an extracellular vesicle having an antigen protein derived from a microorganism or a gene encoding the protein.

[0149] The "antigen protein derived from a microorganism", "gene encoding the protein", "extracellular vesicle", "individual", "administration", "microbial infection", "prevention", "treatment", etc. may be within the ranges described above.

[0150] Furthermore, the present invention provides a method for preventing or treating cancer, which includes administering extracellular vesicles having an antigen protein derived from cancer cells or a gene encoding the protein to an individual in need thereof.

[0151] The “antigen protein derived from cancer cells”, “gene encoding the protein”, “extracellular vesicles”, “individual”, “administration”, “cancer”, “prevention”, “treatment” and the like may be those within the above-described ranges.

[0152] Furthermore, the present invention provides the use of extracellular vesicles having an antigen protein derived from a virus or a gene encoding the protein for the manufacture of a prophylactic or therapeutic agent for viral infections.

[0153] The “antigen protein derived from a virus”, “gene encoding the protein”, “extracellular vesicles”, “viral infection”, “prevention”, “treatment” and the like may be those within the above-described ranges.

[0154] Furthermore, the present invention provides the use of extracellular vesicles having an antigen protein derived from a microorganism or a gene encoding the protein for the manufacture of a prophylactic or therapeutic agent for microbial infections.

[0155] The “antigen protein derived from a microorganism”, “gene encoding the protein”, “extracellular vesicles”, “viral infection”, “prevention”, “treatment” and the like may be those within the above-described ranges.

[0156] In addition, the present invention provides the use of extracellular vesicles having an antigen protein derived from cancer cells or a gene encoding the protein for the manufacture of a prophylactic or therapeutic agent for cancer.

[0157] The “antigen protein derived from cancer cells”, “gene encoding the protein”, “extracellular vesicles”, “viral infection”, “prevention”, “treatment” and the like may be those within the above-described ranges.

[0158] ​ (Mode for Carrying Out the Invention) Hereinafter, preferred examples are presented to assist in the understanding of the present invention. However, the following examples are merely provided to facilitate the understanding of the present invention, and the content of the present invention is not limited by the following examples.

Example

[0159] Example 1. Observation of the phenotype of activated immune cells

[0160] In order to develop a vaccine using extracellular vesicles, the present inventors first selected immune cells, stimulated the immune cells with an antigen, and attempted to produce a vaccine using exosomes isolated from the activated immune cells. For this purpose, as an example of the immune cells, THP-1 cells, which are a human monocyte cell line, were activated, and the phenotype of the activated cells was observed.

[0161] Specifically, THP-1 cells (8×10 5 cells / mL) were treated with lipopolysaccharide (LPS) at 100 ng / mL to activate the cells, and then observed under a microscope together with THP-1 cells that were not activated by LPS treatment. As a result, as shown in Fig. 1a, it was confirmed that the activated THP-1 cells had a changed cell morphology similar to that of macrophages compared to the non-activated THP-1 cells (Resting THP-1).

[0162] Furthermore, based on the above results, in order to confirm whether the activated THP-1 cells were differentiated into M1 macrophages as reported in the art, pro-inflammatory cytokines secreted by M1 macrophages The mRNA levels of IL-1b, IL-6, IL-8, TNF-α, and iNOS, which are markers of [[cytokines]], were measured. As a result, as shown in Fig. 1b, in the case of non-activated THP-1 cells, almost no expression of the markers was observed, whereas in the case of activated THP-1 cells, the expression of the markers increased, and in particular, the mRNA levels of IL-1b, IL-6, and IL-8 increased significantly.

[0163] From the above results, it was found that LPS-treated activated THP-1 cells exhibit the phenotype of M1 macrophages.

[0164] Example 2. Confirmation of the characteristics of exosomes derived from activated immune cells

[0165] In order to confirm the characteristics of the exosomes derived from the activated THP-1 cells, the present inventors isolated exosomes from the cells through the following process. Specifically, the cell culture medium was centrifuged at 1200 rpm for 5 minutes to remove cells and cell debris, and the supernatant was collected. Then, the supernatant was passed through a 0.2-μm filter to remove microparticles larger in size than exosomes, and then the exosomes were concentrated and washed using a tangential flow filtration (TFF) system.

[0166] Next, NTA (Nanosight Tracking Analyis) and cryo-electron microscopy were performed to evaluate the characteristics of the isolated exosomes. As a result, as shown in Figs. 2a and 2b, the average diameter of the isolated exosomes was 128.0 nm, and it was confirmed that they were circular.

[0167] Also, the concentration of the isolated exosomes was 2.6×10 11It was [x] particles / ml, and when the concentration of exosome protein was quantitatively analyzed by the BCA assay, it was confirmed to be 3.5 mg / ml. Furthermore, in order to examine whether the isolated exosomes express exosome markers, Western blotting was performed using cell lysate and the isolated exosomes, and the expression levels of exosome markers CD63, CD81, and flotillin 1 were measured. As a result, as shown in Figure 2c, only CD63 was expressed at a weak level in the cell lysate, whereas in the isolated exosomes, it was confirmed that all of the marker proteins were expressed at high levels.

[0168] From the above results, it was clearly found that the exosomes isolated from the activated THP-1 cells are exosomes having the above-described characteristics.

[0169] Example 3. Confirmation of characteristics of outer membrane vesicles derived from attenuated bacteria

[0170] The present inventors isolated outer membrane vesicles (OMV) from the bacteria through the following process in order to confirm the characteristics of the outer membrane vesicles (OMV) derived from the attenuated bacteria (BL21 strain). Specifically, in order to remove bacteria and bacterial sections, the culture solution was centrifuged at 3000 rpm for 15 minutes to collect the supernatant, and after passing the supernatant through a 0.2-μm filter to remove large-sized microparticles, concentration and washing of the outer membrane vesicles (OMV) were carried out using a tangential flow filtration (TFF) system.

[0171] Next, NTA (Nanosight Tracking Analyis) observation was performed to analyze the characteristics of the isolated outer membrane vesicles. As a result, as shown in Figure 3, 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 particles / ml, and when the protein concentration was quantitatively analyzed by the BCA assay, it was confirmed to be 2.2 mg / ml.

[0172] Example 4. Production of exosomes loaded with recombinant virus-derived proteins

[0173] The inventors loaded the exosomes derived from the activated THP-1 cells isolated in Example 2 above with the H1N1 protein derived from influenza virus or the RSV-F protein derived from respiratory syncytial virus, and attempted to verify the preventive effect as a vaccine against virus infection.

[0174] For this purpose, first, constructs of the H1N1 protein (A / Puerto Rico / 8 / 1934; Accession # ABD77675.1) derived from influenza virus and the RSV-F (fusion glycoprotein; Accession # P03420.1) protein derived from respiratory syncytial virus were prepared. Thereafter subsequently, to load the H1N1 protein or RSV-F protein expressed from the above constructs into the exosomes derived from the activated THP-1 cells, 1.4×10 11 (3 mg) of exosomes and 50 μg of the H1N1 protein or RSV-F protein were prepared. After performing extracorporeal shockwave (ESW) treatment, washing and concentration of the exosomes loaded with the H1N1 protein or RSV-F protein were carried out using an ultracentrifuge (32000 rpm, 16 hours).

[0175] Thereafter, as a result of quantitatively analyzing the amount of the H1N1 protein or RSV-F protein loaded into the exosomes derived from the THP-1 cells using ELISA, it was confirmed that a total of 41.1 μg of the H1N1 protein and a total of 36.43 μg of the RSV-F protein were loaded into the exosomes. The inventors used the exosomes derived from the activated THP-1 cells loaded with the H1N1 protein derived from influenza virus (ImmunExo H1N1) or the exosomes derived from the activated THP-1 cells loaded with the RSV-F protein derived from respiratory syncytial virus (ImmunExo Against RSV-F, the effectiveness against virus infection was evaluated by the following experiment.

[0176] Example 5. Production of extracellular vesicles loaded with mRNA derived from recombinant virus

[0177] The present inventors loaded H1N1 (Influenza AH1N1 Hawaii / 70 / 2019 Hemagglutinin) mRNA derived from influenza virus, RSV-F (Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein) mRNA derived from respiratory syncytial virus, and glycoprotein E (Varicella-Zoster virus glycoprotein E: VZE) mRNA derived from varicella-zoster virus into the exosomes derived from activated THP-1 cells isolated in Example 2 or the outer membrane vesicles derived from bacteria (Bacteria OMV: Bacteria Outer membrane vesicle) isolated from Example 3, and attempted to verify the preventive effect as a vaccine against viral infectious diseases.

[0178] Specifically, the mRNA of each protein was produced by an in vitro transcription (IVT) process from a DNA template by rnageene. The produced mRNA was prepared with 100 μg each of exosomes derived from THP-1 cells or OMV derived from bacteria (6 mg), and after treatment with extracorporeal shock wave (ESW), the extracellular vesicles loaded with the mRNA of each virus-derived protein were washed and concentrated using an ultracentrifuge (32000 rpm, 2 hours). Subsequently, when the amount of each mRNA loaded onto the exosomes derived from the THP-1 cells or OMVs derived from bacteria was quantitatively analyzed using qRT-PCR, it was confirmed that a total of 67.08 μg, 60.3 μg, and 66.3 μg (H1N1, RSV-F, VZE) were loaded into the exosomes derived from the THP-1 cells, and 64.36 μg, 66.5 μg, and 65 μg (H1N1, RSV-F, VZE) were loaded into the OMVs derived from bacteria. The inventors attempted to evaluate each viral infection by the following experiment on the exosomes derived from activated THP-1 cells or OMVs derived from bacteria loaded with the mRNA of each viral protein prepared above.

[0179] Example 6. Confirmation of the vaccine effect of exosomes derived from activated immune cells loaded with viral proteins against viruses

[0180] The inventors attempted to evaluate the efficacy of exosomes derived from activated THP -1 cells loaded with the H1N1 protein derived from influenza virus or the RSV-F protein derived from respiratory syncytial virus prepared in Example 4 above.

[0181] (1) Analysis of the serum antibody response against the virus of exosomes derived from THP-1 cells loaded with the H1N1 protein derived from influenza

[0182] The inventors administered exosomes (ImmunExo) derived from activated THP-1 cells loaded with the H1N1 protein derived from influenza virus to the mice prepared in Example 4 above at a concentration of 5 μg, or collected serum samples from each mouse administered with 500 μg of Al(OH)3 as an adjuvant and 5 μg of the protein together 14 days later, and then diluted them at a ratio of 1:100 to 1:1000 in PBS supplemented with 1% BSA, and measured the absorbance to analyze the antibody binding ability in the serum specific to the H1N1 protein derived from influenza virus.

[0183] As a result, as shown in Fig. 4a, it was confirmed that in the group administered with exosomes together with H1N1 protein, antibody binding in serum specific for influenza virus H1N1 appeared, similar to the group administered with an adjuvant and H1N1 protein together, despite administering a much smaller amount.

[0184] (2) Analysis of the serum antibody response against the virus of exosomes derived from THP-1 cells loaded with RSV-F protein derived from respiratory syncytial virus

[0185] The inventors administered exosomes (ImmunExo) derived from activated THP-1 cells loaded with RSV-F protein derived from respiratory syncytial virus to the mice prepared in Example 4 at a concentration of 8 μg, or collected serum samples 14 days later from each mouse administered with 500 μg of Al(OH)3 as an adjuvant and 8 μg of protein together, and then diluted them at a ratio of 1:100 to 1:1000 in PBS supplemented with 1% BSA, and analyzed the antibody binding in serum specific for RSV-F protein derived from respiratory syncytial virus by measuring the absorbance.

[0186] As a result, as shown in Fig. 4b, it was confirmed that the group administered with exosomes together with RSV-F protein was superior in antibody binding in serum specific for respiratory syncytial virus to the group administered with an adjuvant and RSV-F protein together, despite administering a much smaller amount.

[0187] Example 7. Confirmation of the effectiveness of the vaccine against the virus in extracellular vesicles loaded with mRNA of virus-derived protein

[0188] The inventors attempted to evaluate the efficacy of exosomes derived from activated THP-1 cells loaded with H1N1 mRNA derived from influenza virus, RSV-F mRNA derived from respiratory syncytial virus, or glycoprotein E mRNA derived from varicella-zoster virus, or outer membrane vesicles (OMV) derived from bacteria as vaccines against viruses.

[0189] 7-1. Analysis of serum antibody response to extracellular vesicles loaded with mRNA of virus-derived proteins

[0190] (1) In the case of extracellular vesicles loaded with H1N1 mRNA derived from influenza virus

[0191] The inventors subcutaneously administered 10 μg of exosomes derived from activated THP-1 cells loaded with H1N1 mRNA derived from influenza virus prepared in Example 5 or 10 μg of bacteria-derived outer membrane vesicles (Bacteria OMV) loaded with H1N1 mRNA derived from influenza virus to B6C3F1 / Slc mice (female). After 3 weeks, the same extracellular vesicles were subcutaneously administered in the same amount. Then, 35 days after the first administration, which was 5 weeks after the first administration, the mice were sacrificed and serum samples were collected. Thereafter, the serum samples were diluted at a ratio of 1:100 to 1:1000 in PBS supplemented with 1% BSA, and the absorbance was measured to analyze the antibody binding ability in the serum specific for the H1N1 protein derived from influenza virus (Figure 5).

[0192] As a result, as shown in Figure 6, it was confirmed that in both mice administered with exosomes derived from activated THP-1 cells loaded with H1N1 mRNA derived from influenza virus and bacteria-derived OMV, the antibody binding ability (antibody binding) of antibody (IgG) in the serum specific for the influenza virus H1N1 protein appeared compared with the control group.

[0193] (2) In the case of extracellular vesicles loaded with glycoprotein E mRNA derived from varicella-zoster virus

[0194] The inventors administered 10 μg of exosomes derived from activated THP-1 cells loaded with glycoprotein E mRNA derived from varicella-zoster virus prepared in Example 5 or 10 μg of bacterial outer membrane vesicles (Bacteria OMV) loaded with glycoprotein E mRNA derived from varicella-zoster virus to B6C3F1 / Slc mice (female) by subcutaneous injection. Then, 35 days after the first administration, which was 5 weeks later, the mice were sacrificed and serum samples were collected. Thereafter, the serum samples were diluted at a ratio of 1:100 to 1:1000 in PBS supplemented with 1% BSA, and the absorbance was measured to analyze the antibody binding ability in the serum specific for glycoprotein E derived from varicella-zoster virus (Figure 5).

[0195] As a result, as shown in Figure 7, it was confirmed that in both mice administered with exosomes derived from activated THP-1 cells loaded with glycoprotein E mRNA derived from varicella-zoster virus and bacterial OMV, the antibody binding ability of the antibody (IgG) specific for glycoprotein E derived from varicella-zoster virus appeared in the serum compared to the control group.

[0196] (3) In the case of extracellular vesicles loaded with RSV-F mRNA derived from respiratory syncytial virus

[0197] The inventors administered 10 μg of exosomes derived from activated THP-1 cells loaded with RSV-F mRNA derived from respiratory syncytial virus or 10 μg of bacterial outer membrane vesicles (Bacteria OMV) loaded with RSV-F mRNA derived from respiratory syncytial virus to B6C3F1 / Slc mice (female) by subcutaneous injection. Then, 14 days after the first administration, which was 2 weeks later, serum samples were obtained. Subsequently, the serum samples were diluted in PBS supplemented with 1% BSA at a ratio of 1:100 to 1:1000, and the antibody binding ability in the serum specific for the RSV-F protein derived from respiratory syncytial virus was analyzed by measuring the absorbance. As a result, as shown in Figure 8, in both mice administered with exosomes derived from activated THP-1 cells loaded with RSV-F mRNA derived from respiratory syncytial virus and Bacteria OMV, the antibody binding ability of antibody (IgG) in the serum specific for the RSV-F protein derived from respiratory syncytial virus appeared compared to the control group. This was confirmed.

[0198] 7-2. Analysis of specific T cell responses to extracellular vesicles loaded with viral-derived proteins

[0199] (1) In the case of extracellular vesicles loaded with H1N1 mRNA derived from influenza virus The inventors administered 10 μg of exosomes derived from activated THP-1 cells loaded with H1N1 mRNA derived from influenza virus prepared in Example 5 or 10 μg of viral outer membrane vesicles (Bacteria OMV) loaded with H1N1 mRNA derived from influenza virus to B6C3F1 / Slc mice (female) by subcutaneous injection. Then, 3 weeks later, the same extracellular vesicles were administered by subcutaneous injection in the same amount. After that, 35 days after the first administration, which was 5 weeks later, the mice were sacrificed and splenocytes were isolated. Subsequently, the splenocytes (1×10 7After treating (___ / ml) with 2 μg / mL of H1N1 protein derived from influenza virus for 48 hours, ELISA was performed using the culture solution of the spleen cells to measure the level of IFN-γ protein secreted from Th1 cells.

[0200] As a result, as shown in Fig. 9, in the culture solution of the control group, no IFN-γ protein was detected, while in the spleen culture solution of mice administered with exosomes derived from activated THP-1 cells loaded with H1N1 mRNA derived from influenza virus and OMVs derived from cells, the presence of IFN-γ was confirmed.

[0201] The above results suggest that in the case of the control group, the Th1 cell-mediated immune response was not induced, while the administration of extracellular vesicles according to the present invention strongly induced the Th1 cell-mediated immune response.

[0202] (2) In the case of extracellular vesicles loaded with glycoprotein E mRNA derived from varicella-zoster virus

[0203] The inventors subcutaneously administered 10 μg of exosomes derived from activated THP-1 cells loaded with glycoprotein E mRNA derived from varicella-zoster virus prepared in Example 5 or 10 μg of outer membrane vesicles derived from bacteria (Bacteria OMV) loaded with glycoprotein E mRNA derived from varicella-zoster virus to B6C3F1 / Slc mice (female). After 3 weeks, the same extracellular vesicles were administered subcutaneously in the same amount. Then, 35 days after the first administration, which was 5 weeks after the first administration, the mice were sacrificed and splenocytes were isolated. Then, after treating the splenocytes (1×10 7 / ml) with 2 μg / mL of glycoprotein E derived from varicella-zoster virus for 48 hours, ELISA was performed using the culture solution of the splenocytes to measure the level of IFN-γ protein secreted from Th1 cells.

[0204] As a result, as shown in Fig. 10, no IFN-γ protein was detected in the culture medium of the control group, whereas IFN-γ was confirmed to be present in the spleen cell culture medium of the mice administered with OMVs derived from activated THP-1 cells loaded with varicella-zoster virus-derived glycoprotein E mRNA.

[0205] In the case of the control group, the above results indicate that the Th1 cell-mediated immune response was not induced, whereas the administration of the extracellular vesicles according to the present invention strongly induced the Th1 cell-mediated immune response. This suggests that.

[0206] The disclosure of the present invention described above is merely for illustrative purposes, and those having ordinary knowledge in the technical field to which the present invention pertains can easily understand that it can be easily modified into other embodiments without changing the technical idea and gist of the present invention. Therefore, it should be understood that the above-described embodiments are exemplary in all respects and not restrictive.

Claims

1. Extracellular vesicles having an antigen protein or a gene encoding the protein.

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

3. The extracellular vesicles according to claim 2, wherein the virus is one or more selected from the group consisting of adenovirus, smallpox virus, poliovirus, measles virus, hepatitis C virus, human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), influenza virus, respiratory syncytial virus, herpes simplex virus, human papillomavirus, Zika virus, varicella-zoster virus, and severe fever with thrombocytopenia syndrome virus.

4. The extracellular vesicles according to claim 1, wherein the antigen protein is one or more selected from the group consisting of p24 protein derived from human immunodeficiency virus type 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 microorganism is a microorganism of one or more genera 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, Coccidioides, Cryptococcus, Histoplasma, Rhizopus, Mucor, Cunninghamella, Apophysomyces, Absidia, Saksenaea, Entomophthora, Conidiobolus, Basidiobolus, Sporothrix, Pneumocystis, Talaromyces, Asclepias, Fusarium, Scedosporium, and Mucorales, the extracellular vesicle according to claim 2.

6. The cancer cell is derived from one or more cancers selected from the group consisting of head and neck cancer, melanoma, leukemia, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lung cancer, colorectal cancer, and glioblastoma The extracellular vesicle according to claim 2.

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

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

9. The extracellular vesicle according to claim 1, wherein the extracellular vesicle is derived from one or more cells selected from the group of animal cells, plant cells, or microorganisms.

10. The extracellular vesicle according to claim 9, wherein the animal cell is one or more cells selected from the group consisting of somatic cells, germ cells, immune cells, nerve cells, and tumor cells.

11. The extracellular vesicle according to claim 9, wherein when the extracellular vesicle is derived from an animal cell, the extracellular vesicle expresses one or more proteins selected from the group consisting of CD9, CD63, CD81, Alix, TSG101, syntenin 1, and flotillin 1.

12. The extracellular vesicle according to claim 9, wherein when the extracellular vesicle is derived from a plant cell, the extracellular vesicle expresses one or more proteins selected from the group consisting of syntaxin (PEN1), tetraspanin 8 (Tet8), and heat shock protein (HSP).

13. The extracellular vesicle according to claim 9, wherein when the extracellular vesicle is derived from a microorganism, the extracellular vesicle expresses one or more proteins selected from the group consisting of OmpA, flagellin, HSP70, and β-glucan.

14. A vaccine composition for preventing or treating viral infectious diseases, comprising an extracellular vesicle having a virus-derived antigen protein or a gene encoding the protein.

15. A vaccine composition for preventing or treating microbial infectious diseases, comprising an extracellular vesicle having a microorganism-derived antigen protein or a gene encoding the protein.

16. A vaccine composition for preventing or treating cancer, comprising an extracellular vesicle having a cancer cell-derived antigen protein or a gene encoding the protein.

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

18. A health functional food for preventing or improving viral infectious diseases, comprising an extracellular vesicle having a virus-derived antigen protein or a gene encoding the protein.

19. A health functional food for preventing or improving microbial infectious diseases, comprising an extracellular vesicle having a microbe-derived antigen protein or a gene encoding the protein.

20. A health functional food for preventing or improving cancer, comprising an extracellular vesicle having a cancer cell-derived antigen protein or a gene encoding the protein.

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