Method for constructing nanovesicles derived from outer membrane of gram-negative bacteria and use thereof

Abstract

The present invention relates to a method for preparing nanovesicles (OMNV) derived from the outer membrane of Gram-negative bacteria and uses thereof. In addition, the present invention provides a method of treating or diagnosing cancer using OMNV. Furthermore, the present invention provides a method of delivering therapeutic or diagnostic substances to target cells or target tissues by loading these substances into the OMNV. In addition, the present invention provides a method for preventing and treating various diseases, including cancer and bacterial / viral infections, by co-administering OMNV with an antigen or administering an antigen-expressing OMNV.

Description

Technical Field

[0001] This application claims the Korean Patent Application No. 10-2022-0065067 filed on May 27, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for preparing nanovesicles (OMNV) derived from the outer membrane of Gram-negative bacteria and its use. More specifically, the present invention provides a method for preparing OMNV, and a method for using OMNV to reduce adverse reactions, enhance efficacy, and deliver to target cells or tissues in vitro and in vivo, thereby providing a pharmaceutical composition and method for enhancing the stability and efficacy of therapeutic agents, drug delivery systems, and / or vaccine delivery systems for the treatment or prevention of various diseases including cancer and bacterial / viral infections. Background Art

[0003] As is well known, all cells, including Gram-negative bacteria, naturally secrete extracellular vesicles. Extracellular vesicles secreted by Gram-negative bacteria are also called outer membrane vesicles (OMV). The size range of OMV is 20-200nm, and contains various biologically active substances, such as proteins, lipids, genetic material, lipopolysaccharide (LPS) and metabolites. OMV plays a variety of functions, including delivering proteins or genetic material, removing competing bacteria, improving bacterial survival, delivering toxins to host cells and regulating immune responses.

[0004] Recent research results show that OMVs naturally secreted by various Gram-negative bacteria contain various substances that can activate the immune system. Since these OMVs are inanimate entities without the bacteria themselves, they are safer than the bacteria themselves and have direct therapeutic effects on various diseases, including cancer. They are also being used or developed clinically as drug delivery systems or vaccine delivery systems for the prevention or treatment of various diseases such as cancer, COVID-19, and meningitis.

[0005] It is known that when OMVs naturally secreted by Gram-negative bacteria are administered intravenously into cancer-bearing mice, they induce an anti-cancer immune response that effectively kills cancer tissue without causing significant adverse effects.

[0006] Furthermore, due to their nanosize, OMVs naturally secreted by Gram-negative bacteria can accumulate specifically in cancer tissues via the enhanced permeability and retention (EPR) effect. Therefore, they can be used as drug delivery systems for cancer treatment or diagnosis after loading with drugs.

[0007] On the other hand, it is known that when OMVs naturally secreted by Gram-negative bacteria are intraperitoneally administered to mice three times at intervals of one week, and the corresponding Gram-negative bacteria are introduced into the mouse peritoneum one week after the last administration, the mice can be prevented from dying from the bacterial infection. It is speculated that OMVs prevent death caused by bacterial infection by inducing Th1 and Th17 immune responses. In fact, OMVs are being used or developed as vaccine delivery systems in clinical practice for the prevention or treatment of various diseases such as cancer, COVID-19, and meningitis.

[0008] In addition, various attempts are being made to improve the therapeutic and preventive efficacy of OMVs, such as loading various therapeutic drugs or utilizing bacterial-derived extracellular vesicles expressing various proteins, including cytokines, growth factors, and antibodies (for targeting specific cells or tissues, neutralizing antigen activity, or exerting various functions) as fusion proteins on outer membrane proteins.

[0009] However, Gram-negative bacteria naturally secrete OMVs in very small quantities, and their purification process is complex and difficult, resulting in low yields, making them unsuitable for large-scale production. Moreover, they contain unnecessary intracellular substances, such as periplasmic proteins, inner membrane proteins, cytoplasmic proteins, excess genetic material, and various metabolites, which can reduce drug loading efficiency or cause adverse reactions when administered in vivo. Therefore, it is necessary to develop methods to remove these substances.

[0010] To overcome the above problems, nanovesicles (OMNVs) derived from the outer membrane of Gram-negative bacteria have long been artificially manufactured and developed for use as therapeutic agents, drug delivery systems, and / or vaccine delivery systems for various diseases including cancer.

[0011] Typically, OMNV is prepared by treating Gram-negative bacteria with EDTA and lysozyme, disrupting them by sonication, separating the outer and inner membranes by high-speed centrifugation, removing the inner membrane by treatment with a detergent such as Sarkosyl, and then separating the outer membrane component by high-speed centrifugation, followed by sonication or extrusion.

[0012] However, the above-mentioned method for preparing OMNV has many limitations in large-scale production for clinical use due to its complexity and the need to use various substances such as lysozyme. Therefore, it is necessary to develop a method that can overcome these limitations. Summary of the invention

[0013] Problem to be solved

[0014] The inventors of the present invention conducted research to solve the above conventional problems. As a result, the inventors found that treating Gram-negative bacteria with Sarkosyl removes the inner membrane, allows the outer membrane to be extracted without ultracentrifugation, and utilizes the extracted outer membrane to prepare nanovesicles, thereby completing the present invention.

[0015] The present invention also provides a method for treating or diagnosing cancer using nanovesicles (OMNV) derived from the outer membrane of Gram-negative bacteria, and a method for delivering therapeutic or diagnostic substances by loading them onto the outside, inside or outer membrane of the nanovesicles. In addition, the present invention provides a method for delivering nanovesicles expressing fusion proteins of outer membrane proteins and various proteins such as cytokines, growth factors and antibodies to target cells or target tissues, which enhances the therapeutic effect and reduces adverse reactions, thereby providing a method for improving the effect of preventing / treating various diseases and reducing adverse reactions. Moreover, the present invention also provides a method for preventing and treating various diseases by co-administering OMNV with an antigen or administering OMNV expressing an antigen.

[0016] However, the technical problems to be solved by the present invention are not limited to the above problems, and those skilled in the art will clearly understand other problems not mentioned from the following description.

[0017] Means for solving the problem

[0018] To achieve the above object of the present invention, the present invention provides a method for preparing nanovesicles (OMNV) derived from the outer membrane of Gram-negative bacteria, which comprises the following steps:

[0019] (a) obtaining an outer membrane by removing the inner membrane from a Gram-negative bacterium; and

[0020] (b) preparing nanovesicles from the suspension comprising the outer membrane.

[0021] To achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating a disease, comprising OMNV, wherein the OMNV has therapeutic activity.

[0022] In addition, to achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating a disease, which consists of OMNV, wherein the OMNV has therapeutic activity.

[0023] In addition, to achieve another object of the present invention, the present invention provides a pharmaceutical composition for treating a disease, which is essentially composed of OMNV, wherein the OMNV has therapeutic activity.

[0024] To achieve another object of the present invention, the present invention provides a vaccine composition for preventing or treating a disease, comprising OMNV.

[0025] To achieve another object of the present invention, the present invention provides use of the OMNV of the present invention in preparing a pharmaceutical composition for treating a disease.

[0026] To achieve another object of the present invention, the present invention provides a method for treating a disease, comprising administering an effective amount of a pharmaceutical composition comprising the OMNV of the present invention to a subject in need thereof.

[0027] Hereinafter, the present invention will be described in detail.

[0028] The present invention provides a method for preparing OMNV, comprising the following steps:

[0029] (a) obtaining an outer membrane by removing the inner membrane from a Gram-negative bacterium; and

[0030] (b) preparing nanovesicles from the suspension comprising the outer membrane.

[0031] In the present invention, the term "Gram-negative bacteria" refers to a group of bacteria that cannot retain the color of crystal violet staining in the Gram staining method. Gram-negative bacteria have a cell surface comprising an outer membrane, a periplasmic space containing proteins such as lipoproteins, a peptidoglycan layer, and an inner membrane (or cytoplasmic membrane). Among them, the outer membrane comprises an asymmetric lipid bilayer, the inner layer Composed of phospholipids, and the outer layer It is composed of lipopolysaccharide. In addition, the outer membrane houses outer membrane proteins such as OmpA, OmpE, OmpC, LptD, and BamA.

[0032] In one embodiment of the present invention, the Gram-negative bacteria may be a bacterium selected from the genera Escherichia, Helicobacter, Hemophilus, Neisseria, Cyanobacterium, Klebsiella, Acetobacter, Acinetobacter, Enterobacter, Chlamydia, Vibrio, Pseudomonas, Salmonella, Thiobacter, Borrelia, Burkholderia, Serratia and Treponema, but is not limited thereto. In a preferred embodiment of the present invention, the Gram-negative bacteria may be bacteria belonging to the genus Escherichia, Salmonella or Pseudomonas.

[0033] The term "nanovesicle" in the present invention refers to a structure in which the inside and the outside are separated by a lipid bilayer containing cell membrane components derived from bacteria (including bacterial cell membrane lipids and cell membrane proteins, and having the same topological structure), but intracellular substances such as genetic material and intracellular proteins have been removed, and the size is smaller than the original cell, but is not limited thereto.

[0034] In the present invention, the nanovesicle is not particularly limited as long as its diameter is at the nanometer level. For example, the diameter can be about 20-290nm, 20-280nm, 20-270nm, 20-260nm, 20-250nm, 20-240nm, 20-230nm, 20-220nm, 20-210nm, 20-200nm, 20-190nm, 20-180nm, 20-170nm, 20-160nm, 20-150nm, 20-140nm, 20-130nm, 20-120nm, 20-110nm, 20-100nm, 20-90nm, 20-80nm, 20-70nm, 20-60nm , 20-50nm, 20-40nm, 20-30nm, 30-300nm, 30-290nm, 30-280nm, 30-270nm, 3 0-260nm, 30-250nm, 30-240nm, 30-230nm, 30-220nm, 30-210nm, 30-200nm, 30-190nm, 30-180nm, 30-170nm, 30-160nm, 30-150nm, 30-140nm, 30-130nm , 30-120nm, 30-110nm, 30-100nm, 30-90nm, 30-80nm, 30-70nm, 30-60nm, 30- 50nm, 30-40nm, 40-300nm, 40-290nm, 40-280nm, 40-270nm, 40-260nm, 40-2 50nm, 40-240nm, 40-230nm, 40-220nm, 40-210nm, 40-200nm, 40-190nm, 40- 180nm, 40-170nm, 40-160nm, 40-150nm, 40-140nm, 40-130nm, 40-120nm, 40 -110nm, 40-100nm, 40-90nm, 40-80nm, 40-70nm, 40-60nm, 40-50nm, 50-300n m, 50-290nm, 50-280nm, 50-270nm, 50-260nm, 50-250nm, 50-240nm, 50-230 nm, 50-220nm, 50-210nm, 50-200nm, 50-190nm, 50-180nm, 50-170nm, 50-160 nm, 50-150nm, 50-140nm, 50-130nm, 50-120nm, 50-110nm, 50-100nm, 50-90 nm, 50-80nm, 50-70nm, 50-60nm, 60-300nm, 60-290nm, 60-280nm, 60-270nm,60-260nm, 60-250nm, 60-240nm, 60-230nm, 60-220nm, 60-210nm, 60-200nm, 60-190nm, 60-180nm, 60-170nm, 60-160nm, 60-150nm, 60-140nm, 60-130nm, 60-120nm, 60-110nm, 60-100nm, 60-90nm, 60-80nm, 60-70nm, 70-300nm, 70-290nm, 70-280nm, 70-270nm, 70-2 60nm, 70-250nm, 70-240nm, 70-230nm, 70-220nm, 70-210nm, 70-200nm, 70-190nm, 70-180nm, 70-170nm, 70-160nm, 70-150nm, 70-1 40nm, 70-130nm, 70-120nm, 70-110nm, 70-100nm, 70-90nm, 70-80nm, 80-300nm, 80-290nm, 80-280nm, 80-270nm, 80-260nm, 80-250n m, 80-240nm, 80-230nm, 80-220nm, 80-210nm, 80-200nm, 80-190nm, 80-180nm, 80-170nm, 80-160nm, 80-150nm, 80-140nm, 80-130n m, 80-120nm, 80-110nm, 80-100nm, 80-90nm, 90-300nm, 90-290nm, 90-280nm, 90-270nm, 90-260nm, 90-250nm, 90-240nm, 90-230nm, 90-220nm, 90-210nm, 90-200nm, 90-190nm, 90-180nm, 90-170nm, 90-160nm, 90-150nm, 90-140nm, 90-130nm, 90-120nm, 90-110nm, 90-100nm, 100-300nm, 110-290nm, 120-280nm, 130-270nm, 140-260nm, 150-250nm, 160-240nm, 170-230nm, 180-220nm or 190-210nm.

[0035] In the present invention, the term "Gram-negative bacteria outer membrane-derived nanovesicles (OMNV)" refers to nanovesicles artificially prepared using the outer membrane of Gram-negative bacteria according to the method comprising steps (a) and (b).

[0036] In the method for preparing nanovesicles of the present invention, step (a) is a step of removing the inner membrane from Gram-negative bacteria to obtain the outer membrane.

[0037] In one embodiment of the present invention, step (a) can be carried out by treating Gram-negative bacteria with an anionic surfactant. The term "anionic surfactant" refers to a substance that, when dissociated in water, produces anions that are adsorbed to the surface of the aqueous solution, thereby reducing surface tension. Non-limiting examples of anionic surfactants include sodium dodecyl sulfate (SDS), Sarkosyl, lithium dodecyl sulfate, and sodium 1-octane sulfonate, but are not limited thereto. In a preferred embodiment of the present invention, the anionic surfactant can be Sarkosyl.

[0038] In one embodiment of the present invention, in step (a), the anionic surfactant can be treated in a culture medium or suspension containing Gram-negative bacteria at a final concentration of 0.1%-5% (w / v), preferably 0.1%-4% (w / v), more preferably 0.1%-3% (w / v), even more preferably 0.1%-2% (w / v), and most preferably 0.5%-2% (w / v).

[0039] In the present invention, the term "culture medium" refers to the inclusion of Gram-negative bacteria in a medium containing nutrients in which the Gram-negative bacteria can survive and proliferate; and the term "suspension" refers to a culture medium containing Gram-negative bacteria, wherein the bacteria are separated only by means such as centrifugation, and the separated Gram-negative bacteria are then suspended in a culture medium or buffer. In a preferred embodiment of the present invention, the terms "culture medium" and "suspension" can be interpreted interchangeably.

[0040] In one embodiment of the present invention, in step (a), the anionic surfactant may be treated at a temperature of 4°C-60°C for 5-120 minutes, preferably at a temperature of 4°C-60°C for 5-60 minutes, more preferably at a temperature of 4°C-60°C for 10-60 minutes, even more preferably at a temperature of 4°C-60°C for 10-50 minutes, and most preferably at a temperature of 4°C-60°C for 20-40 minutes, but is not limited thereto.

[0041] In one embodiment of the present invention, in step (a), anionic surfactant can be treated once or repeatedly. When anionic surfactant is repeatedly treated in step (a), anionic surfactant is treated in a culture medium or suspension containing Gram-negative bacteria for a certain period of time, residual anionic surfactant is removed in the supernatant by means such as centrifugation, Gram-negative bacteria are suspended in culture medium or buffer, and then the process of treating anionic surfactant again can be repeated. In the present invention, the term "repeat" can mean 2-10 times, preferably 2-5 times, more preferably 2-4 times, and most preferably 2-3 times. When anionic surfactant is repeatedly treated in step (a), the treatment conditions (i.e., treatment concentration, treatment time, treatment temperature, etc.) of each treatment can be the same or different.

[0042] In step (a), after treating the Gram-negative bacteria with an anionic surfactant, an operation of separating the outer membrane from the inner membrane, cytoplasmic components, etc. may be additionally performed. For example, after step (a), any method known in the art for separating and purifying the outer membrane may be additionally used, such as centrifugation, filtration, dialysis, size exclusion chromatography, Capto Core chromatography, tangential flow filtration, etc., but not limited thereto.

[0043] In the present invention, step (b) is a process of forming nanovesicles by applying energy to the outer membrane of the Gram-negative bacteria separated in step (a). In one embodiment of the present invention, step (b) can be performed by applying one or more means selected from the following to the culture medium or suspension containing the outer membrane of Gram-negative bacteria: microfluidics, ultrasonic treatment, extrusion, treatment with an alkaline solution, treatment with a homogenizer or disperser, and nitrogen cavitation, but is not limited thereto.

[0044] In one embodiment of the invention, after step (b), an operation of purifying or separating and collecting the nanovesicles derived from the outer membrane of Gram-negative bacteria contained in the culture medium or suspension may be additionally performed. Purification or separation may be performed by any method known in the art without limitation. For example, it may be separated or purified by a method selected from the following: ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, Capto Core chromatography, immunoaffinity separation, microfluid separation, aqueous two-phase system, polymer-based precipitation, ultrasonic treatment and extrusion, but is not limited thereto.

[0045] According to the method of the present invention, compared with the conventional technology of separating the outer membrane vesicles of natural secretion, an improved yield can be provided. For example, based on the same volume of bacterial culture medium, the yield of OMNV obtained can be at least about 2-1000 times of the bacterial outer membrane vesicles of natural secretion in terms of particle number. For example, based on the same volume of bacterial culture medium, the level of OMNV obtained can be at least about 2-100 times of the bacterial outer membrane vesicles of natural secretion in terms of total protein. For example, based on the same volume of bacterial culture medium, the purity of OMNV obtained (total particle number / total protein amount) can be at least about 2-100 times of the bacterial outer membrane vesicles of natural secretion. In addition, the OMNV prepared according to the present invention is unlikely to contain components of non-outer membrane origin, such as surrounding cytoplasm, inner membrane and cytoplasm.

[0046] In one embodiment of the present invention, the OMNV prepared according to the method is characterized by lacking at least one component derived from the bacteria from which it is derived that is not derived from the outer membrane. The term "components not derived from the outer membrane" includes peptidoglycan, surrounding cytoplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins and ribosomes.

[0047] In one embodiment of the invention, the OMNVs prepared according to the method are characterized in that they lack at least 50%, preferably at least 70%, more preferably at least 90%, and most preferably 100% of at least one non-outer membrane derived component compared to the outer membrane vesicles naturally secreted by the bacteria from which they are derived.

[0048] In one embodiment of the invention, the Gram-negative bacteria may be transformed.

[0049] In the present invention, the term "transformation" refers to the modification of the genotype of bacteria by the introduction of exogenous polynucleotides, which means that no matter what transformation method is used, the exogenous polynucleotides are introduced into the bacteria. The exogenous polynucleotides introduced into the bacteria can be integrated into the bacterial genome, or not integrated and maintained, and the present invention covers both cases. The exogenous polynucleotides can be inserted into a vector and used for the transformation of bacteria.

[0050] In the present invention, the term "vector" is used for the purpose of replication or expression of exogenous polynucleotides, and generally comprises one or more signal sequences, replication origins, one or more marker genes, enhancers, promoters and transcription termination sequences. Plasmid, as a type of vector, refers to a linear or circular double-stranded DNA molecule to which an external polynucleotide fragment can be connected. Other forms of vectors include viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which can introduce additional DNA fragments into the viral genome. Certain vectors can replicate autonomously in introduced bacteria (e.g., bacterial vectors and episomal mammalian vectors comprising bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the bacterial genome by introducing bacteria and replicated along with the bacterial genome.

[0051] In the present invention, vector can be understood to have the same meaning as "expression vector", which is a type of vector capable of expressing polynucleotides. A polynucleotide sequence is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (e.g., expression level, time, or location) of a polynucleotide sequence. A regulatory sequence is a sequence that affects the expression (e.g., expression level, time, or location) of a nucleic acid to which it is operably linked. A regulatory sequence can exert an influence on the regulated nucleic acid directly, or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or nucleic acid). Regulatory sequences include promoters, enhancers, and other expression control elements.

[0052] The vector can be introduced into the bacteria by methods known in the art, such as but not limited to transient transfection, microinjection, transduction, fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into bacteria, thereby achieving transformation of the bacteria.

[0053] In the present invention, the term "expression" means the production of a protein or polypeptide in bacteria.

[0054] In the present invention, the term "exogenous protein" refers to a protein or peptide that is not endogenously expressed in bacteria, but is expressed in the cytoplasm and / or cell membrane of bacteria through artificial transformation. By transforming bacteria with a vector into which a polynucleotide encoding a protein is inserted, the expression of the exogenous protein in the bacteria can be induced.

[0055] In a preferred embodiment of the present invention, the exogenous protein may include one or more proteins selected from the following: antigenic proteins and / or antigenic peptides that can be expressed or exposed on the outside of the bacterial outer membrane, antigenic proteins or antigenic peptides that can be exposed on the inside of the outer membrane, immunostimulatory proteins, immunosuppressive proteins, cell adhesion molecules, antibodies, targeting proteins, cell membrane fusion proteins, cytokines, enzymes, growth factors, the extracellular domain of membrane receptors, marker proteins, fragments thereof and fusion proteins thereof, but are not limited thereto.

[0056] In one embodiment of the invention, the Gram-negative bacteria may be characterized by reduced expression or knockout of an endogenous protein.The term "endogenous protein" refers to a protein that is inherent to or naturally expressed in the Gram-negative bacteria.

[0057] In the present invention, the endogenous protein whose expression can be reduced or knocked out can include non-essential proteins and / or secreted proteins that are functionally redundant with other endogenous proteins (e.g., for bacterial growth and / or vitality, such as in a biomanufacturing environment). In one embodiment, the endogenous protein whose expression can be reduced or knocked out can be non-essential or related to secretory function. In one embodiment, the endogenous protein whose expression can be reduced or knocked out can be related to immune function, disease resistance, bacterial communication and / or secretion. In a preferred embodiment, the endogenous protein whose expression can be reduced or knocked out can be a protein related to bacterial toxicity.

[0058] In some embodiments, reducing the expression of bacterial endogenous proteins includes removing gene copies encoding endogenous proteins, for example, knocking them out. In some embodiments, reducing the expression of bacterial endogenous proteins includes removing all (e.g., all two) copies of genes encoding endogenous proteins from the bacterial genome, for example, knocking them out. In some embodiments, reducing the expression of bacterial endogenous proteins includes removing regulatory nucleic acid sequences operably connected to genes encoding endogenous proteins, for example, promoters or enhancers, for example, knocking them out. In some embodiments, reducing the expression of bacterial endogenous proteins includes removing all (e.g., all two) copies of regulatory nucleic acid sequences operably connected to genes encoding endogenous proteins, for example, knocking them out.

[0059] In the present invention, the expression of endogenous proteins is eliminated, such as knocking them out, including introducing deletion, substitution or insertion mutation into the gene encoding the endogenous protein or the regulatory nucleic acid sequence (such as a promoter or enhancer) operably linked to the gene encoding the endogenous protein, which can reduce the expression of the endogenous protein.

[0060] Removing a gene copy encoding an endogenous protein or a regulatory element operably connected to a gene (e.g., knocking them out) can be achieved by any gene editing system known in the art. Exemplary gene editing systems include regular clustered short palindromic repeats (CRISPR) systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). In some embodiments, removing a gene copy encoding an endogenous protein or a regulatory element operably connected to a gene (e.g., knocking them out) includes using a CRISPR-Cas9 molecule (e.g., Cas9 molecule) in conjunction with a regulatory nucleic acid sequence (e.g., promoter or enhancer) specifically for a gene encoding an endogenous protein or operably connected to a gene encoding an endogenous protein (e.g., gRNA, sgRNA, and / or trans-activating crRNA).

[0061] In some embodiments, reducing the expression of endogenous bacterial proteins includes reducing the expression of mRNA transcripts encoding endogenous proteins, such as by lack in bacteria. In some embodiments, reducing the expression of mRNA transcripts encoding endogenous proteins, such as by lack, including using siRNA that can be combined with mRNA, the mRNA encoding endogenous proteins or the regulatory nucleic acid sequence operably connected thereto, such as promoter or enhancer. Those skilled in the art know the tools and techniques for designing siRNA and delivering it to bacteria. Such tools and / or techniques include but are not limited to Dharmacon HorizonsiDesign tools, InvivoGen siRNA Wizard, GenScript siRNA construct services, IDT CustomDicer-Substrate siRNA, Sigma-Aldrich siRNA design services or those taught by www.rnaiweb.com / RNAi / siRNA_Design / .

[0062] In one embodiment of the present invention, the transformed bacteria can be transformed to reduce toxicity, target specific cells or tissues, fuse with the cell membrane of target cells, express substances for treating and / or diagnosing diseases, and simultaneously inhibit specific substances and express specific substances, but are not limited thereto.

[0063] In a preferred embodiment of the present invention, the Gram-negative bacteria may be characterized in that they are transformed to reduce toxicity. For example, the bacteria may be transformed to reduce the toxicity of lipopolysaccharides that mediate host inflammatory responses, or the expression of endotoxin-producing genes may be reduced or deleted. Specifically, the bacteria are transformed to have one or more genotypes selected from the following: ΔmsbB, ΔkdsA, ΔkdsB, ΔlpxB, ΔkdtA, ΔlpxC, ΔlpxD, Δssc, ΔlpxA and ΔhtrB, but are not limited thereto.

[0064] In one embodiment of the present invention, Gram-negative bacteria can be transformed by substance treatment or gene introduction, and the transformation can be performed twice or more.

[0065] In one embodiment of the present invention, the membrane of OMNV may also include components other than the bacterial outer membrane.

[0066] Components other than the bacterial outer membrane may include targeting substances, cell membrane fusion substances (fusogens), cyclodextrin, polyethylene glycol, hyaluronic acid, etc. In addition, components other than the outer membrane may be added by various methods, including chemical modification of the outer membrane.

[0067] For example, the membrane components of OMNV can be chemically modified using sulfhydryl (-SH) or amino (-NH2), or by chemically binding proteins, polyethylene glycol or hyaluronic acid to OMNV. To this end, the method for preparing OMNV of the present invention may further include a step of chemically modifying the membrane components of OMNV after step (b).

[0068] The present invention also provides OMNV prepared by the above method.

[0069] In one embodiment of the invention, the OMNV of the invention may be characterized in that it lacks at least one non-outer membrane component of the bacteria from which it is derived."Non-outer membrane components" include peptidoglycan, peripheral cytoplasmic proteins, inner membrane proteins, nucleic acids, cytoplasmic proteins and ribosomes.

[0070] In one embodiment of the invention, the OMNVs of the invention may be characterized by lacking at least 50%, preferably at least 70%, more preferably at least 90%, and most preferably 100% of at least one non-outer membrane component compared to the outer membrane vesicles naturally secreted by the bacteria from which they are derived.

[0071] In one embodiment of the present invention, the OMNV of the present invention may be characterized in that it is processed in the following manner to reduce adverse reactions when administered in vivo:

[0072] (1) OMNV can be prepared using genetically transformed bacteria to reduce toxicity. For example, OMNV can be prepared using transformed bacteria to reduce the toxicity of lipopolysaccharide, which mediates host inflammatory responses.

[0073] (2) The toxicity of bacteria can be reduced by using drugs that inhibit endotoxin activity. An example of such a drug is polymyxin B. The drug can be co-administered with OMNV, or OMNV can be prepared from bacteria treated with the drug during culture.

[0074] (3) Adverse reactions can be reduced by using drugs with anti-inflammatory and / or anticoagulant activity. Such drugs include aspirin. Co-administration of OMNV with aspirin can prevent adverse reactions caused by OMNV, such as inflammatory reactions and blood coagulation. In addition, OMNV can be prepared from bacteria treated with drugs during culture.

[0075] (4) The membrane components of OMNV can be chemically modified. For example, the membrane components of OMNV can be chemically modified using thiol or amino groups, or by chemically binding proteins, polyethylene glycol or hyaluronic acid to OMNV.

[0076] (5) The use of sterilized OMNV can prevent infection caused by live bacteria. For example, sterilized OMNV can be obtained by sterilizing with ultraviolet rays and gamma rays or removing bacteria by filtration.

[0077] The method for reducing the adverse reaction of OMNV of the present invention is not limited to the above examples, and each method may be used alone or in combination.

[0078] In one embodiment of the present invention, the OMNV of the present invention may be characterized in that it exhibits therapeutic activity against a disease itself.

[0079] Therefore, the present invention provides a pharmaceutical composition for treating a disease, comprising the OMNV of the present invention.

[0080] In addition, the present invention provides a pharmaceutical composition for treating a disease, which consists of the OMNV of the present invention.

[0081] The present invention provides a pharmaceutical composition for treating a disease, which consists essentially of the OMNV of the present invention.

[0082] In the present invention, "disease" can include various diseases, including cancer. In one embodiment of the present invention, cancer can be selected from thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colon cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, stomach cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penis cancer, prostate cancer, ovarian cancer and cervical cancer, but not limited to this. In one embodiment of the present invention, the disease can be selected from hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, atherosclerosis, obesity, peptic ulcer, Alzheimer's disease,, emphysema, skin disease, skin infection, respiratory tract infection, genitourinary infection, bone and joint infection, central nervous system infection and sepsis, but not limited to this.

[0083] In one embodiment of the present invention, the composition may further comprise substances that enhance the therapeutic effect or alleviate adverse reactions.

[0084] The substance that enhances the therapeutic effect or reduces the adverse reaction can be selected from anticancer agents, immunostimulants, STING agonists, anti-inflammatory agents, endotoxin inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles and nanoparticles, but is not limited thereto.

[0085] "Anticancers" block the replication, transcription and translation of DNA in cancer cells. The types of anticancer agents that can be used as therapeutic substances in the present invention are not particularly limited. Anticancer agents can be selected based on general principles considered when selecting anticancer agents, such as the type of cancer cells, the absorption rate of the anticancer agent (treatment cycle and administration route), the location of the tumor, and the size of the tumor. Anticancer agents that can be used in the present invention include DNA cross-linking agents such as cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozotocin, busulfan, thiotepa, cisplatin and carboplatin; anticancer antibiotics such as dactinomycin (actinomycin D), doxorubicin (adriamycin), epirubicin, idarubicin, mitoxantrone, plicamycin, daunorubicin, mitomycin C and bleomycin; plant alkaloids such as vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan and irinotecan; antibodies such as herceptin and rituximab, but are not limited thereto.

[0086] The "anti-inflammatory agent" may be selected from dexamethasone, indomethacin, ibuprofen, clobetasol propionate, diflorasone diacetate, halobetasol propionate, amcinonide, fluocinolone acetonide, mometasone furoate, desoximetasone, diclofenac and piroxicam, but is not limited thereto.

[0087] "Peptides" and "proteins" may include various growth factors, such as VEGF and EGF; cytokines, such as IL-1, IFN-γ and IL-10; various antibody therapeutics, various peptides or proteins, DNase, and various proteins and peptides that can inhibit cancer growth and metastasis and inhibit inflammatory responses, but are not limited to these.

[0088] "Toxin" is a general term, referring to substances derived from various organisms that can be toxic after being absorbed by the body, and can induce cell death. The type of toxin in the present invention is not particularly limited.

[0089] The "nucleic acid" may be selected from DNA, RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino nucleic acid, but is not limited thereto.

[0090] "Nanoparticles" may be selected from iron oxide, gold, carbon nanotubes and magnetic beads, but are not limited thereto.

[0091] In one embodiment of the present invention, substances that enhance therapeutic effects or alleviate adverse reactions can be loaded into OMNV.

[0092] In the present invention, the term "loading" means displaying a desired substance on the surface of the OMNV membrane, or encapsulating the substance inside the OMNV, but is not limited thereto.

[0093] The pharmaceutical composition of the present invention may also include a pharmaceutically acceptable carrier in addition to the active ingredient. That is, it may be used by mixing one or more of physiological saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, glucose solution, maltodextrin solution, glycerol, ethanol, liposomes and other ingredients, and may also include other conventional additives such as antioxidants and buffers as needed. In addition, diluents, dispersants, detergents, adhesives and / or lubricants may be additionally added to prepare injection preparations (aqueous solutions, suspensions and emulsions), pills, capsules, granules or tablets. Moreover, it may be preferably prepared according to each component using the method disclosed in the relevant technical field. The pharmaceutical composition of the present invention is not particularly limited in terms of preparation, but is preferably formulated into an injection or inhalation preparation.

[0094] The administration of the pharmaceutical composition of the present invention is not particularly limited, but can be administered orally or parenterally according to the expected method, such as intravenous, subcutaneous, intraperitoneal, intranasal, inhaled or topical administration. The dosage varies due to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and disease severity. The daily dose represents the amount sufficient to treat and alleviate the disease state when the therapeutic substance of the present invention is administered to the subject in need of treatment. The effective amount of the therapeutic substance varies according to the specific compound, the disease condition and its severity and the subject in need of treatment, and can be determined by a conventional method by a person skilled in the art. As a non-limiting example, the dosage of the composition of the present invention to people can vary according to the patient's age, weight, sex, administration form, health status and disease severity, and for an adult patient weighing 70 kg, the usual range is 0.1-1000 mg per day, preferably 1-500 mg per day, and can be administered once or more per day at fixed intervals.

[0095] In one embodiment of the present invention, the pharmaceutical composition may further comprise a drug that inhibits the toxicity caused by OMNV. In addition, the drug may be loaded into OMNV. The drug may include a drug that inhibits the toxicity caused by endotoxin, such as polymyxin B.

[0096] The present invention also provides a vaccine composition for preventing or treating a disease, comprising the OMNV of the present invention. The term "vaccine" means artificially injecting an antigen into a human body to activate the immune system to prevent or treat a disease.

[0097] According to one embodiment of the present invention, it has been demonstrated that the OMNV of the present invention itself has excellent activity in inducing innate immunity and, when administered together with an antigen, can serve as an immune adjuvant (ie, a vaccine adjuvant) that enhances the immune response in vivo.

[0098] The terms "immune adjuvant", "vaccine adjuvant" or "adjuvant" refer to a drug or immunological substance that is administered to enhance the immune response of a vaccine, ie, to enhance the immune response elicited by an antigen in the body.

[0099] In one embodiment of the present invention, the vaccine composition may further comprise an antigen.

[0100] The term "antigen" can be interpreted synonymously with "immunogen", indicating that it can induce antibody formation and / or cellular immune response, in particular can activate a specific immune response, and includes proteins, recombinant proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides or polynucleotides of pathogens.

[0101] In one embodiment of the present invention, the antigen may be derived from one or more infectious pathogens selected from bacteria, viruses and fungi. For example, the antigen can be glycoprotein E (gE) antigen of varicella zoster virus; glycoprotein E (gE) antigen of Japanese encephalitis virus; inactivated antigen of seasonal influenza virus; hemagglutinin antigen and neuraminidase antigen of influenza virus; spike antigen of coronavirus; pertussis toxin antigen, filamentous hemagglutinin antigen and pertactin antigen of Bordetella pertussis; antigen of human papillomavirus (HPV); capsular polysaccharide antigens of groups A, B, C, Y and W-135 of Helicobacter pylori; tetanus toxoid antigen of Clostridium tetani; diphtheria toxoid antigen of Corynebacterium diphtheriae; type 3 capsular polysaccharide antigen of Streptococcus pneumoniae; Mycobacterium tuberculosis; tuberculosis); GP-120 and GP-160 antigens of human immunodeficiency virus (HIV); cholera toxin B subunit antigen of Vibrio cholerae; staphylococcal enterotoxin B antigen of Staphylococcus; Shigella polysaccharide antigen of Shigella; vesicular stomatitis virus glycoprotein antigen of vesicular stomatitis virus; antigen of cytomegalovirus (CMV); antigen of hepatitis A / B / C / D / G virus; antigen of respiratory syncytial virus (RSV); or antigen of herpes simplex virus, but are not limited thereto.

[0102] In one embodiment of the present invention, the antigen may be a cancer antigen. For example, the cancer antigen may be an antigen derived from HPV, carcinoembryonic antigen, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), mucin-1 (MUC-1), BCR / ABL, alpha-fetoprotein (AFP), an antigen derived from Epstein-Barr virus (EBV), an antigen derived from HBV, an antigen derived from HCV, cancer antigen-125 (CA-125), cancer antigen-72-4 (CA-72-4), cancer antigen-15-3 (CA-15-3), cancer antigen-19-9 (CA-19-9), or a mutant Ras protein, a Raf protein, a Src protein, a Myc protein, EGFR, PDGFR, VEGFR, p53, PTEN or HER2 / neu, but is not limited thereto.

[0103] In one embodiment of the invention, the antigen may be loaded into the OMNV of the invention, or included in a composition with a separate carrier.

[0104] The vaccine composition can be administered in combination with common adjuvants, such as aluminum hydroxide, aluminum phosphate, alum, MF59, virosomes, AS04 (a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)), AS03 (a mixture of DL-α-tocopherol, squalene and polysorbate 80), CpG DNA, flagellin, Poly I:C, AS01 (a mixture of QS-21, MPL and liposomes), AS02 (a mixture of QS-21, MPL and oil-in-water emulsion), immunostimulatory complexes (ISCOMs) and immunostimulatory complex matrices, but are not limited thereto. In addition, the vaccine composition can also be administered in combination with other drugs to enhance the efficacy of the vaccine or reduce adverse reactions.

[0105] In one embodiment of the present invention, the vaccine composition can be used to prevent or treat infectious diseases caused by bacteria, viruses or fungi. In the present invention, the infection can be selected from skin infection, respiratory tract infection, urogenital infection, bone and joint infection, central nervous system infection and sepsis, but is not limited thereto.

[0106] In one embodiment of the present invention, the vaccine composition can be used to prevent or treat cancer. In the present invention, the cancer can be selected from thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colon cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, stomach cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penis cancer, prostate cancer, ovarian cancer and cervical cancer, but is not limited thereto.

[0107] In one embodiment of the present invention, the vaccine composition can be used to prevent or treat a disease selected from the following: hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, atherosclerosis, obesity, peptic ulcer, Alzheimer's disease, chronic obstructive pulmonary disease, asthma, skin diseases and autoimmune diseases, but not limited thereto.

[0108] According to one embodiment of the present invention, the route of administration of the vaccine adjuvant or the vaccine composition comprising the adjuvant can be any conventional route, as long as it can reach the target tissue. Such routes of administration can include transdermal administration, mucosal administration (e.g., oral, nasal, rectal or vaginal) or parenteral administration, such as intraperitoneal, intravenous, intramuscular, subcutaneous or intrasynovial administration, but are not limited thereto.

[0109] The vaccine composition of the present invention can be formulated into a suitable dosage form together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can include, for example, carriers for parenteral administration, such as water, suitable oils, physiological saline, water-soluble glucose and ethylene glycol, and can also include stabilizers and preservatives. Suitable stabilizers include antioxidants, such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl or propyl parabens and chlorobutanol. In addition, according to the needs of the administration method or preparation, the composition of the present invention can appropriately include suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorbents, detergents, diluents, excipients, pH regulators, analgesics, buffers, antioxidants, etc. Pharmaceutically acceptable carriers and preparations suitable for the present invention include those exemplified above, which are described in detail in the document [Remington's Pharmaceutical Sciences, latest edition].

[0110] The dosage of the vaccine composition for a patient may vary depending on many factors, including the patient's height, body surface area, age, specific compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously. The amount of pharmaceutically active DNA administered may be 100 ng / kg body weight-10 mg / kg body weight, more preferably 1-500 μg / kg body weight, most preferably 5-50 μg / kg body weight, and the dosage will be adjusted taking into account the above factors.

[0111] Furthermore, the vaccine composition of the present invention is administered in a pharmaceutically effective amount.

[0112] The present invention also provides use of the OMNV of the present invention in preparing a pharmaceutical composition for treating a disease.

[0113] The present invention also provides a method for treating a disease, comprising administering an effective amount of the OMNV pharmaceutical composition of the present invention to a subject in need thereof.

[0114] The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors such as the type and severity of the disease, age, gender, drug activity, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The vaccine composition of the present invention can be administered at a dose of 0.1 mg / kg-1 g / kg, and more preferably at a dose of 1 mg / kg-500 mg / kg. At the same time, the dosage can be appropriately adjusted according to the patient's age, gender and disease condition.

[0115] The "effective amount" of the present invention refers to an amount that, when administered to a subject, shows the effect of improving, treating, detecting, diagnosing, inhibiting or alleviating cancer and the diseases described in the present invention, and the "subject" can be an animal, preferably a mammal, especially an animal including a human, or a cell, tissue, organ, etc. derived from an animal. The subject can be a patient in need of the effect.

[0116] In the present invention, "treatment" refers in a broad sense to improving symptoms caused by cancer or the above diseases, which may include curing, substantially preventing or improving the disease condition, and includes alleviating, curing or preventing one or most of the symptoms caused by the disease, but is not limited thereto.

[0117] As used herein, the term "comprising" is synonymous with "including" or "characterized by," and does not exclude additional components or steps not specifically mentioned in the compositions or methods of the invention. The term "consisting of" indicates the exclusion of additional elements, steps or components not specifically described. The term "consisting essentially of" indicates that the scope of the composition or method may include substances or steps that do not materially affect the basic characteristics of the substances or steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 A schematic diagram showing a method for preparing Gram-negative bacteria outer membrane-derived nanovesicles (OMNVs).

[0119] Figure 2a-2i Characterization of Gram-negative outer membrane vesicles (OMV) and nanovesicles OMNV is shown. (a) Transmission electron microscopy image, (b) size distribution determined by dynamic light scattering analysis, (cd) yield ((c) total number of particles per culture volume, and (d) total protein amount per culture volume), (e) total number of particles per total protein amount, (f) SDS-PAGE results, and (g) Western blotting results using anti-OmpA antibodies. In addition, there are (h) results of measuring AmCyan fluorescence values ​​per culture volume and (i) total nucleic acid amounts of whole cell lysates and OMNV. a: Scale bar = 100nm.

[0120] Figure 3a-3d (a) HEK-Blue treated with OMV and OMNV TM hTLR2 and (b) HEK-Blue TM Results for hTLR4 are shown, and the extent of the response is depicted in a graph. In addition, there are results for measuring (c) TNF-α and (d) IL-6 concentrations in the conditioned medium by ELISA after treatment of mouse macrophages RAW264.7 with OMVs and OMNVs.

[0121] Figure 4a-4e Characterization of OMNV prepared from various Gram-negative bacteria is shown. (a) Transmission electron microscopy image, (b) size distribution determined by dynamic light scattering analysis, (cd) yield (c-total number of particles per culture volume, d-total amount of protein per culture volume), and (e) total number of particles per total amount of protein. a: Scale bar = 200 nm.

[0122] Figure 5a-5b (a) HEK-Blue treated with OMNV prepared from various Gram-negative bacteria TM hTLR2 and (b) HEK-Blue TM Results for hTLR4 are shown, and the extent of the response is depicted in a graph. In addition, there are results of measuring the concentrations of (c) TNF-α and (d) IL-6 in the conditioned medium by ELISA after RAW264.7 cells were treated with OMNVs derived from various Gram-negative bacteria.

[0123] Figure 6 Shown are the results of adding polyethylene glycol to the surface of OMNV.

[0124] Figure 7a-7b Figure 2 shows the OMNVs (OMNVs) loaded with the anticancer drug daunorubicin (DNR) during preparation as confirmed by (a) fluorescence microscopy and (b) flow cytometry. DNR ) The results of whether daunorubicin can be delivered to target cells. a: Scale bar = 20 μm.

[0125] Figure 8a-8b The results of (a) fluorescence microscopy and (b) flow cytometry confirm that OMNVs loaded with daunorubicin (OMNV DNR-Inc ) The results of whether daunorubicin can be delivered to target cells. a: Scale bar = 20 μm.

[0126] Figure 9a-9b The results show that the OMNVs loaded with daunorubicin by sonication after preparation were confirmed by (a) fluorescence microscopy and (b) flow cytometry. DNR-Son) The results of whether daunorubicin can be delivered to target cells. a: Scale bar = 20 μm.

[0127] Figure 10a-10b The results show that the OMNVs loaded with daunorubicin after freeze-thawing after preparation were confirmed by (a) fluorescence microscopy and (b) flow cytometry. DNR-FT ) The results of whether daunorubicin can be delivered to target cells. a: Scale bar = 20 μm.

[0128] Fig.11 OMNV (OMNV DEX ) After RAW264.7 cells were treated with ELISA to measure the release of IL-6.

[0129] Figure 12a-12b The results show that OMNVs loaded with GFP (OMNV GFP ) The results of whether GFP can be delivered to target cells. a: Scale bar = 20 μm.

[0130] Fig.13 The results of confirming adverse reactions after intraperitoneal administration of OMNV to mice are shown.

[0131] Figures 14a-14e The results show that OMNV labeled with Cy7 was administered intraperitoneally to mice. Cy7 Results of the in vivo distribution of OMNV over time after administration. (a, b) observed the distribution of the whole body of mice over time by IVIS, (c) observed the distribution in major organs 24 hours after administration, and (de) showed the distribution degree in major organs in the form of graphs.

[0132] Figure 15a-15b Graphical results showing the change in cancer tissue size over time after the mouse bladder cancer cell line MB49 was introduced into mice and OMNV was administered to the cancer.

[0133] Figures 16a-16d Shown are the results of (a) determination of cell viability following treatment of dendritic cells with OMNV, and measurement of (b) TNF-α, (c) IL-6, and (d) IL-12p40 concentrations in conditioned media by ELISA.

[0134] Figure 17a-17b Alexa 594-labeled OMNV ( Alexa 594 The results of whether dendritic cells take up OMNV were analyzed by treating dendritic cells with OMNV. a: Scale bar = 5 μm.

[0135] Figures 18a-18e Shown are (a) the experimental procedure for intraperitoneal administration of OMNV with a mixture of antigen (GFP or SARS-CoV-2 S protein) and adjuvant (alum) or CuSO4 to mice; (be) graphical results of serum IgG titers against GFP or S protein 7, 14 and 21 days after administration.

[0136] Figures 19a-19e Shown are (a) the experimental procedure of intraperitoneal administration of OMNV with a mixture of antigen (GFP or SARS-CoV-2 S protein) and adjuvant (Alum 4) or CuSO to mice; (b) graphical results of serum IgG titers against GFP or S protein 21 and 49 days after administration.

[0137] Figures 20a-20e Shown are (a) the experimental procedure for intraperitoneal administration of a mixture of OMNV with an antigen (GFP or SARS-CoV-2 S protein) and an adjuvant (alum) or CuSO4 to mice; (be) graphical results of T cell responses (IFN-γ secretion) against GFP or S protein 49 days after administration.

[0138] Figures 21a-21e Shown are (a) the experimental procedure of intraperitoneal or intramuscular administration of OMNV with a mixture of antigen (H5N1 hemagglutinin) and adjuvant (alum) to mice; (b) graphical results of serum IgG titers against H5N1 hemagglutinin 7, 14, 21 and 45 days after administration (b, c - intraperitoneal administration; d, e - intramuscular administration).

[0139] Figures 22a-22e Figure 2 shows (a) a plasmid map for expressing antigens (S1 RBD; SARS-CoV-2 spike protein 1 receptor binding domain or H5N1 hemagglutinin) in bacteria based on an outer membrane protein display system; (b) a plasmid map for isolating OMNV expressing antigens ( S1RBD- OmpA OMNV, S1 RBD-LolB OMNV, H5N1-LolB (c-e) Results of Western blotting of OMNV antigen using anti-His6 antibody.

[0140] Figures 23a-23f The figures show the results of measuring the concentrations of TNF-α (a, d), IL-6 (b, e), and IL-12p40 (c, f) in the conditioned medium by ELISA after dendritic cells were treated with OMNV expressing antigens based on the outer membrane protein display system.

[0141] Figures 24a-24eShown are (a) the experimental process of intraperitoneal administration of OMNV expressing antigen to mice based on the outer membrane protein display system; (b, c) serum IgG titers against S1 RBD 5, 11 and 17 days after administration; (d) serum IgG titers against S1 RBD 49 days after administration, and (e) T cell response (IFN-γ secretion) against S1 RBD 49 days after administration.

[0142] Figures 25a-25c Shown are (a) the experimental procedure of intramuscular administration of OMNV expressing antigens into mice based on the outer membrane protein display system; (b, c) serum IgG titers against S1 RBD or H5N1 hemagglutinin 5, 11 and 17 days after administration.

[0143] Figures 26a-26f The graph shows the survival rate and body weight change (df) of mice when OMNV expressing antigens were intraperitoneally administered to mice based on the outer membrane protein display system.

[0144] Figure 27a-27b Shown are (a) a schematic diagram of the process of loading proteins into OMNVs; and (b) the amount of proteins loaded into OMNVs.

[0145] Figures 28a-28e OMNV isolated by various methods are shown for characterization. (a) Transmission electron microscopy image. (b) Size distribution determined by dynamic light scattering analysis. (c, d) Yield ((c) - total number of particles per culture volume, (d) - total amount of protein per culture volume). (e) Total number of particles per total amount of protein. a: Scale bar = 200 nm.

[0146] Figures 29a-29d OMNV isolated by various methods are shown to (a) HEK-Blue TM hTLR2 and (b) HEK-Blue TM Also shown are the concentrations of (c) TNF-α and (d) IL-6 in the conditioned medium measured by ELISA after RAW264.7 cells were treated with OMNV isolated by various methods.

[0147] Fig.30 The graph shows the changes in tumor tissue size over time when OMNV isolated by various methods was intratumorally administered after the mouse bladder cancer cell line MB49 was introduced into mice.

[0148] Fig.31 Schematic diagram showing various preparation methods and applications of OMNV. Example

[0149] The present invention is described in more detail below by way of examples. These examples are only used to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

[0150] Example 1. Preparation of Gram-negative bacterial outer membrane nanovesicles (OMNV)

[0151] To prepare OMNV, E. coli BL21 (DE3) ΔmsbB-AmCyan was cultured in a lysogenic culture broth (VP-LB) based on plant peptone, and the culture was then centrifuged at 6,000 x g for 20 minutes at 4°C, and the bacteria were obtained by removing the supernatant. The obtained bacteria were suspended in Tris-based saline (TBS; 50 mM Tris-HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl) at a ratio of 100 mL per 1 L of culture medium. The suspension was centrifuged at 6,000 x g for 20 minutes at 4°C, and the bacterial cells were washed by removing the supernatant.

[0152] The washed bacterial cells were quickly frozen with liquid nitrogen, then suspended in 30 mL of buffer A (10 mM Tris-HCl (pH 8.0), 5% sucrose), and treated with Sarkosyl at a final concentration of 1% for 30 minutes at room temperature. The suspension was centrifuged at 10,000 x g for 20 minutes at 4°C to remove the supernatant. Next, the precipitate was suspended in 30 mL of buffer A and treated with Sarkosyl at a final concentration of 0.25% for 10 minutes at 4°C. The suspension was centrifuged at 10,000 x g for 20 minutes at 4°C to remove the supernatant. The precipitate was suspended in 50 mL of buffer A, and the operation of centrifuging at 10,000 x g for 20 minutes at 4°C and removing the supernatant was repeated three times to remove Sarkosyl and separate the outer membrane of Gram-negative bacteria.

[0153] The outer membrane of Gram-negative bacteria was suspended in 50 mL of buffer A and then circulated through a microfluidizer (Microfluidics, MA) at 10,000 psi for 4 times to prepare OMNV. The solution passed through the microfluidizer was centrifuged at 6,000 x g for 20 minutes at 4 ° C to obtain the supernatant, and substances greater than 500 kDa were concentrated by tangential flow filtration and dialyzed using buffer A to obtain about 7 mL of sample. Finally, 3 mL of 2.5 M sucrose, 3 mL of 0.8 M sucrose, and 7 mL of sample were placed in an ultracentrifuge tube in sequence, ultracentrifuged at 200,000 x g for 2 hours at 4 ° C, and OMNV was finally obtained from the layer between 2.5 M sucrose and 0.8 M sucrose.

[0154] Schematic diagram of the method for preparing OMNV Figure 1 shown.

[0155] Example 2. Isolation of Gram-negative bacterial outer membrane vesicles (OMV)

[0156] The bacteria used to separate OMVs were Escherichia coli BL21 (DE3) ΔmsbB-AmCyan. The bacteria were cultured in VP-LB and the culture was centrifuged at 6,000 x g for 20 minutes at 4 ° C. The supernatant was filtered using a filter with a pore size of 0.45 μm, and the filtrate was concentrated to substances greater than 100 kDa using tangential flow filtration. The concentrate was ultracentrifuged at 100,000 x g for 2 hours at 4 ° C, and the precipitate was suspended in 50% iodixanol. In an ultracentrifuge tube, 2 mL of 50% iodixanol suspension, 1.5 mL of 40% iodixanol, and 1 mL of 10% iodixanol were added in sequence, and ultracentrifuged at 200,000 x g for 2 hours at 4 ° C to separate OMVs from the layer between 40% iodixanol and 10% iodixanol.

[0157] Example 3. Characterization of OMVs and OMNVs

[0158] 3-1. Analysis of the morphology and size of OMVs and OMNVs.

[0159] In order to analyze the morphology of the OMNV prepared by the method of Example 1 and the OMV separated by the method of Example 2, they were observed using a transmission electron microscope. OMV and OMNV were adsorbed on a glow-discharged, carbon-coated copper grid for 20 minutes. The grid was washed with distilled water, stained with 1% uranyl acetate for 2 seconds, and observed using a transmission electron microscope (JEM1011, JEOL, Japan). As a result, Figure 2a As shown, it was confirmed that both OMVs and OMNVs contain a lipid bilayer with a diameter of 50-100 nm.

[0160] In addition, to measure the size of OMVs and OMNVs, they were diluted to a concentration of 1 μg / mL in HEPES-buffered saline (HBS, 20 mM HEPES (pH 7.4), 150 mM NaCl) and analyzed using a dynamic light scattering particle size analyzer (Zetasizer NanoZS, Malvern Instruments, UK). Figure 2b As shown, it was confirmed that the average diameters of OMVs and OMNVs were 70.1±11.4 nm and 74.3±15.8 nm, respectively.

[0161] 3-2. Analysis of total particle number and total protein content of OMV and OMNV

[0162] To measure the total particle number of OMV and OMNV, they were diluted to a concentration of 1 μg / mL in HBS, placed in a sample chamber, and analyzed using a nanoparticle tracking analyzer (Nanosight LM-10, Malvern Instruments, UK). Figure 2c As shown, it was confirmed that in each 1L culture, an average of (1.33±0.24) x 10 11 particles and (7.35±3.54) x 10 13 This indicates that the amount of OMNV obtained is about 550 times that of OMV in terms of total particle number.

[0163] In addition, the total protein amount of OMV and OMNV was quantified using Bradford assay. Figure 2d As shown, it was confirmed that an average of 0.812±0.001 mg and 14.200±0.283 mg of OMV and OMNV were obtained per 1 L of culture, respectively, which indicated that in terms of total protein amount, the OMNV obtained was approximately 17 times that of OMV.

[0164] Furthermore, to confirm the purity of OMVs and OMNVs, the total number of particles per total protein amount was calculated. Figure 2e As shown in Figure 2, the purity of OMV and OMNV was (0.164±0.024) x 10 9 Particles / μg and (5.177±0.220)x 10 9 This indicates that the purity of OMNV is about 32 times that of OMV.

[0165] 3-3. Analysis of protein distribution and the presence of outer membrane proteins in OMVs and OMNVs

[0166] First, to prepare whole cell lysate, E. coli BL21 (DE3) ΔmsbB-AmCyan culture was centrifuged at 6,000 x g for 20 minutes at 4°C. After removing the supernatant, the pellet was suspended in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF). Then, the suspension was sonicated to obtain whole cell lysate.

[0167] To verify the protein distribution of whole cell lysates, OMVs, and OMNVs, a total of 5 μg of whole cell lysates, OMVs, and OMNVs were subjected to non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue. Figure 2fAs shown, whole cell lysates and OMVs exhibited various protein bands, whereas OMNVs showed only a limited number of protein bands.

[0168] To verify the presence of outer membrane proteins in whole-cell lysates, OMVs and OMNVs, a total amount of 1 μg of whole-cell lysates, OMVs and OMNVs were subjected to non-reducing electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes using the semi-dry transfer method. Subsequently, the membrane was blocked with 3% skim milk / TBS-T (0.05% Tween 20) solution for 1 hour, and then incubated with a primary antibody specific for OmpA for 2 hours at room temperature. After washing the membrane three times with TBS-T (0.05% Tween 20), a secondary antibody labeled with horseradish peroxidase was added and incubated at room temperature for 1 hour. The membrane was washed three times again with TBS-T (0.05% Tween 20), and the expression of OmpA was detected using an enhanced chemiluminescence (ECL) substrate. Figure 2g As shown, OmpA was more highly expressed in OMVs and OMNVs compared to whole cell lysates.

[0169] 3-4. Measurement of AmCyan fluorescence and total nucleic acid content in whole cell lysates and OMNV

[0170] To verify the presence of cytoplasmic proteins in whole cell lysates and OMNV, the relative amount of the cytoplasmic fluorescent protein AmCyan was measured. Fluorescence was measured using a spectrophotometer at an excitation wavelength of 457 nm and an emission wavelength of 491 nm. Figure 2h As shown, OMNVs contain almost no cytoplasmic fluorescent proteins compared to whole-cell lysates.

[0171] To verify the total nucleic acid content in whole cell lysates and OMNVs, the absorbance at a wavelength of 260 nm was measured using a micro-volume spectrophotometer. Figure 2i As shown, OMNV contained almost no nucleic acid compared to whole cell lysates.

[0172] In summary, a method for producing and purifying high-purity OMNV was established, which increased the production yield by tens to hundreds of times compared with OMV and effectively removed unnecessary substances such as cytosolic proteins and nucleic acids by disrupting the bacterial inner membrane.

[0173] Example 4. Determination of innate immunity inducing activity of OMV and OMNV

[0174] To measure the innate immune induction activity of the OMNV prepared by the method of Example 1 and the OMV isolated by the method of Example 2, the OMV and OMNV were used to treat HEK-Blue TM hTLR2 and HEK-Blue TMhTLR4 and the extent of the response was determined. In addition, the release of cytokines was measured by treating mouse macrophages RAW264.7 with it.

[0175] To measure HEK-Blue TM hTLR2 and HEK-Blue TM The response of hTLR4 to OMV and OMNV was firstly measured by adding 2x10 5 Cells / well) were inoculated onto the well plate and kept for 24 hours. Then, various concentrations (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10000 ng / mL) of OMV and OMNV were used to treat each cell for 18 hours, and conditioned medium was collected. The collected conditioned medium was reacted with a chemiluminescent substrate to measure RLU, and the RLU of the conditioned medium of the cells not treated with OMV or OMNV was normalized.

[0176] To quantify the extent of RAW264.7 response to OMV and OMNV, RAW264.7 (2x10 4 Cells / well) were inoculated onto the plate for 24 hours. Then, various concentrations (0, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 ng / mL) of OMV and OMNV were used to treat RAW264.7 for 24 hours, and the conditioned medium was collected. mTNF-α and mIL-6 in the collected conditioned medium were quantified by ELISA.

[0177] like Figure 3a As shown in Figure 3, OMV and OMNV can induce innate immunity in a dose-dependent manner, and at the same concentration, OMV induces innate immunity to a greater extent than OMNV. These results indicate that OMNV induces innate immunity with fewer adverse reactions than OMV.

[0178] Example 5. Characterization of OMNV prepared from various types of Gram-negative bacteria

[0179] 5-1. Morphology and size determination of OMNV prepared from various types of Gram-negative bacteria

[0180] OMNVs were prepared by culturing Escherichia coli BL21 (DE3) wild type, Pseudomonas aeruginosa PAO1 wild type, Salmonella enterica wild type and S. enterica ΔmsbB according to the method of Example 1. In order to analyze the morphology and size of OMNVs prepared from various Gram-negative bacteria, transmission electron microscopy and dynamic light scattering analysis were performed according to Example 3-1. Figure 4a As shown in Figure 2, all OMNVs prepared from various Gram-negative bacteria were confirmed to contain a lipid bilayer. Figure 4b As shown, the average diameters of OMNVs from Escherichia coli BL21 (DE3) wild type, Pseudomonas aeruginosa PAO1 wild type, Salmonella Enteritidis wild type and Salmonella Enteritidis ΔmsbB were 125.5±40.2 nm, 124.0±39.3 nm, 144.6±48.9 nm and 154.8±32.1 nm, respectively.

[0181] 5-2. Determination of the total particle number and total protein amount of OMNV prepared from various types of Gram-negative bacteria

[0182] In order to determine the total particle number and total protein amount of OMNV prepared from various types of Gram-negative bacteria, the analysis and quantification were performed using a nanoparticle tracking analyzer and Bradford assay with reference to Example 3-2. Figure 4c As shown in Figure 2, the average number of OMNV particles from wild-type Escherichia coli BL21 (DE3), wild-type Pseudomonas aeruginosa PAO1, wild-type Salmonella Enteritidis, and wild-type Salmonella Enteritidis ΔmsbB per liter of culture medium was (2.10 ± 0.22) x 10 13 Particles, (2.56±0.09) x 10 13 Particles, (3.52±0.23)x 10 13 particles and (3.36±0.59)x 10 13 In addition, Figure 4d As shown, the average total protein amounts of OMNV from Escherichia coli BL21 (DE3) wild type, Pseudomonas aeruginosa PAO1 wild type, Salmonella Enteritidis wild type and Salmonella Enteritidis ΔmsbB per liter of culture medium were 12.733±1.803 mg, 11.043±1.866 mg, 24.151±3.077 mg and 25.479±3.619 mg, respectively.

[0183] In addition, to determine the purity of OMNV prepared from various types of Gram-negative bacteria, the total number of particles per total protein amount was calculated. Figure 4eAs shown in Figure 2, the purity of OMNV from wild-type Escherichia coli BL21 (DE3), wild-type Pseudomonas aeruginosa PAO1, wild-type Salmonella Enteritidis, and wild-type Salmonella Enteritidis ΔmsbB were (1.657 ± 0.112) x 10 9 Particles / μg, (2.353±0.349)x 10 9 Particles / μg, (1.467±0.140)x 10 9 Particles / μg and (1.320±0.193)x 10 9 Particles / μg.

[0184] Example 6. Analysis of the innate immunity inducing activity of OMNV prepared from various Gram-negative bacteria

[0185] The innate immunity inducing activity of OMNV prepared from various Gram-negative bacteria by the method of Example 5 was analyzed using the method of Example 4. Figure 5a-5d As shown, OMNV prepared from various Gram-negative bacteria were confirmed to be able to induce innate immunity in a dose-dependent manner.

[0186] Example 7. Preparation of OMNV containing polyethylene glycol

[0187] Polyethylene glycol was incorporated into the surface of the OMNV prepared by the method of Example 1 to prepare OMNV containing polyethylene glycol on the surface.

[0188] The OMNV prepared by the method of Example 1 was diluted to 1 mg / mL with HEPES buffered saline (20 mM HEPES (pH 7.4), 150 mM NaCl) and then mixed so that the final concentrations of cholesterol-PEG and cholesterol-PEG-biotin were 20 μg / mL and 2 μg / mL, respectively. Subsequently, the mixture was incubated at 4° C. for 30 minutes, and PEGylated OMNV ( 聚乙二醇化 OMNV).

[0189] To measure 聚乙二醇化 OMNV was pegylated by adding various concentrations (0, 100, 300, 1000 ng / mL) of OMNV to the 聚乙二醇化 OMNV were seeded into 96-well plates at room temperature for 2 hours. Then, they were blocked with 1% BSA / PBS at room temperature for 1 hour and treated with streptavidin-HRP for 30 minutes at room temperature. Finally, luminescence was measured using a multi-mode microplate reader equipped with BM chemiluminescent substrate to confirm PEGylation.

[0190] Results, such as Figure 6 As shown, it was found 聚乙二醇化In the case of OMNV, cholesterol-polyethylene glycol-biotin binds to OMNV and shows high RLU. On the other hand, OMNV shows low RLU.

[0191] Example 8. Drug delivery using OMNV

[0192] 8-1. Using OMNV-1 to deliver anticancer drugs: OMNV loaded with anticancer drugs during preparation

[0193] In order to determine whether the anticancer drug daunorubicin (DNR) can be delivered using the OMNV prepared by the method of Example 1, nanovesicles loaded with daunorubicin were prepared according to the method of Example 1. In the step of treating with Sarkosyl to prepare the outer membrane of Gram-negative bacteria, 500 μg / mL of daunorubicin was added. Subsequently, continuous extrusion was performed to prepare OMNV loaded with daunorubicin (OMNV DNR ).

[0194] To verify whether OMNV can be used DNR To deliver daunorubicin to target cells, Alexa 488 NHS ester was conjugated to OMNV DNR Incubate with the cells and separate only the fluorescently labeled OMNVs by size exclusion chromatography DNR ( Alexa488 OMNV DNR RAW264.7 cells were cultured on glass slides treated with gelatin and treated with 1 μg / mL Alexa488 OMNV DNR The cells were treated for 24 hours, and then the cell nuclei were stained with Hoechst dye and observed under a fluorescence microscope. In addition, various concentrations (0, 10, 100, 1000 ng / mL) of Alexa 488 OMNV DNR RAW264.7 cells were treated and flow cytometry was performed 24 hours later.

[0195] Results, such as Figure 7a-7b As shown, OMNV DNR Delivery of daunorubicin to target cells.

[0196] 8-2. Delivery of anticancer drugs using OMNV-2: Preparation of OMNV loaded with anticancer drugs by various methods

[0197] In order to determine whether daunorubicin (DNR) can be delivered to target cells even after OMNVs were prepared by the method of Example 1 and then loaded by various methods, OMNVs were prepared by the method of Example 1 and then loaded with daunorubicin by incubation, ultrasonic treatment and freeze-thaw methods.

[0198] First, in order to load daunorubicin into OMNV by incubation, OMNV (1 mg / mL) was mixed with daunorubicin (500 μg / mL) and incubated at 37 ° C for 6 hours. In addition, to load daunorubicin into OMNV by ultrasonic treatment, OMNV (1 mg / mL) was mixed with daunorubicin (500 μg / mL), and then ultrasonic treatment was performed 10 times under the conditions of 50% amplitude, 0.5 cycle, and 4 ° C. Finally, to load daunorubicin into OMNV by freeze-thaw, OMNV (1 mg / mL) was mixed with daunorubicin (500 μg / mL), frozen in liquid nitrogen for 3 minutes, and then thawed for 3 minutes at 37 ° C, and repeated 10 times. The OMNV loaded with daunorubicin was separated from the mixture of incubation, ultrasonic treatment and freeze-thaw treatment by size exclusion chromatography. Finally, OMNV was prepared, and the OMNV loaded with daunorubicin by incubation, ultrasonic treatment and freeze-thaw method was named OMNV DNR-Inc OMNV DNR-Son and OMNV DNR- FT.

[0199] In order to verify whether OMNV can be used DNR-Inc OMNV DNR-Son and OMNV DNR-FT ,OMNV DNR-Inc Daunorubicin is delivered to target cells and OMNV DNR-Inc OMNV DNR-Son and OMNV DNR-FT Incubate with Alexa 488 NHS ester and separate the fluorescently labeled ones by the method of Example 8-1 ( Alexa 488 OMNV DNR-Inc , Alexa488 OMNV DNR-Son , Alexa 488 OMNV DNR-FT ). RAW264.7 was treated with these OMNVs by the method of Example 8-1, and then flow cytometry and fluorescence microscopy were performed. Figure 8a-8b , 9a-9b and 10a-10b, OMNV DNR-Inc OMNV DNR-Son and OMNV DNR-FT Delivery of daunorubicin to target cells.

[0200] 8-3. Using OMNV-2 to deliver anti-inflammatory drugs

[0201] In order to determine whether the anti-inflammatory drug dexamethasone (DEX) can be delivered using the OMNV prepared by the method of Example 1, OMNV loaded with dexamethasone was prepared by the method of Example 1. In the step of treating with Sarkosyl to prepare the outer membrane of Gram-negative bacteria, 500 μg / mL of dexamethasone was added, and then extrusion was performed to prepare OMNV loaded with dexamethasone (OMNV DEX ).

[0202] In order to verify whether OMNV can be used DEX Inducing anti-inflammatory response, RAW264.7 (5x10 4 Cells / well) were inoculated on the plate for 24 hours. Subsequently, various concentrations (10, 30, 100, 300, 1000 ng / mL) of OMNV were used. DEX and OMNV prepared in Example 1 DEX The cells were treated for 18 hours and the conditioned medium was obtained. mIL-6 in the obtained conditioned medium was quantified by ELISA.

[0203] Results, such as Fig.11 As shown, OMNV DEX It can inhibit the secretion of IL-6 by macrophages in a dose-dependent manner.

[0204] Example 9. Protein delivery using OMNV

[0205] In order to determine whether the OMNV prepared by the method of Example 1 can be used to deliver proteins, OMNVs of GFP were prepared according to the method of Example 1. In the step of treating with Sarkosyl to prepare the outer membrane of Gram-negative bacteria, 500 μg / mL of GFP was added, and then continuous extrusion was performed to prepare OMNVs loaded with GFP (OMNV GFP ).

[0206] To verify whether OMNV can be used GFP To deliver GFP to target cells, Alexa 594 NHS ester was conjugated to OMNV GFP Incubate with the fluorescently labeled ( Alexa 594 OMNV GFP ). RAW264.7 were cultured on slides treated with gelatin and Alexa 594 OMNV GFP The cells were treated with 1 μg / mL for 24 h, and then the nuclei were stained with Hoechst and observed under a fluorescence microscope. In addition, various concentrations (0, 10, 100, 1000 ng / mL) of Alexa 594OMNV GFP RAW264.7 cells were treated and flow cytometry was performed 24 hours later.

[0207] Results, such as Figure 12a-12b As shown, OMNV GFP Delivery of GFP to target cells.

[0208] In summary, OMNVs can be used to deliver various drugs and proteins to target cells.

[0209] Example 10. Analysis of adverse reactions to OMNV administration

[0210] In order to verify the adverse reactions of OMNV administration prepared by the method of Example 1, OMNV (50 μg / mouse) was intraperitoneally administered to C57BL / 6 (male, 6 weeks old) mice, and serum was prepared from the mouse blood 24 hours later. Then, the serum was subjected to blood biochemical analysis using an automatic chemical analyzer (BS-390, Mindray, China).

[0211] Results, such as Fig.13 As shown, there was no significant difference between the mice administered with OMNV and the control group in most indicators, with only a slight significant increase in the concentration of C-reactive protein.

[0212] In summary, OMNV has few adverse reactions, which makes it extremely valuable in developing therapeutic and diagnostic methods for cancer, methods of delivering substances for disease treatment and diagnosis, and vaccine delivery methods for the prevention and treatment of diseases including cancer and infectious diseases.

[0213] Example 11. In vivo distribution analysis after OMNV administration.

[0214] In order to analyze the in vivo distribution of OMNV prepared by the method of Example 1 after administration, Cy7-labeled OMNV ( Cy7 OMNV) and free Cy7 was removed by size exclusion chromatography. Cy7 OMNV was intraperitoneally administered to mice, and systemic distribution was measured over time using an in vivo imaging system (IVIS). In addition, 24 hours after administration, major organs (thymus, spleen, heart, liver, lung, kidney, skin at the injection site, small intestine) were collected and analyzed for distribution within these organs using an IVIS device. Cy7 Dye controls were used as controls.

[0215] Results, such as Fig.14a As shown, in the whole body distribution, it is not detectable Cy7 Dye control; Fig.14b As shown, over time, Cy7OMNV spreads around the peritoneal cavity. Fig.14c and 14d As shown, in the organ distribution, no Cy7 Dye control; Cy7 OMNV showed strong signals in the liver and small intestine, and when the signals were normalized to organ weight, strong signals were detected in the thymus, spleen, heart, and liver.

[0216] Example 12. Analysis of the anti-tumor activity of OMNV

[0217] In order to determine the antitumor activity of OMNV prepared by the method of Example 1, MB49 (mouse bladder cancer cells, 1×10 6 10 cells / mouse) were subcutaneously introduced into C57BL / 6 (male, 7-week-old) mice, and the size of the tumor tissue was measured over time using a vernier caliper (size = 0.5 × width × length × length). When the size of the tumor tissue reached approximately mm 3 OMNV was intratumorally administered three times at 3-day intervals (0, 1, or 10 μg / mouse each time), and the size of tumor tissue was measured over time.

[0218] Results, such as Fig.15a and 15b As shown, OMNV exhibited antitumor activity.

[0219] Example 13. Analysis of the interaction between OMNV and dendritic cells

[0220] 13-1. Analysis of the effect of OMNV on the survival rate and cytokine release of dendritic cells

[0221] In order to determine the effects of OMNV prepared by the method of Example 1 on the survival rate and cytokine release of dendritic cells, bone marrow-derived dendritic cells (BMDCs) were isolated and treated with OMNV to confirm the survival rate and cytokine release of dendritic cells.

[0222] Femurs and tibias of C57BL / 6 (male, 4-week-old) mice were collected, and DMEM (2.5% FBS) was administered to the bone marrow to separate bone marrow cells, which were cultured with RPMI1640 (10% FBS, 50 μM 2-mercaptoethanol, 20 ng / mL rmGM-CSF) to separate bone marrow-derived dendritic cells. The isolated bone marrow-derived dendritic cells (1×10 5Cells / well) were seeded onto well plates for 24 hours and treated with OMNV at various concentrations (0, 1, 3, 10, 30, 100, 300, 1000 ng / mL) for 24 hours. Cell viability was confirmed by MTT assay, and mTNF-α, mIL-6, and mIL-12p40 in conditioned medium were quantified using ELISA.

[0223] Results, such as Fig.16a As shown in Figure 2, OMNV did not affect the survival of DCs; and as Fig.16b , 16c As shown in Figure 16d, they induced the release of mTNF-α, mIL-6, and mIL-12p40 in a dose-dependent manner.

[0224] 13-2. Analysis of OMNV uptake by dendritic cells

[0225] In order to verify whether dendritic cells can effectively absorb OMNV prepared by the method of Example 1, Alexa594-labeled OMNV ( Alexa 594 OMNV) and free Alexa 594 was removed by size exclusion chromatography. Alexa 594 OMNV was treated with dendritic cells, and uptake by dendritic cells was determined by fluorescence microscopy and flow cytometry. Alexa 594 The dye control was used as a control group.

[0226] Results, such as Fig.17a and 17b As shown, dendritic cells were determined to take up Alexa 594 OMNV, and Alexa 594 The dye control has no absorption.

[0227] Example 14. Vaccination using OMNV and antigens

[0228] 14-1. Analysis of short-term immune-inducing activity of OMNV

[0229] To determine the short-term immune-inducing activity of OMNV prepared by the method of Example 1, antigen (GFP or SARS-CoV-2S protein) and adjuvant (alum or CuSO4) were mixed with OMNV and intraperitoneally administered to C57BL / 6 (male, 4 weeks old) mice three times at weekly intervals. Blood was collected on days 7, 14, and 21 after the first administration (day 0), and serum was separated. The experimental process is as follows Fig.18a shown.

[0230] The antigen (GFP or SARS-CoV-2S protein) was incubated overnight (100ng / well) at room temperature in a 96-well black plate and washed three times with PBS-T (0.05% Tween 20). Then, it was blocked with 1% BSA / PBS for 1 hour at room temperature and then washed three times with PBS-T (0.05% Tween 20). Mouse serum was diluted in 1% BSA / PBS and then added to the plate for treatment for 2 hours at room temperature, and then washed three times with PBS-T (0.05% Tween 20). Subsequently, the secondary antibody (anti-mouse IgG-HRP) coupled to peroxidase was diluted in 1% BSA / PBS and added to the plate for treatment for 1 hour at room temperature, and then washed three times with 1% BSA / PBS. Finally, luminescence was measured using a multi-mode microplate reader equipped with BM chemiluminescent substrate to confirm the antigen-specific IgG titer.

[0231] Results, such as Figure 18b-18e As shown, the group administered with antigen, adjuvant (alum or CuSO4) and OMNV showed higher antigen-specific IgG titers than the control group and the group administered with antigen and adjuvant (alum or CuSO4) only. This result confirms that the co-administration of OMNV with antigen and adjuvant (alum or CuSO4) can more effectively induce short-term immunity.

[0232] 14-2. Analysis of long-term immune-inducing activity of OMNV

[0233] To confirm the long-term immune-inducing activity of OMNV, antigen (GFP or SARS-CoV-2S protein) and adjuvant (alum or CuSO4) were mixed with OMNV and intraperitoneally administered to C57BL / 6 (male, 4 weeks old) mice three times at weekly intervals. Blood was collected 21 and 49 days after the first administration (day 0), and serum was separated. The experimental process is as follows Fig.19a shown.

[0234] The antigen-specific IgG titers of the isolated sera were measured as described in Example 14-1. Figure 19b-19e As shown, the co-administration of antigen, adjuvant (alum) or CuSO4 and OMNV showed higher antigen-specific IgG titers than the control group and the case of only administering antigen and adjuvant (alum) or CuSO4. This result confirms that the co-administration of OMNV with antigen and adjuvant (alum) or CuSO4 can more effectively induce long-term antigen-specific IgG titers.

[0235] In addition, to confirm the effect of OMNV on long-term T cell activation, antigen (GFP or SARS-CoV-2S protein) and adjuvant (alum) or CuSO4 were mixed with OMNV and administered intraperitoneally to C57BL / 6 (male, 4 weeks old) three times at weekly intervals. The first administration was on day 0, and spleens were obtained from mice on day 49. T cells were isolated from the isolated spleen, and conditioned medium was obtained by treating the cells with antigen for 72 hours. mIFN-γ in the obtained conditioned medium was quantified by ELISA. The experimental process is as follows Fig.20a shown.

[0236] Results, such as Figure 20b-20e As shown, compared with the control group and the case where only antigen and adjuvant (alum) or CuSO4 were administered, the case where antigen, adjuvant (alum) or CuSO4 were co-administered with OMNV showed higher antigen-specific mIFN-γ secretion. This result confirms that co-administration of OMNV with antigen and adjuvant can more effectively induce long-term antigen-specific Th1 response.

[0237] In conclusion, co-administration of OMNV with antigen and adjuvant (alum) or CuSO4 can more effectively induce long-term immunity. 14-3. Analysis of the immune-inducing activity of OMNV by administration route

[0238] To determine the immune-inducing activity of OMNV by route of administration, antigen (H5N1 hemagglutinin), adjuvant (alum) and OMNV were mixed and administered intraperitoneally or intramuscularly to C57BL / 6 (male, 4 weeks old) three times at weekly intervals. The first administration was on day 0, and blood was collected on days 21 and 45, and serum was separated. Fig.21a shown.

[0239] The antigen-specific IgG titers of the isolated sera were measured as described in Example 14-1. Figure 21b-21e As shown in Figure 2, both routes of administration showed higher antigen-specific IgG titers when antigen, adjuvant and OMNV were co-administered compared to the control group and the case of antigen and adjuvant alone. This result confirms that intraperitoneal or intramuscular co-administration of OMNV with antigen and adjuvant can effectively induce long-term antigen-specific IgG titers.

[0240] In summary, OMNV has few adverse reactions, which makes it extremely valuable in developing treatments and vaccines for various diseases including cancer and infectious diseases, methods for cancer treatment and diagnosis, methods for delivering therapeutic and diagnostic substances, and methods for vaccine delivery for disease prevention and treatment.

[0241] In addition, OMNV is able to effectively induce both short-term and long-term production of neutralizing antibodies, which makes it useful as a vaccine to prevent viral infection, the development of cancers caused by viral infection (such as cervical cancer), or bacterial infection.

[0242] Example 15. Preparation of OMNV expressing antigen based on outer membrane protein display system

[0243] OMNV expressing antigens is prepared based on the outer membrane protein display system, such as Fig.22a As shown, the signal sequence of the outer membrane protein (OmpA or LolB), the antigen (S1-RBD (SARS-CoV-2 spike protein 1 receptor binding domain) or H5N1 hemagglutinin) and the His6-tag are inserted into the multiple cloning site of the pHCE vector, and the Escherichia coli BL21 (DE3) ΔmsbB is transformed to overexpress the antigen on the outer membrane of Gram-negative bacteria. Using the transformed Gram-negative bacteria, OMNV expressing the antigen is prepared by the method of Example 1. S1 RBD-OmpA OMNV, S1 RBD-LolB OMNV, H5N1-LolB OMNV).

[0244] In order to analyze the characteristics of OMNV expressing antigens, the size, yield and purity of OMNV expressing antigens were measured by the methods of Examples 3-1 and 3-2. Figure 22b At the same time, in order to confirm the presence of antigens in OMNV, Western blotting was performed using a His6-tag specific primary antibody according to the method of Example 3-3. Fig.22c , 22d As shown in Figure 22e, the His6-tag specific band was confirmed, indicating the presence of the antigen in OMNV.

[0245] Example 16. Analysis of the activity of cytokine release induced by dendritic cells using OMNV expressing antigens based on the outer membrane protein display system

[0246] In order to confirm the activity of OMNV expressing antigen based on outer membrane protein display system prepared by the method of Example 15 in inducing dendritic cells to release cytokines, bone marrow-derived dendritic cells were treated with OMNV expressing antigen by the method of Example 13-1 to obtain conditioned medium. mTNF-α, mIL-6 and mIL-12p40 in the obtained conditioned medium were quantified by ELISA.

[0247] like Figures 23a-23f As shown, OMNV expressing antigens based on the outer membrane protein display system can induce dendritic cells to release mTNF-α, mIL-6 and mIL-12p40 in a dose-dependent manner.

[0248] Example 17. Vaccination using OMNV expressing antigens based on outer membrane protein display system

[0249] 17-1. Intraperitoneal administration model

[0250] To determine the short-term and long-term immune induction activity of OMNV expressing antigens based on the outer membrane protein display system prepared by the method of Example 15, OMNV expressing antigens was intraperitoneally administered three times to C57BL / 6 (male, 4 weeks old) at intervals of 5-6 days. The first administration was on day 0, and blood was collected on days 5, 11, and 17, and serum was separated. The experimental process is as follows Fig.24a shown.

[0251] Then, the antigen-specific IgG titer was measured by the method of Example 14-1. Figure 24b and 24c As shown, the case where OMNV expressing the antigen was administered showed higher antigen-specific IgG titer compared with the control group.

[0252] In addition, the long-term immune induction activity of OMNV expressing antigens based on the outer membrane protein display system was determined by collecting serum and spleen from mice on day 49 using the method of Example 14-2. Fig.24d and 24e As shown, the administration of OMNV expressing the antigen showed higher antigen-specific IgG titer and mIFN-γ release compared with the control group.

[0253] 17-2. Intramuscular drug administration model

[0254] To verify the short-term and long-term immune-inducing activities of OMNV expressing antigens, OMNV was intramuscularly administered three times at intervals of 5-6 days to C57BL / 6 (male, 4 weeks old) mice.

[0255] These outer membrane nanovesicles were administered intramuscularly to 4-week-old male C57BL / 6 mice, once every 5 to 6 days, for a total of three injections. The first administration was on day 0, and blood was collected on days 5, 11, and 17, and serum was separated. Fig.25a shown.

[0256] Next, the antigen-specific IgG titer was analyzed according to the method of Example 14-1. Fig.25b and 25c As shown, the case where OMNV expressing the antigen was administered showed higher antigen-specific IgG titer compared with the control group.

[0257] Example 18. Safety analysis of OMNV expressing antigens based on outer membrane protein display system

[0258] To determine the safety of OMNV expressing antigens based on the outer membrane protein display system prepared by the method of Example 15, OMNV (0, 10, 50, 100 μg / mouse, single administration) was intraperitoneally administered to mice. The survival and body weight of the mice were observed for 48 hours after administration. Figures 26a-26f As shown, no significant difference was observed between the control group and the experimental group administered with OMNV expressing the antigen.

[0259] Combined with the results of Examples 15, 16, 17 and 18, OMNVs expressing various proteins such as peptides, cytokines, growth factors, antibodies, antigenic peptides and antigenic proteins using outer membrane proteins (such as OmpA, LolB, OmpF and ClyA) of Gram-negative bacteria using an outer membrane protein display system are extremely valuable in developing methods for treating and diagnosing cancer, methods for delivering substances for treating and diagnosing diseases, and methods for delivering vaccines for preventing and treating diseases, and have minimal adverse reactions, and are therefore very useful in developing treatments and vaccines for various diseases including cancer and infectious diseases. These various proteins can be used to enhance the therapeutic and preventive efficacy of outer membrane vesicles and the efficacy of vaccine delivery vectors.

[0260] Example 19. Methods for loading proteins into OMNV

[0261] To develop a method for loading therapeutic substances, diagnostic substances, immunopotentiating substances or vaccine antigens into OMNV, according to Fig.27a Schematic diagram in, OMNVs were mixed with GFP as a model substance. Control, electroporation, sonication, and freeze-thaw methods were used to load GFP, and the amount of loaded GFP was analyzed by size exclusion chromatography (using a standard curve drawn with purified GFP). Figure 27b As shown, 1 μg of OMNV was loaded with approximately 8.4 ng, 14.8 ng, 20.4 ng and 24.4 ng of GFP by control, electroporation, sonication and freeze-thaw methods, respectively.

[0262] Furthermore, to improve the efficiency of loading therapeutic substances, diagnostic substances, immunostimulatory substances or vaccine antigens into OMNVs, the Sarkosyl-treated outer membrane or purified OMNVs can be treated with a known alkaline solution to open the membrane structure, load the therapeutic substances, diagnostic substances, immunostimulatory substances or vaccine antigens, and then reseal the membrane structure.

[0263] Example 20. Preparation of OMNV by various methods

[0264] In order to prepare OMNV without ultracentrifugation, E. coli BL21 (DE3) ΔmsbB-AmCyan was cultured in VP-LB using the method of Example 1, and the outer membrane of Gram-negative bacteria was separated using Sarkosyl. Subsequently, OMNV was prepared using a microfluidic processor, and centrifugation, tangential flow filtration and dialysis were performed to obtain about 7 mL of sample. Finally, OMNV (OMNV (UC-free)) was obtained from the portion obtained by CaptoCore 700. At the same time, OMNV (OMNV (UC)) was prepared using E. coli BL21 (DE3) ΔmsbB using the method of Example 1.

[0265] Example 21. Comparison of characterizations according to methods for preparing OMNV

[0266] 21-1. Analysis of morphology and size according to the method of preparing OMNV

[0267] In order to analyze the morphology and size of OMNV (UC) and OMNV (without UC) prepared by the method of Example 20, with reference to Example 3-1, OMNV (UC) and OMNV (without UC) were observed and analyzed by transmission electron microscopy and dynamic light scattering analyzer. Fig.28a As shown, both OMNV (UC) and OMNV (no UC) include a lipid bilayer. Fig.28b As shown, the average diameters measured for OMNV (UC) and OMNV (no UC) were 74.3 ± 15.8 nm and 105.1 ± 10.7 nm, respectively.

[0268] 21-2. Analysis of total particle number and total protein amount according to the method of preparing OMNV

[0269] In order to quantify the total number of particles and total protein amount of OMNV (UC) and OMNV (without UC) prepared by the method of Example 20, OMNV (UC) and OMNV (without UC) were analyzed and quantified using a nanoparticle tracking analyzer and Bradford assay with reference to Example 3-2. Fig.28c As shown in Figure 2, for OMNV (UC) and OMNV (no UC), an average of (7.35 ± 0.35) x 10 13 and (7.45±0.21) x 10 13 In addition, Fig.28d As shown, an average of 14.200 ± 0.283 mg and 4.400 ± 0.566 mg were obtained per liter of culture medium for OMNV (UC) and OMNV (no UC), respectively.

[0270] In addition, to determine the purity of OMNV(UC) and OMNV(no UC), the total number of particles per total protein amount was calculated. Fig.28e As shown in Figure 2, the purity of OMNV (UC) and OMNV (without UC) were (5.353 ± 0.107) x 10 9 particles / μg and (15.183±0.921)x 10 9 Particles / μg.

[0271] Example 22. Analysis of innate immunity inducing activity according to the method for preparing OMNV

[0272] The innate immune induction activity of OMNV (UC) and OMNV (without UC) prepared by the method of Example 20 was determined by the method of Example 4. Figures 29a-29d As shown, OMNV(UC) and OMNV(no UC) were able to induce innate immunity in a dose-dependent manner and to a similar extent.

[0273] Example 23. Analysis of antitumor activity according to the method for preparing OMNV

[0274] To determine the antitumor activity of OMNV (UC) and OMNV (no UC) prepared by the method of Example 20, MB49 (mouse bladder cancer cells, 5×10 5 10 cells / mouse) were subcutaneously introduced into C57BL / 6 (male, 7-week-old) mice, and the size of the tumor tissue was measured over time using a vernier caliper (size = 0.5 × width × length × length). When the size of the tumor tissue reached approximately mm 3 OMNV(UC) and OMNV(without UC) were intratumorally administered at 3-day intervals, and the size of tumor tissues was measured over time.

[0275] Results, such as Fig.30 As shown, OMNV(UC) and OMNV(no UC) exhibited antitumor activity.

[0276] In summary, various substances or their complexes, such as anticancer compounds, anti-inflammatory compounds, various immunopotentiators including STING agonists, peptides, proteins, nucleic acids (mRNA, siRNA, DNA, plasmids, etc.), aptamers, toxins, and various forms of antigens, can be loaded into OMNV or displayed on the surface of OMNV, either alone or in combination, in order to enhance in vivo and in vitro stability, including in blood, reduce adverse reactions, and improve therapeutic efficacy, drug delivery efficacy, and vaccine delivery efficacy. In this way, the stability and efficacy of OMNV as a therapeutic agent, drug delivery carrier, and / or vaccine delivery carrier can be effectively enhanced while reducing adverse reactions.

[0277] The OMNV of the present invention can be prepared by various methods and applied in various fields ( Fig.31 ). The stability and efficacy of OMNV as a therapeutic agent, drug delivery vehicle and / or vaccine delivery vehicle can be effectively enhanced while reducing adverse reactions by additionally adopting the following various methods: 1) regulating and homogenizing the size; or 2) by loading various substances or their complexes, such as anti-cancer compounds, anti-inflammatory compounds, various immunopotentiators including STING agonists, peptides, proteins, nucleic acids (mRNA, siRNA, DNA, plasmids, etc.), aptamers, toxins and various forms of antigens, alone or in combination, into OMNV and / or displaying them on the surface of OMNV to enhance in vivo and in vitro stability including in blood, reduce adverse reactions, and improve therapeutic efficacy, drug delivery efficacy and vaccine delivery efficacy; or 3) combining the above methods.

[0278] Furthermore, the method for preparing OMNV of the present invention, in which substances for enhancing therapeutic efficacy, drug delivery vector efficacy and vaccine delivery vector efficacy can be loaded and / or displayed alone or in combination, can be used for therapeutic, drug delivery or vaccine purposes, or for in vitro or in vivo experimental purposes.

[0279] In addition, in general, OMNV or OMNV loaded with therapeutic, diagnostic and vaccine substances can be administered at dosages well known to those skilled in the art of medicine or veterinary medicine, taking into account factors such as age, sex, species, breed and physical condition of the recipient animal and the route of administration. The route of administration can be transdermal, mucosal (e.g., oral, nasal, rectal, vaginal) or parenteral (intramuscular, subcutaneous, intravenous, intraperitoneal or intraarticular). Therapeutic, diagnostic and vaccine compositions can be administered alone, or co-administered or sequentially with other treatments or therapies. Dosage forms can include suspensions, syrups or elixirs, as well as preparations for oral, parenteral, subcutaneous, intradermal, intramuscular or intravenous administration, such as sterile suspensions or emulsions. Therapeutic, diagnostic and vaccine compositions can be administered in the form of a spray, or mixed with food and / or water, or delivered by a suitable carrier, diluent or excipient (e.g., sterile water, saline, glucose, etc.). The composition may contain auxiliary substances, such as wetting agents or emulsifiers, pH buffers, adjuvants, gelling agents or thickening agents, preservatives, flavoring agents, coloring agents, etc., depending on the desired formulation and route of administration. Suitable formulations may be prepared without undue experimentation by reference to standard pharmaceutical textbooks such as "Remington's Pharmaceutical Sciences, 1990".

Claims

1. A method for preparing nanovesicles (OMNV) derived from the outer membrane of Gram-negative bacteria, comprising the following steps: (a) Obtaining the outer membrane by removing the inner membrane from Gram-negative bacteria; and (b) preparing nanovesicles from the suspension comprising the outer membrane.

2. The method of claim 1, wherein the gram-negative bacteria is selected from the group consisting of Escherichia, Helicobacter, Hemophilus, Neisseria, Cyanobacterium, Klebsiella, Acetobacter, Acinetobacter, Enterobacter, Chlamydia, Vibrio, Pseudomonas, Salmonella, Thiobacter, Borrelia, Burkholderia, Serratia, and Treponema.

3. The method of claim 1, wherein the Gram-negative bacteria is transformed.

4. The method of claim 3, wherein the Gram-negative bacteria are transformed Gram-negative bacteria whose toxicity of the nanovesicles derived from the outer membrane is attenuated.

5. The method of claim 3, wherein the Gram-negative bacterium is a genetically modified Gram-negative bacterium having one or more genotypes selected from the group consisting of ΔmsbB, ΔkdsA, ΔkdsB, ΔlpxB, ΔkdtA, ΔlpxC, ΔlpxD, Δssc, ΔlpxA, and ΔhtrB.

6. The method of claim 3, wherein the Gram-negative bacterium is a transformed Gram-negative bacterium that expresses a cell membrane fusion substance.

7. The method of claim 3, wherein the Gram-negative bacteria is a transformed Gram-negative bacteria that is targeted to specific cells or tissues.

8. The method of claim 3, wherein the Gram-negative bacteria is a transformed Gram-negative bacteria that expresses one or more of the following: antigenic proteins, antigenic peptides, immunostimulatory proteins, immunosuppressive proteins, cell adhesion molecules, antibodies, targeting proteins, cell membrane fusion proteins, cytokines, enzymes, growth factors, the extracellular domains of membrane receptors, marker proteins, fragments thereof, and fusion proteins thereof.

9. The method of any one of claims 3 to 8, wherein the Gram-negative bacteria is Gram-negative bacteria transformed twice or more.

10. The method of claim 1, wherein the membrane of the OMNV further comprises components other than the outer membrane of the Gram-negative bacteria.

11. The method of claim 10, wherein the component other than the cell membrane of the Gram-negative bacteria is selected from the group consisting of a targeting substance, a cell membrane fusion substance, cyclodextrin, polyethylene glycol, and hyaluronic acid.

12. The method of claim 1, wherein the membrane component of the OMNV is chemically modified.

13. The method of claim 12, wherein the membrane component of OMNV chemically modified with thiol or amino groups, or protein, polyethylene glycol or hyaluronic acid is chemically bound to the nanovesicles derived from the outer membrane of Gram-negative bacteria.

14. The method of claim 1, wherein the inner membrane of the Gram-negative bacteria is removed by treating the Gram-negative bacteria with Sarkosyl.

15. The method of claim 1, further comprising, after step (b), isolating the OMNV by a method selected from the group consisting of ultracentrifugation, density gradient ultracentrifugation, ultrafiltration, size exclusion chromatography, ion exchange chromatography, Capto Core chromatography, immunoaffinity separation, microfluidic separation, aqueous two-phase system, polymer-based precipitation, sonication, and extrusion.

16. OMNV prepared by the method of claim 1.

17. A pharmaceutical composition for treating a disease, comprising the OMNV of claim 16.

18. The pharmaceutical composition of claim 17, wherein the disease is selected from the group consisting of thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colon cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, stomach cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penile cancer, prostate cancer, ovarian cancer, and cervical cancer.

19. The pharmaceutical composition of claim 17, wherein the disease is selected from the group consisting of hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, atherosclerosis, obesity, peptic ulcer, Alzheimer's disease, emphysema, chronic obstructive pulmonary disease, asthma, skin diseases, and autoimmune diseases.

20. The pharmaceutical composition of claim 17, wherein the composition further comprises a substance that enhances therapeutic effects or reduces adverse reactions.

21. The pharmaceutical composition of claim 20, wherein the substance that enhances therapeutic effects or reduces adverse reactions is loaded into the OMNV.

22. The pharmaceutical composition of claim 20, wherein the substance that enhances the therapeutic effect or reduces adverse reactions is one or more selected from the following: anticancer agents, immunostimulants, STING agonists, anti-inflammatory agents, endotoxin inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles and nanoparticles.

23. The pharmaceutical composition of claim 22, wherein the nucleic acid is selected from the group consisting of DNA, RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino nucleic acid.

24. The pharmaceutical composition of claim 22, wherein the nanoparticles are selected from the group consisting of iron oxides, gold, carbon nanotubes, and magnetic beads.

25. A vaccine composition for preventing or treating a disease, comprising the OMNV of claim 16.

26. The vaccine composition of claim 25, wherein the vaccine composition further comprises an antigen.

27. The vaccine composition of claim 26, wherein the antigen is loaded into the OMNV.

28. The vaccine composition of claim 26, wherein the antigen is a bacteria-derived antigen, a virus-derived antigen, a fungus-derived antigen, a cancer-derived antigen, or a mutant of Ras protein, Raf protein, Src protein, Myc protein, EGFR, PDGFR, VEGFR, p53, PTEN or HER2 / neu.

29. The vaccine composition of claim 25, wherein the disease is an infection caused by bacteria, viruses or fungi.

30. The vaccine composition of claim 29, wherein the infection is selected from the group consisting of skin infections, respiratory tract infections, genitourinary infections, bone and joint infections, central nervous system infections, and sepsis.

31. The vaccine composition of claim 25, wherein the disease is selected from the group consisting of thyroid cancer, liver cancer, osteosarcoma, oral cancer, brain tumor, gallbladder cancer, colon cancer, lymphoma, bladder cancer, leukemia, small intestine cancer, tongue cancer, esophageal cancer, kidney cancer, stomach cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer, testicular cancer, penile cancer, prostate cancer, ovarian cancer, and cervical cancer.

32. The vaccine composition of claim 25, wherein the disease is selected from the group consisting of hypertension, osteoporosis, irritable bowel syndrome, acute coronary syndrome, stroke, diabetes, atherosclerosis, obesity, peptic ulcer, Alzheimer's disease, chronic obstructive pulmonary disease, asthma, skin diseases, and autoimmune diseases.

33. The vaccine composition of claim 25, wherein the vaccine is used in combination with a drug or adjuvant that increases efficacy or reduces adverse reactions.

34. Use of the OMNV of claim 16 in the preparation of a pharmaceutical composition for treating a disease.

35. A method of treating a disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the OMNV of claim 16.

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