Use of polypeptides with superoxide dismutase activity and extracellular vesicles for the treatment or prevention of respiratory viral infections - Patents.com

Abstract

The present invention relates to the use of a polypeptide having superoxide dismutase (SOD) activity and / or extracellular vesicles containing SOD for the prevention and / or treatment of a respiratory virus infection or a disease caused by a respiratory virus infection in a subject. The present invention also relates to the use of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain for the prevention and / or treatment of a respiratory virus infection or a disease caused by a respiratory virus infection in a subject.

Description

[Technical field]

[0001] The present invention relates to the use of a polypeptide having superoxide dismutase (hereinafter simply referred to as "SOD") activity obtained from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD obtained from a Bacillus subtilis strain (hereinafter simply referred to as "SOD-EVs") for the prevention and / or treatment of a respiratory viral infection in a subject. [Background technology]

[0002] Respiratory viruses, which are one of the causes of respiratory infections, are known to induce enzymes that generate reactive oxygen species (e.g., nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), xanthine oxidase, etc.).

[0003] Furthermore, coronaviruses, which are representative respiratory viruses, mainly infect the respiratory and digestive tracts of mammals and birds. These coronaviruses are known to be one of the pathogens that cause the common cold, along with rhinoviruses, influenza, and respiratory syncytial viruses.

[0004] Meanwhile, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a severe acute respiratory disease coronavirus 2 that was first known in 2019 and is classified as a positive-sense single-stranded RNA virus. The disease caused by this virus is called coronavirus disease 2019, abbreviated as COVID-19. The World Health Organization (WHO) has officially declared the coronavirus a pandemic, and as of November 2020, the number of infected people in South Korea is approaching 30,000, and approximately 55.6 million people worldwide are known to be infected, and COVID-19 continues to spread. When infected with COVID-19, symptoms such as fever, cough, difficulty breathing, and pneumonia appear after an incubation period of about 2 to 14 days.

[0005] COVID-19 is a type of virus that causes respiratory tract infection and was first discovered in Wuhan, Hubei Province, China in 2019. It belongs to the Coronaviridae family and is known to be similar to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV).

[0006] Clinical trials for COVID-19 have been conducted on drugs such as Kaletra (active ingredient: lopinavir), a drug for treating human immunodeficiency virus (HIV), and Remdesivir, a drug for treating Ebola virus, and in October 2020, the U.S. Food and Drug Administration (FDA) approved Remdesivir for use in treating COVID-19, but it is not a drug with therapeutic effects that can both prevent and treat COVID-19. Therefore, it is important to find new therapies that can prevent and treat respiratory viral infections such as coronaviruses, including COVID-19. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection, the method comprising administering a therapeutically effective amount of a polypeptide having superoxide dismutase (SOD) activity and / or extracellular vesicles containing SOD.

[0008] Another object of the present invention is to provide a method for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection, the method comprising administering a therapeutically effective amount of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain.

[0009] Yet another object of the present invention is to provide a method for the prevention and / or treatment of a coronavirus infection or a disease caused by a coronavirus infection, the method comprising administering a therapeutically effective amount of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain.

[0010] Yet another object of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of respiratory viral infections, the composition comprising a polypeptide having SOD activity and / or extracellular vesicles containing SOD.

[0011] Yet another object of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of respiratory viral infections, the composition comprising a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain.

[0012] Yet another object of the present invention is to provide a pharmaceutical composition for the prevention and / or treatment of coronavirus infection, the composition comprising a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain.

[0013] Yet another object of the present invention is to provide a food composition for preventing and / or ameliorating respiratory viral infections, the composition comprising a polypeptide having SOD activity and / or extracellular vesicles containing SOD.

[0014] Yet another object of the present invention is to provide a food composition for preventing and / or ameliorating respiratory viral infections, the composition comprising a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain.

[0015] Yet another object of the present invention is to provide a food composition for preventing and / or ameliorating coronavirus infection, the composition comprising a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain.

[0016] Yet another object of the present invention is to provide a use of the pharmaceutical composition for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection.

[0017] Yet another object of the present invention is to provide a use of the pharmaceutical composition for preparing a medicament for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection.

[0018] Yet another object of the present invention is to provide a use of the food composition for the prevention and / or amelioration of respiratory viral infections or diseases caused by respiratory viral infections. [Means for solving the problem]

[0019] The inventors of the present invention have confirmed that a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain, when administered to a mammal alone or together with extracellular vesicles containing SOD derived from a Bacillus subtilis strain, exhibits the effect of preventing, treating, or ameliorating coronavirus infection, and have completed the present invention. Effect of the Invention

[0020] The polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain or the extracellular vesicles containing SOD derived from a Bacillus subtilis strain according to the present invention, when used alone or in combination, exhibit excellent effects in the prevention, treatment, or amelioration of respiratory viral infections or diseases caused thereby. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows the results of particle size analysis of extracellular vesicles containing SOD derived from a Bacillus subtilis strain. [Diagram 2] FIG. 1 shows the results of particle size analysis of extracellular vesicles containing SOD derived from a Bacillus subtilis strain. [Diagram 3] FIG. 1 shows an animal experiment design for identifying the preventive and therapeutic effects of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain against coronavirus infection. [Figure 4] FIG. 1 shows the therapeutic effect of extracellular vesicles containing a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or SOD derived from a Bacillus subtilis strain on lung damage caused by coronavirus infection. [Diagram 5] FIG. 1 shows the preventive effect of extracellular vesicles containing a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or SOD derived from a Bacillus subtilis strain against lung damage caused by coronavirus infection. [Figure 6] FIG. 13 shows that the lung damage observed in Syrian hamsters administered with a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain is significantly different from that observed in Syrian hamsters not administered with the polypeptide and / or extracellular vesicles containing SOD. [Figure 7] FIG. 1 shows histopathological results of observing the lungs after administration of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain. [Figure 8]FIG. 1 shows histopathological scores of the lungs after therapeutic administration of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain. [Figure 9] FIG. 1 shows histopathological scores of the lungs after prophylactic administration of a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In one aspect of the present invention, there is provided a method for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection, the method comprising administering a therapeutically effective amount of (i) a polypeptide having superoxide dismutase (SOD) activity, (ii) an extracellular vesicle containing SOD, or (iii) a polypeptide having SOD activity and an extracellular vesicle containing SOD.

[0023] In this case, the respiratory virus infection may be a coronavirus infection. The coronavirus infection is selected from the group consisting of cold, pneumonia, interstitial pneumonia, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), sinusitis, COVID-19 infection, otitis, or pharyngitis. Furthermore, the disease caused by the respiratory virus infection may be viral lung injury or lung disease.

[0024] Furthermore, the coronavirus may be an alphacoronavirus, a betacoronavirus, a gammacoronavirus, or a deltacoronavirus. In particular, the coronavirus may be a human coronavirus.

[0025] The polypeptide having SOD activity of the present invention has the amino acid sequence of SEQ ID NO: 1. The polypeptide having SOD activity of the present invention is also understood to include an amino acid sequence substantially identical to this amino acid sequence. "Substantially identical" means an amino acid sequence having at least 80% homology, more preferably 96% homology, and most preferably 98% homology, when the amino acid sequence of the present invention is aligned with another sequence as closely as possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0026] The polypeptide can be isolated from Bacillus amyloliquefaciens GF423 strain, which was internationally deposited at the Korea Institute of Bioscience and Biotechnology under the accession number KCTC 13222BP on March 6, 2017, and the characteristics and culture method of the strain are described in Korean Patent No. 1762199.

[0027] The extracellular vesicles containing SOD derived from the Bacillus subtilis strain according to the present invention refer to extracellular vesicles having SOD activity. The extracellular vesicles refer to a heterogeneous collection of membrane-enclosed nano-sized vesicles containing complex hydrates including proteins, lipids, and nucleic acids.

[0028] Extracellular vesicles containing SOD can be isolated from a Bacillus subtilis SOD strain.

[0029] In the present invention, the polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or the extracellular vesicles containing SOD derived from a Bacillus subtilis strain can be used to suppress the proliferation of viruses in individuals already infected with respiratory viruses. Preferably, the polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or the extracellular vesicles containing SOD derived from a Bacillus subtilis strain can suppress the proliferation of viruses in individuals already infected with coronaviruses. More preferably, the polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or the extracellular vesicles containing SOD derived from a Bacillus subtilis strain can suppress the proliferation of viruses in individuals infected with SARS or COVID-19.

[0030] Furthermore, the polypeptide having SOD activity and the extracellular vesicle containing SOD can be administered simultaneously or sequentially.

[0031] In another aspect of the present invention, there is provided a method for preventing and / or treating a respiratory viral infection or a disease caused by a respiratory viral infection, the method comprising administering a therapeutically effective amount of a polypeptide having SOD activity and / or extracellular vesicles containing SOD and / or a pharma-ceutically acceptable salt thereof. The respiratory viral infection may be a coronavirus infection. Furthermore, the coronavirus infection may be a SARS-CoV-2 infection.

[0032] In yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing and / or treating a respiratory virus infection or a disease caused by a respiratory virus infection, the pharmaceutical composition comprising a polypeptide having SOD activity and / or an extracellular vesicle containing SOD, in which the respiratory virus infection may be a coronavirus infection.

[0033] In yet another aspect of the present invention, there is provided a pharmaceutical composition for the prevention and / or treatment of a respiratory virus infection or a disease caused by a respiratory virus infection, the pharmaceutical composition comprising a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or an extracellular vesicle containing SOD derived from a Bacillus subtilis strain, in which the respiratory virus infection may be a coronavirus infection.

[0034] In yet another aspect of the present invention, there is provided the use of the composition for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection, wherein the composition is the same as described above.

[0035] In yet another aspect of the present invention, there is provided the use of the composition for the preparation of a medicament for the prevention and / or treatment of a respiratory viral infection or a disease caused by a respiratory viral infection, wherein the composition is the same as described above.

[0036] The polypeptide having SOD activity and / or extracellular vesicles containing SOD of the present invention can be used together with a compound, polypeptide, etc. known to have a preventive or therapeutic effect against respiratory virus infections or diseases caused by respiratory virus infections. Furthermore, the polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain of the present invention can be used together with a compound, polypeptide, etc. known to have a preventive or therapeutic effect against respiratory virus infections or diseases caused by respiratory virus infections.

[0037] In yet another aspect of the present invention, there is provided a method for preventing and / or ameliorating a respiratory viral infection or a disease caused by a respiratory viral infection, the method comprising the step of administering a biologically effective amount of a food composition comprising a polypeptide having SOD activity and / or extracellular vesicles containing SOD.

[0038] In yet another aspect of the present invention, there is provided a food composition for preventing or ameliorating a disease caused by a respiratory virus infection, the composition comprising a polypeptide having SOD activity and / or extracellular vesicles containing SOD. More specifically, there is provided a food composition for preventing or ameliorating a disease caused by a respiratory virus infection, the composition comprising a polypeptide having SOD activity derived from a Bacillus amyloliquefaciens strain and / or extracellular vesicles containing SOD derived from a Bacillus subtilis strain. In this case, the respiratory virus infection may be a coronavirus infection.

[0039] An aspect of the present invention provides the use of a food composition for the prevention and / or amelioration of a respiratory viral infection or a disease caused by a respiratory viral infection.

[0040] The pharmaceutical composition of the present invention can be formulated according to conventional methods into the form of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, aerosols, injection solutions, etc. and used. For example, the carrier, excipient, or diluent that may be included in the pharmaceutical composition of the present invention includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, crystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When the pharmaceutical composition is formulated, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0041] The pharmaceutical composition of the present invention can be administered orally or parenterally by any desired method. When administered parenterally, it is preferable to select nasal drops, topical application to the skin, or intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, or intrathoracic injection.

[0042] In this case, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the complex composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are generally used as simple diluents, various excipients such as wetting agents, sweeteners, flavorings, and preservatives can be used.

[0043] Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solutions or suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used.

[0044] Furthermore, the pharmaceutical composition according to the present invention can be administered by inhalation. The drug is delivered directly to the lungs, but is not toxic and can exert a long-lasting effect even in small doses. Administration for inhalation can be administration using a pharmaceutical formulation that contains inhalable particles or droplets containing the drug and can be inhaled through the airways, nasal passages, etc. For example, inhalation administration can be, but is not limited to, a dry powder inhaler (DPI) or a pressurized metered dose inhaler (pMDI). For example, the drug particles are lightly compressed into a friable matrix contained within a delivery device (dry powder inhaler). Upon operation, the delivery device scrapes off some of the drug particles from the matrix and disperses them into the inhaled air to deliver the drug particles to the airways. Alternatively, the drug particles can be a free-flowing powder contained within a reservoir within the delivery device (dry powder inhaler). The reservoir can be an integral compartment within the device or a capsule, blister, or reservoir with similar functionality that is inserted into the device prior to operation. Upon operation, the device disperses some of the drug particles from the reservoir and disperses them into the inhaled air to deliver the drug particles to the airways.

[0045] Furthermore, the pharmaceutical composition of the present invention may further comprise a carrier, excipient, or diluent. Lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or minerals such as silicon dioxide may be used as the carrier, excipient, or diluent.

[0046] The pharmaceutical composition of the present invention should be administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined according to factors including the patient's weight, sex, age, condition, severity of the disease, activity of the drug, sensitivity to the drug, timing of administration, route of administration and excretion rate, duration of treatment, drugs used in combination with the composition, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, in combination with other therapeutic agents, or sequentially or simultaneously with conventional therapeutic agents. Such administration may be a single dose or multiple doses. It is important to administer an amount that can obtain maximum effect with a minimum amount without side effects, and such an amount can be easily determined by one skilled in the art. EXAMPLES

[0047] Hereinafter, the present invention will be described in detail with reference to examples in order to better understand the present invention. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0048] Example 1. Isolation / purification of superoxide dismutase (SOD) polypeptide from Bacillus amyloliquefaciens GF423 1.1 Cultivation of Bacillus amyloliquefaciens GF423 To cultivate Bacillus amyloliquefaciens GF423 strain, a single colony formed on LB agar medium (Luia-Bertani (LB) agar; tryptophan 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) was inoculated into 30 mL of LB medium and cultured at 37°C for 12 hours. This seed culture was re-inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37°C for 20 hours.

[0049] 1.2 Isolation and purification of superoxide dismutase The cell culture solution was centrifuged at 3,578×g for 20 min at 4°C to obtain the supernatant, and then the supernatant was concentrated 10-fold using ultrafiltration (hereafter, UF (MWCO 10,000)). Ammonium sulfate was added to 300 mL of the concentrated supernatant at 4°C while stirring, up to 60% saturation, and the resulting mixture was stirred for 30 min. The mixture was then centrifuged at 3,578×g for 30 min to obtain the supernatant, and the supernatant was loaded onto a HiPrepTM Phenyl HP16 / 10 column equilibrated with 50 mM potassium phosphate (pH 7.5) containing 2 M ammonium sulfate. Elution was then performed using 50 mM potassium phosphate (pH 7.5) containing 2 M to 0.1 M ammonium sulfate.

[0050] The SOD-containing fractions (#35-#40) were collected, concentrated using a UF (MWCO 10,000) and dialyzed against 50 mM potassium phosphate (pH 7.5) to remove salts. Protein concentration was measured by Bradford assay (Bradford M, Anal Biochem, 72, 248-254, 1976).

[0051] The activity of superoxide dismutase was confirmed using a superoxide dismutase assay kit (Cayman Chemical, Michigan, USA). One unit of superoxide dismutase activity is defined as the amount of enzyme that inhibits superoxide radical by 50%. The activity of the purified SOD was 2231.12 ± 269 U / mg, and the molecular weight on SDS was approximately 22,000 daltons.

[0052] Example 2. Isolation / purification of extracellular vesicles containing SOD from Bacillus subtilis strains 2.1. Cultivation of Bacillus subtilis SOD strain The Bacillus subtilis SOD strain, which the present inventors had frozen and stored, was used as a strain for the isolation and purification of superoxide dismutase. 1 mM DSM (nutrient medium 8 g / L, KCl 1 g / L, MgSO4·7H2O 0.12 g / L, Ca(NO3)2, 0.01 mM MnCl2·4H2O, and 0.001 mM FeSO4) was used as the basal medium. Round-bottom tube and flask cultures were performed using a shaking incubator, with the stirring speed set to 200 rpm and the temperature set to 37°C. A 5 L jar fermenter (Fermentec Co., Ltd., FMT-ST-M05) was used as the fermentation tank, with the stirring speed set to 200 rpm, temperature set to 37°C, aeration volume set to 1.0 vvm, and pH set to 6.8.

[0053] Preculture of Bacillus subtilis strain was performed by picking a single colony on an SOD plate, inoculating the colony into a round-bottom tube containing 3 mL of basal medium, and culturing for 9 hours. In addition, 3 mL of the medium cultured in the round-bottom tube was inoculated into a 500 mL flask containing 30 mL of basal medium, and after culturing for 16 hours, it was used for the main culture inoculation. In the fermenter, the culture was performed in 3.0 L, the inoculum amount was 1%, and the culture was performed for 24 hours.

[0054] 2.2.SOD-EV isolation As described above, Bacillus subtilis SOD strain was cultured in a jar fermenter for 24 hours, and the medium was centrifuged at 12,000 rpm at 4°C for 30 minutes to obtain the culture supernatant. The culture supernatant was then used to remove bacteria remaining in the medium using a 0.22 μm cassette in the TFF system, and only the pure culture supernatant was isolated. The pure culture supernatant was concentrated 200-fold using a 100 kDa cutoff membrane in the TFF system to obtain SOD-containing extracellular vesicles (SOD-EVs).

[0055] 2.3. Quantification and identification of SOD-EVs Protein electrophoresis was performed on the SOD-EVs obtained by the above-mentioned method, and the protein amount of the SOD-EVs was measured using Bradford analysis and BCA analysis.

[0056] [Protein electrophoresis] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 4-20% gradient gel (Biorad, Mini-protean TGX precast gel) was used. 25 mM Tris base, 192 mM glycine, and 0.1% SDS (pH 8.3) were used as SDS-PAGE buffer, and SOD-EV was mixed with 5x sample buffer and heated at 100°C for 10 minutes before use. After protein electrophoresis, proteins were identified by Coomassie staining and Western blotting. Proteins were stained with Coomassie brilliant blue for 30 minutes, and then reacted with decolorizing buffer for 3 hours.

[0057] [Western Blot] After SDS-PAGE of SOD-EV, the proteins were transferred to a 0.22 μm PVDF membrane, and then the membrane was blocked in 5% skim milk for 1 h. After that, it was washed three times with washing buffer, and then anti-SOD antibody (1:10,000 skim milk) was bound to it for 3 h at room temperature. Then, it was washed three times using washing buffer, and secondary antibody (1:100,000 skim milk) was bound to it and the reaction was carried out at room temperature for 1 h. Then, it was washed three times with washing buffer and confirmed by ECL reaction.

[0058] Mini-Protean II (Bio-Rad, Inc.) was used as the protein electrophoresis device, and Chemi-doc MP imaging system (Bio-Rad, Inc.) was used as the confirmation device.

[0059] [Determination of the size of SOD-containing extracellular vesicles (SOD-EVs)] Dynamic light scattering (DLS) was used to measure the size of purified SOD-EVs. 8 μL of SOD-EV sample was placed in a well and placed in the chamber of the DLS device, and the particle size was measured using laser light. Figures 1 and 2 show the particle size analysis results. The protein concentration of SOD-EVs was 0.5 mg / mL by Bradford analysis and 0.124 mg / mL by BCA analysis (see Table 1 below). [Table 1]

[0060] Example 3. Preventive and therapeutic effects of SOD and SOD-EV against coronavirus infection Example 3-1. Visual assessment of the lungs following prophylactic and therapeutic administration of SOD and SOD-EV To confirm the preventive and therapeutic effects of SOD and SOD-EV against coronavirus infection, 40 6-week-old Syrian hamsters (male) were purchased for animal experiments from Central Lab. Animal Inc. The purchased Syrian hamsters were divided into 8 groups as shown in Table 2 below, and SOD, SOD-EV, and SOD and SOD-EV were administered to the hamsters, respectively, and Sars-Cov2, a respiratory virus, was used as the challenge virus. [Table 2]

[0061] Groups for testing the efficacy of treatment of coronavirus infection were divided into G1 to G3 based on treatment mode. SOD-EV was administered to G1, SOD to G2, and SOD and SOD-EV to G3 daily from the day of Sars-Cov2 challenge until 4 days after challenge.

[0062] Groups were divided into G4 to G6 based on the preventive mode to test the preventive effect against coronavirus infection. SOD-EV was administered to G4, SOD to G5, and SOD and SOD-EV to G6, once a day from 2 days before Sars-Cov2 challenge to 4 days after challenge.

[0063] G7 was challenged with virus without the use of test substances, and G8 was administered SOD-EV and SOD alone without viral challenge. The experimental design for each of the therapeutic and preventive modes is shown in Figure 3.

[0064] Lung injury was observed after Sars-Cov2 challenge in all groups, G1 to G7, except for G8, which was not challenged with Sars-Cov2.

[0065] However, the lung injury scores of G1 to G3, which were administered SOD and / or SOD-EV after Sars-Cov2 challenge, were lower than that of G7 (see FIG. 4). In particular, the lung injury shown in G2, which was administered only SOD, and G3, which was administered SOD-EV together with SOD, showed a statistically significant difference compared to the lung injury observed in G7 (p<0.05), indicating that the use of SOD alone or SOD and EV together had a superior therapeutic effect on infections caused by respiratory viruses such as Sars-Cov2.

[0066] Furthermore, G4, G5, and G6, which were administered SOD and / or SOD-EV 2 days before challenge, showed less lung damage than G7, which was not administered SOD and / or SOD-EV (see Figures 5 and 6). In particular, G6, which was administered SOD-EV and SOD together, showed a statistically significant difference in lung damage compared to G7 (p<0.05). These experimental results indicate that when SOD and EV are used together, the treatment effect of infection caused by respiratory viruses such as Sars-Cov2 is excellent.

[0067] Furthermore, pulmonary edema and inflammation in G1 to G3, which received SOD-EV and / or SOD, were less than those observed in G7 (see treatment mode in FIG. 7).

[0068] On the other hand, pulmonary edema and inflammation in G4 to G6, in which SOD-EV and / or SOD were administered prior to Sars-Cov2 challenge, were less than those in G7 (see prevention mode in Figure 7).

[0069] Example 3-2. Histopathological evaluation of lungs after prophylactic and therapeutic administration of SOD and SOD-EV To confirm the preventive and therapeutic effects of SOD and SOD-EV against coronavirus infection, histopathological examination of the lungs of the eight groups evaluated with the naked eye in Example 3-1 was performed.

[0070] In detail, tissue slices were taken from the lungs of the eight groups, and each tissue slice was fixed in 10% neutral buffered formalin solution for 24 hours. The fixed tissue slices were placed in an automatic tissue processor to remove water from the tissue and infiltrate the tissue with paraffin. After the infiltration process was completed, the tissue slices were placed in a frame, and paraffin was poured into the frame to form a block of a given shape. The formed block was cooled and then cut into 4 μm thick slices using a microtome.

[0071] The slides prepared by the above method were subjected to H&E staining as follows.

[0072] First, slides containing paraffin tissue sections were deparaffinized by placing each slide in separate xylenes for 5 min each, and then rehydrated by treatment with 100% ethanol twice for 3 min, 80% ethanol for 2 min, and 70% ethanol for 2 min.

[0073] After washing the sections in tap water for 5 min, the tissue slides were placed in Harris hematoxylin for 10 min and washed in tap water for 3 min. The sections were quickly immersed twice in 0.5% acid alcohol to remove any residues, washed in tap water for 1 min, and the washed sections were placed in 1% ammonia solution for 2 min to develop a blue color. The sections were then briefly immersed in 95% ethanol for 10 s and then stained with eosin for 1 min 30 s. The stained sections were dehydrated in 100% ethanol three times for 50 s and placed in xylene twice for 5 min for clarity.

[0074] A drop of aqueous fixative solution was placed on the coverslip of the H&E stained slide, the slide was then dried, and the results were observed under a light microscope.

[0075] Histopathological examination was performed using Table 3 as the basis for histopathological scores, the results of which are shown in Table 4 below, and in Figures 8 and 9. [Table 3] [Table 4] JPEG0007639136000005.jpg65149

[0076] Histopathological examination showed that the consolidation and bronchial epithelial atrophy observed in G1 to G6, which received SOD and / or SOD-EV, were significantly less than those observed in G7, which did not receive SOD and / or SOD-EV (see Table 4).

[0077] Furthermore, G1, G2, and G3, which were administered SOD and / or SOD-EV after loading, had lower histopathological scores than G7, which was not administered SOD and / or SOD-EV (see FIG. 8). In particular, the histopathological scores shown in G2, which was administered only SOD, and G3, which was administered SOD and SOD-EV together, showed significant differences compared to those shown in G7 (p<0.01).

[0078] Furthermore, G7, which was challenged with virus alone, had severe bronchial epithelial atrophy, such that bronchial epithelium was barely observed, and G4, G5, and G6, which were administered SOD and / or SOD-EV two days before challenge, had lower histopathological scores than G7 (see FIG. 9). In particular, the histopathological scores shown by G6, which was administered SOD-EV together with SOD, showed a significant difference compared to those shown by G7 (p<0.05).

[0079] That is, the histopathological scores showed a consistent pattern with respect to the lung damage scores observed with the naked eye, and it was found that the preventive and therapeutic effects on consolidation and bronchial epithelial atrophy were particularly excellent.

Claims

1. A pharmaceutical composition for the prevention and / or treatment of coronavirus infection, comprising: (i) a polypeptide having superoxide dismutase (SOD) activity; (ii) SOD-containing extracellular vesicles (SOD-EVs), or (iii) Extracellular vesicles containing a polypeptide having SOD activity and SOD (SOD-EV) as an active ingredient, wherein the polypeptide is derived from a Bacillus amyloliquefaciens strain, and the SOD-EV is derived from a Bacillus subtilis strain.

2. A food composition for preventing and / or ameliorating coronavirus infection, comprising: (i) a polypeptide having superoxide dismutase (SOD) activity; (ii) SOD-containing extracellular vesicles (SOD-EVs), or (iii) Extracellular vesicles containing a polypeptide having SOD activity and SOD (SOD-EV) as an active ingredient, wherein the polypeptide is derived from a Bacillus amyloliquefaciens strain, and the SOD-EV is derived from a Bacillus subtilis strain.

3. The pharmaceutical composition of claim 1 or the food composition of claim 2, wherein the coronavirus infection is selected from the group consisting of cold, pneumonia, interstitial pneumonia, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), sinusitis, SARS-Cov2 infection, otitis, and pharyngitis.

4. The pharmaceutical composition of claim 1 , wherein the polypeptide having SOD activity and / or the extracellular vesicles containing SOD are administered by nasal drops.

5. The pharmaceutical composition of claim 1 or the food composition of claim 2, wherein the polypeptide having SOD activity and the extracellular vesicles containing SOD are administered simultaneously or sequentially.

6. Use of the pharmaceutical composition described in claim 1 for preparing a drug for the prevention and / or treatment of coronavirus infection.

7. Use of the food composition described in claim 2 for producing a food for the prevention and / or amelioration of coronavirus infection.

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