Composition for prevention or treatment of coronavirus infectious disease

Abstract

The present invention involves recovering pulmonary inflammation, pulmonary edema or pulmonary capillary vasodilation symptoms caused by infection with viruses, particularly, coronavirus, and furthermore, has the effect of regenerating alveoli, and thus can effectively prevent, ameliorate or treat coronavirus infection or diseases caused by coronavirus infection.

Description

Composition for preventing or treating coronavirus infectious disease

[0001] The present invention relates to the use of an SPP1 (Secreted Phosphoprotein 1) inhibitor for preventing, improving, or treating a coronavirus infection disease.

[0002] Coronaviruses are representative viruses that cause fatal infectious diseases in modern civilization. In April 2003, Severe Acute Respiratory Syndrome (SARS), also known as SARS, became prevalent, killing many people with a mortality rate of 9.6%. In 2015, Middle East Respiratory Syndrome (MERS), also known as MERS, spread from the Middle East to the entire world, causing many deaths with a mortality rate of about 36%. In addition, since December 2019, a novel coronavirus infection (SARS-CoV-2; COVID-19) that originated in Wuhan, China has spread worldwide, resulting in more than 500 million cases of infection and more than 6 million deaths worldwide.

[0003] Although the development of therapeutics against coronaviruses is being promoted, there is a lack of agents that can effectively prevent or treat coronavirus infection or infectious diseases. In addition, previous studies on SARS-CoV-2 infection have primarily focused on peripheral blood mononuclear cells (PBMCs), resulting in a lack of research on the effects of the virus on inflamed tissues.

[0004] Accordingly, the present inventors analyzed the molecular / cellular responses that change during the period of infection using lung tissue of transgenic mice infected with SARS-CoV-2, thereby discovering a key therapeutic target that enables regeneration of damaged alveoli, leading to the present invention.

[0005] One object of the present invention is to provide a composition for various uses that can prevent, improve or treat infection by a virus, particularly a coronavirus, or various diseases caused by such infection.

[0006] Another object of the present invention is to provide a method for screening a composition for treating a coronavirus infectious disease.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0008] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0009] Unless otherwise specifically defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0010]

[0011] According to one embodiment of the present invention, the present invention relates to a composition for preventing, improving or treating a viral infection or infectious disease, comprising an SPP1 (Secreted Phosphoprotein 1) inhibitor as an active ingredient.

[0012] According to another embodiment of the present invention, there is provided a method for preventing, improving or treating a viral infection or infectious disease, comprising administering to a subject an SPP1 (Secreted Phosphoprotein 1) inhibitor.

[0013] In the present invention, the "SPP1 (Secreted Phosphoprotein 1)" is a protein encoded by the SPP1 gene and is a multifunctional glycoprotein involved in various physiological and pathological processes. It is expressed in numerous cell types, including macrophages, endothelial cells, and osteoclasts, and plays a crucial role in cell signaling and the extracellular matrix (ECM). It interacts with integrins and the CD44 receptor to mediate cell adhesion, migration, and survival. It is known to serve as a potential biomarker for cancer diagnosis and prognosis, and is also known to be involved in neuroinflammatory signaling in neurodegenerative diseases. In the present invention, the SPP1 amino acid sequence may be represented by SEQ ID NO: 1, but is not limited thereto.

[0014] In the present invention, the "SPP1 (Secreted Phosphoprotein 1) inhibitor" may be an agent that reduces the expression level of the SPP1 gene or an agent that inhibits the activity of a protein encoded by the SPP1 gene.

[0015] The agent for reducing the expression level of the SPP1 gene of the present invention may include at least one selected from the group consisting of antisense nucleotides, short interfering RNA (siRNA), short hairpin RNA, microRNA (miRNA), guide RNA (gRNA), and ribozyme that complementarily bind to the SPP1 gene, specifically, the mRNA of the gene, and the agent for inhibiting the activity of the protein encoded by the SPP1 gene may include at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the protein encoded by the SPP1 gene. However, as long as it is a means for directly or indirectly acting on the target protein or gene SPP1 to induce an effect of inhibiting its activity or expression, and can be easily derived by a known technique using a method commonly used in the art, it is not limited thereto and may include all of them.

[0016] In the present invention, the "antisense nucleotide" is defined by Watson-Crick base pairing, which binds (hybridizes) to the complementary base sequence of DNA, immature mRNA, or mature mRNA, thereby disrupting the flow of genetic information from DNA to protein. The specificity of antisense nucleotides for their target sequence makes them exceptionally versatile. Because antisense nucleotides are long chains of monomeric units, they can be easily synthesized against target RNA sequences. Recent studies have demonstrated the utility of antisense nucleotides as a biochemical tool for studying target proteins. Recent advances in oligonucleotide chemistry and the synthesis of nucleotides exhibiting enhanced cell adhesion, target binding affinity, and nuclease resistance suggest that the use of antisense nucleotides can be considered a novel form of inhibitor.

[0017] In the present invention, the "siRNA" and "shRNA" are nucleic acid molecules capable of mediating RNA interference or gene silencing, and are used as efficient gene knockdown methods or gene therapy methods because they can suppress the expression of target genes. shRNA forms a hairpin structure by binding between complementary sequences within a single-stranded oligonucleotide, and in vivo, the shRNA is cleaved by Dicer into small RNA fragments of 21 to 25 nucleotides in size, which become siRNA, a double-stranded oligonucleotide, and can specifically bind to mRNA with a complementary sequence to suppress its expression. Therefore, which means between shRNA and siRNA to use can be determined by the choice of those skilled in the art, and if the mRNA sequences they target are the same, a similar expression reduction effect can be expected. For the purpose of the present invention, the expression of SPP1 can be inhibited by specifically acting on the SPP1 gene to cleave the SPP1 gene (e.g., mRNA molecule) and inducing RNA interference (RNAi, RNA interference) phenomenon. siRNA can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and methods known in the art can be used. For example, there may be, but are not limited to, a method of directly chemically synthesizing siRNA, a method of synthesizing siRNA using in vitro transcription, a method of cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme, an expression method through intracellular delivery of an shRNA expression plasmid or viral vector, and an expression method through intracellular delivery of a PCR (polymerase chain reaction)-induced siRNA expression cassette.

[0018] In the present invention, the above "microRNA" refers to a single-stranded RNA molecule that is an oligonucleotide that is not expressed in cells and has a short stem-loop structure and suppresses target gene expression through complementary binding to the mRNA of the target gene.

[0019] In the present invention, the "gRNA (guideRNA)" refers to an RNA molecule used in a gene editing system that recognizes a target gene and induces a nuclease to specifically cleave the recognized region. A representative example of such gene editing systems is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0020] In the present invention, the "ribozyme" refers to an RNA molecule having catalytic activity. Ribozymes with various activities are known, and the ribozyme of the SPP1 gene includes known or artificially created ribozymes. Optionally, a ribozyme having target-specific RNA cleavage activity can be produced using standard, known techniques.

[0021] In the present invention, the "peptide mimetics" are peptides or non-peptides that inhibit the binding domain of the SPP1 protein, which leads to inhibition of the activity of the protein encoded by the SPP1 gene. Key residues of non-hydrolyzable peptide analogs include a β-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th AmeriCan Peptide Symposium) Pierce chemiCal co. Rockland, IL, 1985), azepines (Huffman et al. in Peptides: chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al. in Peptides; chemistry and Biology, GR Marshall ed., EScOM Publisher: Leiden, Netherlands, 1988), and β-aminoalcohols. (Gordon et al. Biochem Biophys Res commun 126:419 1985) and substituted gamma-lactam rings (Garvey et al. in Peptides: chemistry and Biology, GR Marshell ed., EScOM Publisher: Leiden, Netherlands, 1988).

[0022] In the present invention, the "aptamer" is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and has the characteristics of being able to bind to a target molecule with high affinity and specificity. Since the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) was first developed (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), many aptamers that can bind to various target molecules, including small-molecule organic compounds, peptides, and membrane proteins, have continued to be discovered. Because of their unique characteristics of being able to bind to target molecules with high affinity (usually pM level) and specificity, aptamers are comparable to single antibodies, and in particular, they have high potential as a substitute antibody to the point of being called a "chemical antibody."

[0023] In the present invention, the "antibody" may be either one manufactured by injecting a protein encoded by the SPP1 gene or one purchased commercially. Furthermore, the antibody includes polyclonal antibodies, monoclonal antibodies, and fragments capable of binding to an epitope.

[0024] Here, the polyclonal antibody can be produced by a conventional method, including injecting an animal with a protein encoded by the SPP1 gene, collecting blood from the animal, and obtaining serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art, and can be produced from any animal species host, such as a goat, rabbit, sheep, monkey, horse, pig, cow, or dog.

[0025] Additionally, the monoclonal antibody can be produced using any technique that allows for the production of antibody molecules through continuous cell line culture. Such techniques include, but are not limited to, hybridoma technology, human B-cell line hybridoma technology, and EBV hybridoma technology.

[0026] Additionally, antibody fragments containing a specific binding site for a protein encoded by the SPP1 gene can be produced. For example, but not limited to, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries can be miniaturized to rapidly and easily identify monoclonal Fab fragments with desired specificity.

[0027] In the present invention, the antibody may be bound to a solid substrate to facilitate subsequent steps, such as washing or complex separation. Examples of solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres, and microbeads. Furthermore, synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, and nylon.

[0028] In the present invention, the agent that inhibits the activity may be an antibody that specifically binds to a part or the entire sequence of the SPP1 protein, but is not limited thereto.

[0029] In the present invention, the virus may be an RNA virus. In the present invention, the "RNA virus" refers to any virus that uses RNA as its genetic material. For example, the RNA viruses include Coronaviridae, Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Totiviridae, Quadriviridae, Arteriviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Caliciviridae, Flaviviridae, Luteoviridae, Togaviridae, Pneumoviridae, Arenaviridae, Deltavirus,Or it could be a virus of the Orthomyxoviridae family, but more specifically it could be a coronavirus of the Coronaviridae family, and more specifically a coronavirus belonging to the Coronaviridae family.

[0030] In the present invention, the above "coronavirus" is a virus of the Coronaviridae family and the Coronavirinae subfamily, which has four genera (alpha, beta, gamma, and delta), and is an RNA virus with a gene size of 27 to 32 kb, and is known to cause respiratory and digestive infections in humans and animals. It is easily infected mainly through mucosal transmission and droplet transmission, and although it generally causes mild respiratory infections in humans, it can rarely cause fatal infections, and it can cause diarrhea in cows and pigs, and respiratory diseases in chickens. It is divided into the classifications in Table 1 below (Korea Centers for Disease Control and Prevention, 2020). Among the four genera, alpha and beta are reported to infect humans and animals, while gamma and delta are reported to infect only animals.

[0031] Genus Human-coronavirus Non-human coronavirus Alpha coronavirus 229, NL63 Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), canine coronavirus (CCoV), feline coronavirus (FCoV), Miniopterus bat coronavirus 1, Miniopterus bat coronavirus HKU8, Rhinolophus bat coronavirus HKU2, Scotophilus bat coronavirus 512 Beta coronavirus OC43, HKU1, SARS-CoV, MERS-CoV Porcine hemagglutinating encephalomyelitis virus (PHEV), bovine coronavirus (BCoV), equine coronavirus (EqCoV), murine coronavirus (MuCoV), Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Gamma coronavirus - Avian coronavirus, Beluga whale - coronavirus SW1, Delta coronavirus - Bulbul - coronavirus HKU11, Thrush - coronavirus HKU12, Munia - coronavirus HKU13

[0032] In particular, there are currently seven types of human-infecting coronaviruses, as shown in Table 2 below, and they are divided into types that cause colds (229E, OC43, NL63, HKU1) and types that cause severe pneumonia (SARS-CoV, MERS-CoV, SARS-CoV2).

[0033] Virus nameGenusHostSymptomHCoV-229EAlphaHumanMild respiratory symptomsHCoV-NL63AlphaHumanMild respiratory symptomsSARS-CoVBetaHumanSevere respiratory symptomsMERS-CoVBetaHumanSevere respiratory symptomsHCoV-OC43BetaHumanMild respiratory symptomsHCoV-HKU1BetaHumanPneumonia symptomsSARS-CoV-2BetaHumanMild respiratory symptomsSevere cases may cause breathing difficulties

[0034] In the present invention, the "beta coronavirus" is one of the four genera of coronaviruses in the coronavirus subfamily and corresponds to a zoonotic infectious disease. Examples of beta coronaviruses are known to exist, such as Severe Acute Respiratory Syndrome virus (SARS; SARS-CoV), Severe Acute Respiratory Syndrome virus-2 (COVID-19; Severe Acute Respiratory Syndrome virus-2, SARS-CoV-2), Middle East Respiratory Syndrome (MERS; MERS-CoV), human coronavirus OC43 (HCoV-OC43), or human coronavirus HKU1 (HCoV-HKU1).

[0035] In the present invention, the "Severe Acute Respiratory Syndrome virus-2 (SARS-CoV-2)" corresponds to an enveloped, positive-sense single-stranded RNA beta coronavirus belonging to the Coronaviridae family. Historically, coronaviruses that infect humans are severe common cold viruses, including hCoV-OC43, HKU, and 229E5. Analysis of the sequence of the SARS-CoV-2 isolate reveals that the 30-kb genome encodes 14 open-reading frames (ORFs), and the 13 ORFs at the 3' end of the viral genome are expressed from nine predicted subgenomic RNAs, which are expressed from four structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N), and nine putative additional elements. Here, the nucleocapsid protein comprises N1, N2 and N3 segments.

[0036] In the present invention, the coronavirus infectious disease may be at least one selected from the group consisting of coronavirus enteritis, coronavirus diarrhea, coronavirus hepatitis, coronavirus encephalitis, coronavirus gastritis, coronavirus pulmonary inflammation, coronavirus pulmonary fibrosis, coronavirus pulmonary edema, coronavirus telangiectasia, severe acute respiratory syndrome coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome (MERS), but any disease that can be caused by the coronavirus infection may be included without limitation.

[0037] In the present invention, the above-mentioned "subject" refers to a subject that has symptoms of or is suspected of being infected with a virus, and needs to improve or treat a virus infection or a disease caused by the infection by suppressing viral activity, etc., and all animals, including humans, that are already infected or can be infected with the virus.

[0038] In the present invention, “prevention” means any act of delaying viral infection or a disease caused by viral infection by administering the composition of the present invention.

[0039] In the present invention, “improvement” means any act of eliminating or reducing symptoms caused by a viral infection or an infectious disease caused by a viral infection by administering the composition of the present invention.

[0040] As used herein, "treatment" refers to a series of activities performed to alleviate or improve a viral infection or an infectious disease caused by a viral infection by administering the composition of the present invention. For the purposes of the present invention, "treatment" includes activities that suppress or delay the onset of a coronavirus-related infectious disease, and may include, without limitation, any action that alleviates or benefits symptoms caused by a coronavirus-related infectious disease.

[0041] The composition of the present invention can be applied to the prevention or treatment of coronavirus infection or an infectious disease caused by coronavirus by regenerating alveoli damaged by coronavirus infection or an infectious disease caused by coronavirus, improving inflammation, and suppressing edema or capillary expansion.

[0042] The composition of the present invention can be applied as a pharmaceutical composition for regenerating damaged alveoli, a pharmaceutical composition for suppressing lung inflammation, a pharmaceutical composition for suppressing pulmonary edema, or a pharmaceutical composition for suppressing the expansion of pulmonary capillaries, which contains an SPP1 (Secreted Phosphoprotein 1) inhibitor as an active ingredient, and specifically, it can be used for the purpose of regenerating damaged alveoli due to coronavirus infection or an infectious disease, suppressing pulmonary edema due to coronavirus infection or an infectious disease, and suppressing the expansion of pulmonary capillaries due to coronavirus infection or an infectious disease, but is not limited thereto.

[0043] The composition of the present invention can be used as a pharmaceutical composition or a food composition, but is not particularly limited thereto.

[0044] The pharmaceutical composition of the present invention may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans.

[0045] The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections. In the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0046] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0047] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0048] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0049] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0050] The food composition of the present invention can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. It can also be used as a food additive composition necessary for manufacturing the food composition.

[0051] In the present invention, when the inhibitor included as an active ingredient is included in a food composition, the amount may be added at a ratio of 0.1 to 50% of the total weight. Here, when the food composition is manufactured in the form of a beverage, there is no special limitation other than containing the food composition at the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as additional ingredients like a typical beverage. That is, as a natural carbohydrate, it may include a monosaccharide such as glucose, a disaccharide such as fructose, a polysaccharide such as sucrose, a dextrin, a cyclodextrin, and a typical sugar such as dextrin, a cyclodextrin, and a sugar alcohol such as xylitol, sorbitol, and erythritol. As the flavoring agent, there may be mentioned a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).

[0052] In addition, the food composition or food composition additive of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0053] These ingredients can be used independently or in combination. The proportions of these additives are not particularly critical, but are typically selected within the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention.

[0054]

[0055] According to another embodiment of the present invention, there is provided a method for preventing, improving or treating a viral infection disease, comprising administering to a subject in need of administration an effective amount of a composition comprising a Secreted Phosphoprotein 1 (SPP1) inhibitor as an active ingredient.

[0056] In the method for preventing, improving or treating the viral infection disease of the present invention, the contents regarding the SPP1 inhibitor, the virus, the viral infection disease, etc. are the same as those described above, and thus are omitted to avoid excessive complexity of the present specification.

[0057] In the present invention, the term “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0058] In the present invention, the "subject" requiring the administration may include both mammals and non-mammals. Here, examples of mammals include, but are not limited to, humans, non-human primates such as chimpanzees, other apes, or monkey species; livestock animals such as cows, horses, sheep, goats, and pigs; farmed animals such as rabbits, dogs, or cats; and laboratory animals such as rodents such as rats, mice, or guinea pigs. In addition, examples of non-mammals in the present invention may include, but are not limited to, birds or fish.

[0059] The inhibitor included as an active ingredient in the composition of the present invention can be combined with a carrier to enhance the efficacy of treating inflammatory diseases. Any carrier that is biocompatible and capable of achieving the desired effects of the present invention may be used without limitation. Examples include, but are not limited to, serum albumin, peptides, immunoglobulins, hemocyanins, and polysaccharides.

[0060] In the present invention, the formulation of the composition administered as described above is not particularly limited, and may be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, and may be administered as a solid formulation intended to be converted into a liquid formulation for oral or parenteral administration immediately before use, and may be administered by being formulated in the form of, for example, oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, but is not limited thereto.

[0061] In addition, in the present invention, an adjuvant may be further added together with the composition of the present invention during the administration, and any adjuvant known in the art as a pharmaceutical or immunological substance may be used without limitation. For example, sugars or amino acids may be used as stabilizers, and mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, etc. may be used as adjuvants.

[0062] In addition, in the present invention, a pharmaceutically acceptable carrier may be additionally administered together with the composition of the present invention during the administration. Here, the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a fragrance, etc. for oral administration, and may include a mixture of a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. for injections, and may include a base, an excipient, a lubricant, a preservative, etc. for topical administration. The formulation of the composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, the composition may be prepared in the form of a tablet, troche, capsule, elixir, suspension, syrup, wafer, etc. for oral administration, and may be prepared in the form of a unit dosage ampoule or a multiple dosage form for injections. It can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

[0063] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0064] Routes of administration of the composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0065] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0066] As used herein, a "pharmaceutically effective amount" refers to a sufficient amount of an agent to produce a desired biological result. This result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration in a biological system. For example, an "effective amount" for prophylactic or therapeutic use is the amount of a composition disclosed herein required to produce a clinically significant reduction in a disease. The appropriate "effective" amount in any individual case can be determined by one skilled in the art using routine experimentation. Accordingly, the expression "effective amount" generally refers to an amount of an active agent that has a therapeutic effect. In the present invention, the active agent is an SPP1 inhibitor and an agent for the prevention, improvement, or treatment of a viral infection disease.

[0067] The composition of the present invention may vary depending on various factors including the activity of the active ingredient used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 100 mg / kg or 0.001 to 100 mg / kg per day. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0068] The composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, chemotherapy, and biological response modifiers.

[0069]

[0070] According to another embodiment of the present invention, there is provided a method for screening a composition for treating a coronavirus infectious disease.

[0071] The method of the present invention may include a step of contacting a candidate substance with a biological sample containing cells expressing SPP1 (Secreted Phosphoprotein 1); and a step of measuring the expression level of SPP1 in the sample.

[0072] The method of the present invention may further include a step of determining that the candidate substance is a composition for treating a coronavirus infectious disease when the expression level of SPP1 in the sample decreases.

[0073] The "biological sample" of the present invention means any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood including plasma and serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate Examples of biological fluids may include, but are not limited to, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0074] In the screening method of the present invention, descriptions of SPP1 and viruses are omitted below to avoid excessive complexity of the specification as they overlap with those described above.

[0075] The composition provided in the present invention can effectively prevent, improve or treat coronavirus infection or diseases caused by coronavirus infection by restoring symptoms of lung inflammation, pulmonary edema or pulmonary capillary dilation caused by viruses, and further by having an alveolar regeneration effect.

[0076] Figure 1 is a longitudinal single-cell lung atlas of K18-hACE2 transgenic mice infected with SARS-CoV-2 according to one embodiment of the present invention, (a) study design overview, (b) 1Х10 5 Hematoxylin and eosin staining of lung sections from K18-hACE2 mice after mock infection or intranasal infection with pfu SARS-CoV-2, (c) UMAP plot of all 55,340 QC-positive cells sorted by cell type, (d) UMAP plot showing temporal changes in cell types during SARS-CoV-2 infection, (e) proportion of cells over time, (f) heatmap showing proportion of cells with viral RNA by cell type (columns) and time (rows) after correction for viral peripheral RNA (top: cumulative proportion of cells with viral RNA by cell type over time. Left: cumulative proportion of cells with viral RNA by cell type over time), (g) UMAP plot with contour plot showing density of cells with viral RNA.

[0077] FIG. 2 illustrates cellular / molecular changes induced by SARS-CoV-2 infection in the myeloid compartment according to one embodiment of the present invention, wherein (a) UMAP plots of 25,936 myeloid compartments colored by cell type, (b) relative proportions of myeloid compartment cells over time, (c) enrichment of SARS-CoV 2 RNA by myeloid compartment cell type with significance determined by binomial test (inset: UMAP plots of SARS-CoV-2 RNA+ myeloid compartment cells colored red, (d) heatmap showing expanded signature scores of gene sets over myeloid compartment cell type and time, (e) heatmap showing normalized enrichment scores (NES) over myeloid compartment cell type and time, where the sign of the NES indicates increased (positive sign) or decreased (negative sign) expression of the corresponding gene set at a given dpi compared to 0 dpi. Non-significant NES (FDR ≥ 0.05) are shown in gray. (f) Relative proportion of myeloid cell types contributing to the UMI counts of genes associated with cytokine storm over time (top: cumulative proportion of UMI counts of genes associated with cytokine storm over time), (g) Genes associated with type I, II, and III interferons.

[0078] FIG. 3 illustrates cellular and molecular changes in lymphocytes due to SARS-CoV-2 infection according to one embodiment of the present invention, including (a) a UMAP plot of 20,878 lymphocytes colored by cell type, (b) relative proportions of lymphocyte cell types over time, (c) a heatmap showing scaled signature scores of gene sets over lymphocyte cell type and time, (d-f) t-SNE plots of CD8+ T cells subtype-specifically categorized using Palantir (d), inferred pseudotime (e), and differentiation trajectories inferred by dpi (f), (g) a heatmap showing gene expression trends of genes associated with activation of CD8+ T cells along two trajectories, and (h) a violin plot of signature scores of genes associated with T cell exhaustion (left) and type I and type II interferon signaling (right) over time.

[0079] [Revised 21.01.2025 by Rule 91] Figure 4 relates to the impairment of the alveolar regeneration program due to SARS-CoV-2 infection according to one embodiment of the present invention, (a) in situ hybridization results of SARS CoV-2 and sfrpc RNA on lung sections of K18-hACE2 mice, (b) UMAP plots of 5,352 epithelial cells sorted by cell type (inset: Palantir t-SNE plot of AT cells showing differentiation trajectories during alveolar regeneration), (c to g) violin plots of signature scores of genes associated with differentiation potential of AT cells and normalized expression of Etv5, (h) heatmap showing expanded expression of genes associated with AT subtypes, (i and j) t-SNE plots of AT cells colored over virtual time using differentiation trajectories inferred by Palantir (i), AT subtypes (i) using RNA velocity inferred by scVelo, and branch probability (i). A diagram showing the sampled cells (j) assigned to the trajectory according to probabilities.

[0080] FIG. 5 shows the results of SARS-CoV-2 infection leading to impaired cell communication and failure to repair lung damage according to one embodiment of the present invention, (a) a violin plot of PROGENy regulon scores for genes involved in TGFβ signaling, (b) a dot plot showing the communication probability of ligand-receptor pairs (Tgfb1-Tgfbr1 / Tgfbr2) of TGFβ signaling from myeloid subtypes to AT subtypes over time (wherein the dot size represents the P value and the color represents the communication probability), (c) Siglech and Tgfbr1 RNA in situ hybridization results for lung sections from K18-hACE2 mice, (d) a circular plot showing the inferred TGFβ signaling network transmitted to pDCs over time (wherein the circle size is proportional to the number of cells of a given cell type and the edge width represents the communication probability between cell types), (e) a circular plot showing the inferred TGFβ signaling network transmitted to pDCs over time and across myeloid cell types. The figure shows a violin plot showing the expression of Tgfb1, Tgfbr1, and Tgfbr2.

[0081] Figure 6 is about the effect of SPP1 target inhibition to alleviate SARS-CoV-2 infection pathogenesis according to one embodiment of the present invention, (a) study design overview (K18 hACE2 transgenic female mice were injected with 1Х10 2After infection with SARS-CoV-2 at 10 PFU, cells were treated with either aIgG (n=21) or aSPP1 (n=14) in three independent experiments. (a) Survival assessed at the indicated DPI (*p < 0.05, statistical significance was determined by log-rank test), (c) and (d) hematoxylin and eosin staining. Images are shown at low magnification (top; scale bar, 250 μm) and high magnification (bottom; scale bar, 50 μm). Statistical significance is indicated as ***p < 0.001 and **p < 0.005 and was determined by two-way ANOVA.

[0082] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0083] Example

[0084] Experimental method

[0085] Preparation of experimental animals

[0086] Mice (8-week-old, male) used in this study were obtained from the Jackson Laboratory (B6. Cg-Tg(K18-ACE2)2Prlman / J) and have the same genetic background as C57BL / 6. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Seoul National University Bundang Hospital, and all protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (IACUC No. BA-2008-301-071-05). The Ji Seok-Young Research Center is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. All animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition of the Laboratory Animal Care and Use Guidelines, and the Institutional Biosafety Committee of Seoul National University Bundang Hospital approved the specimen handling, inactivation, and transport procedures in an Animal Biosafety Level 3 (ABSL-3) isolation facility.

[0087]

[0088] Cell lines and virus culture

[0089] SARS-CoV-2 strain S virus (BetaCoV / Korea / KCDC03 / 2020, NCCP 43326) was provided by the Korea Centers for Disease Control and Prevention (KDCDC03 / 2020), and Vero E6 cells (CRL-1586) were provided by the Korea Centers for Disease Control and Prevention (KDCDC03 / 2020). Vero E6 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, CA, USA) containing 10% fetal bovine serum (FBS) (Life Technologies, CA, USA) without mycoplasma contamination, and regularly monitored using the LookOut® Mycoplasma qPCR Detection Kit (Merck). Vero E6 cells were inoculated with SARS-CoV-2, and the cytopathic effect was confirmed on day 3. Virus titers were measured by plaque assay and stored at -70°C. Mycoplasma contamination was regularly monitored using a mycoplasma detection kit (Lonza). Mice were anesthetized with ketamine (20 mg / kg) / xylazine (10 mg / kg). All mice received 10 5 Mice were infected intranasally with PFU, and their body weight, body temperature, and health status were monitored daily. For the SPP1 blockade experiment, mice were administered 10 2 Mice were intranasally infected with PFU and treated five times with 100 μg of αIgG (MOPC-21) or αSPP1 (MPIIIB10), as shown in Figure 6a. Mice were sacrificed in a CO2 chamber on day 0 post-infection (dpi) or on days 1, 2, or 5. All experiments with SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory at Seoul National University Bundang Hospital.

[0090]

[0091] Virus titer by plaque assay

[0092] Plaque assays for SARS-CoV-2 were performed using conventional methods. Vero E6 cells were seeded at 3 x 10 per well in 12-well plates. 5 Cells were seeded at a concentration of 10 cells / mL and cultured to form a monolayer 1 day before plaque assay. Cells were infected in duplicate with 10-fold serial dilutions of SARS-CoV-2 for 1 h and treated with 0.3% SeaPlaque (LONZA, Basel, Switzerland) agarose medium containing 2% FBS. After 72 h of incubation, virus-infected cells were fixed with 4% (v / v) paraformaldehyde (Biosesang, Seongnam, Korea) for 1 h and then stained using crystal violet solution (Sigma-Aldrich, 548-62-9). Infectious virus titers were measured as PFU per ml.

[0093]

[0094] cell culture

[0095] Air-liquid interface (ALI) culture was established using human adenocarcinoma A549 (ATCC# CCL-185) cell line, and the membrane of Millicell inserts (0.4 mm pore, polycarbonate, Millipore, USA) was pre-coated with 70 μg / mL rat tail collagen type I (BD Biosciences, USA). Then, single-cell suspension of A549 cells was seeded onto the apical surface of the collagen-precoated membrane. The seeding density was 3 х 10 per well for inserts with diameters of 30 mm and 12 mm, respectively. 6 The cells were canine. 24 hours after inoculation, the culture medium on the apical side was removed to establish ALI conditions. ALI cultured cells were supplied with fresh medium under the insert every two days.

[0096]

[0097] Histopathology

[0098] Lung tissues were fixed in 10% neutral buffered formalin for 1 day and processed using standard methods. 3-μm paraffin-embedded sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome for collagen. Lesions were graded using a semiquantitative scale based on the percentage of tissue affected as follows: 0, absent; 1, minimal, if less than 10% of the tissue was affected; 2, mild, if 10% to less than 25% of the tissue was affected; 3, moderate, if 25% to less than 50% of the tissue was affected; 4, moderately severe, if 50% to less than 75% of the tissue was affected; or 5, severe, if 75% or more of the tissue was affected. Lung lesions were assessed using the following criteria. (1) Pulmonary inflammation, involving the perivascular / peribronchial spaces and the interstitial space with a moderate number of inflammatory cells surrounding the surrounding area and more than five inflammatory cells in each alveolar space; (2) Pulmonary edema, involving the perivascular space with an edematous palate and alveolar edematous spaces. To assess splenic lesions, apoptosis of the white pulp was scored based on the extent of the affected area. White pulp atrophy was scored based on the extent of size reduction compared to the normal splenic white pulp, and the degree of villus atrophy in the duodenum was scored based on changes in the shape (kinking) and length of the villi. Histopathologic scores were determined by at least two pathologists.

[0099]

[0100] Immunostaining and in situ hybridization (ISH)

[0101] To perform immunofluorescence staining on tumor tissues, 3 μm cross-sectioned paraffin sections were dewaxed, rehydrated, and then heated in 0.01 M citrate buffer (pH 6.0) at 100 °C for 20 min to allow antigen retrieval for immunostaining. The sections were blocked with phosphate-buffered saline (PBS) containing 2.5% BSA and then incubated overnight at 4 °C with goat anti-Osteopontin / OPN antibody (AF808; R&D Systems) and rabbit anti-F4 / 80 (70076T; Cell signaling). Secondary antibodies conjugated with Alexa Fluor 488 donkey anti-mouse IgG (A-21202; Thermo Fisher Scientific) and Alexa Fluor 568 donkey anti-rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A10042; Thermo Fisher Scientific) were used. Slides were mounted with Vectashield mounting media (H-1200; Vector Laboratories, Burlingame, CA, USA). mRNAs of the S gene encoding ly6a, sftpc, siglech, tgfbr1, and the viral spike protein were detected by in situ hybridization (ISH) using RNAscope Probes (ACD, Bio-Techne) using RNAscope® 2.5 HD Duplex Assay (ACD, Bio-Techne). ISH was performed according to the manufacturer's protocol. Paraffin sections were dewaxed, rehydrated, recovered in heated buffer, and then treated with protease for 15 minutes. The probes were applied to the slides and incubated in a HybEZ oven (ACD) at 40°C for 2 hours. After signal amplification, the slides were developed with HRP-based Green and AP-based Fast Red. Mayer hematoxylin was used for counterstaining.

[0102]

[0103] Generation of single-cell suspensions from mouse lung tissue

[0104] After cutting the jugular vein, PBS was injected into the heart using a 10 ml syringe. Dispase and low-melt agarose were injected into the trachea using a catheter. The lungs were removed and placed in dispase and incubated at room temperature for 45 minutes. The lung tissue was pulled out using forceps and then incubated in DMEM containing 100 μl DNase for 10 minutes at room temperature. Single-cell suspensions were formed using 100 μm, 70 μm, and 40 μm strainers. The suspension was centrifuged at 60 g for 15 minutes, and the supernatant was removed. The pellet was resuspended in RBC lysis buffer and diluted with DMEM. After centrifugation at 160 g for 15 minutes, the pellet was resuspended in DMEM to form a single-cell suspension. To increase viability, dead cells were filtered from the single cell suspension using the MACS® kit.

[0105]

[0106] single-cell RNA sequencing

[0107] For each scRNA-seq, two mice were used from each group, and dead cells were removed from the single-cell suspension using the Dead Cell Removal Kit according to the manufacturer's instructions before submitting for scRNA-seq. After isolating single cells from SARS-CoV-2-infected mice, 6,000 cells were split into particles containing barcoded beads, single cells, and a reverse transcriptase mixture using a GemCode instrument. Single-cell libraries were constructed using the 10X Genomics Single Cell 3' v3.1 Reagent Kit according to the manufacturer's instructions.

[0108]

[0109] scRNA-seq data analysis

[0110] Data preprocessing and quality control

[0111] Raw reads were aligned to the GRCm39 mouse reference genome (Ensembl release 104), which concatenates the SARS-CoV-2 RNA genome (Wuhan-Hu-1, GenBank NC_045512.2) and the human ACE2 gene sequence (GenBank NG_012575.3), and processed using Cell Ranger software (v5.0.1). After constructing a gene-wise cell UMI count matrix using default parameters, empty droplets were removed using the emptyDrops function in the DropletUtils (v1.14.2) R package with an FDR < 0.01. Additionally, low-quality cells with fewer than 1,000 total UMIs and more than 10% of their UMIs mapping to mitochondrial genes were filtered using the perCellQCMetrics function in the scater (v1.18.6) R package. The aggregated count matrix across all samples was processed using the Seurat (v4.0.0) R package for normalization, feature selection, scaling, and visualization. The raw count matrix was normalized using the NormalizeData function, and the top 500 highly variable genes (HVGs) were selected using the FindVariableGenes function for data aggregation and visualization. The normalized expression matrix of the HVGs was further scaled using the ScaleData function.

[0112] Data Integration and Visualization

[0113] Principal component analysis (PCA) was performed on the scaled expression matrix of HVG using the RunPCA function of the Seurat package. Batch effects were then corrected using the RunHarmony function of the Harmony (v1.0) R package. Each sample was considered a batch. For visualization, the 20 Harmony-corrected principal components (PCs) were used as input to the RunUMAP function of the Seurat package.

[0114] Correcting for ambient viral reads

[0115] A binomial test was used to determine whether cells were infected based on the total number of UMIs and the number of viral UMIs mapped to the SARS-CoV-2 RNA genome in each cell. The probability of capturing a neighboring viral UMI was estimated by calculating the proportion of vial UMIs in cells discarded during the quality control step. The binomial test was performed at the sample level, and the raw P-values ​​across all samples were pooled and adjusted using the Benjamini-Hochberg method. Cells were considered infected if the adjusted P-value was <0.01.

[0116] Gene set enrichment analysis and signature scoring of signaling pathways

[0117] We used the fgsea (v1.16.0) R package to perform gene set enrichment analysis (GSEA). Pairwise differential expression analysis was performed between each dpi and the control group (0 dpi) using the FindMarkers function of the Seurat package, and genes were ranked based on the sign of the log2 (fold change) multiplied by the -log10 (P-value). We then used the fgsea function on 10,000 permutations and gene sets obtained from the msigdbr (v7.4.1) R package. The signature scores of gene sets or signaling pathways were calculated using the AddModuleScore function of the Seurat package and z-scaled. In addition, g:Profiler was used for gene transformation in Figure 3h to analyze only genes with one-to-one mapping between human and mouse.

[0118] Partial correlation analysis

[0119] To identify transcriptional signatures associated with SARS-CoV-2 infection at the single-cell level, we performed a partial correlation analysis between viral transcription levels and pathway activity, controlling for the total number of UMIs per cell. We then calculated the Pearson correlation coefficient to assess the association between viral transcription levels and pathway activity, and results with an adjusted P-value of less than 0.05 were considered significant.

[0120] Trajectory inference

[0121] To infer the differentiation trajectories of CD8+ T and alveolar (AT) epithelial cells, we used the Python package Palantir (v1.0.0). For CD8+ T cell analysis, the raw count matrix was normalized and log-transformed, and high-variable-gene clustering (HVG) was selected using the pp.highly_variable_genes function of the scanpy (v1.8.1) Python package, with n_top_genes=1500 and flavor='cell_ranger'. Diffusion components (DCs) were calculated using the run_diffusion_maps function with 100 PCs calculated from the HVG using the run_pca function. For AT cells, DCs were calculated using the run_diffusion_maps function with 30 harmonic-corrected PCs to correct for batch effects. We then inferred pseudotimes and branching probabilities using the run_palantir function with default options. We selected the cells with the highest expression of Tcf7 in CD8+ T cells as the starting cells for pseudotime construction. Similarly, we selected the cells with the highest expression of Etv5 in AT cells as the starting cells. For both cell types, we used the decide_multiscale function followed by the run_tsne function with default options for visualization. To support the inferred trajectories of AT cells, we estimated RNA velocities using the scVelo (v0.2.4) Python package. We quantified spliced ​​and unspliced ​​counts in each sample using the run option of the velocyto (v0.17.17) Python package. After aggregating the output across all samples, we normalized and log-transformed the spliced ​​and unspliced ​​count matrix using the pp.filter_and_normalize function with n_top_genes=500. We then computed moments using the pp.moments function with n_pcs=30 and n_neighbors=30, and used the probabilistic mode to tl.The velocity was calculated using the velocity function.

[0122] Intercellular interactions

[0123] To analyze differential cell-to-cell interactions induced by SARS-CoV-2 infection in the mouse lung, we used the CellChat (v1.0.0) R package. Significantly upregulated genes encoding ligands and receptors for each cell type were identified using the identifyOverExpressedGenes function, based on the ligand-receptor (LR) interaction database loaded by the CellChatDB.mouse function. The communication probabilities between the two cell types were then calculated using the ComputeCommunProb and ComputeCommunProbPathway functions. After inferring cell-to-cell interactions in each sample, we compared and visualized the information flow for specific signaling pathways associated with the lung injury response to SARS-CoV-2 infection at four time points (0, 1, 2, and 5 dpi) using the rankNet function with stacked=FASLE. The signaling pathway network showing interacting cell type pairs was visualized using the netVisual_aggregate function, and the communication probabilities of interacting ligand-receptor pairs were visualized using the netVisual_bubble function.

[0124]

[0125] statistics

[0126] Statistical analysis was performed using Prism 10 software (GraphPad Software). A P value of less than 0.05 was considered statistically significant. Unless otherwise stated, all data are expressed as mean ± standard deviation (SD).

[0127]

[0128] Experimental results

[0129] Time-resolved single-cell lung atlas of K18-hACE2 mice

[0130] To study the dynamic changes associated with severe SARS-CoV-2 infection, 8-week-old K18-hACE2 TG mice were injected with 1 x 10 5Mice were intranasally inoculated with SARS-CoV-2 (strain NCCP43326) at a dose of plaque-forming units (PFU), and lung tissues were collected at 0, 1, 2, and 5 days post-infection (dpi) (see Fig. 1a). After SARS-CoV-2 infection, mice lost weight starting at 5 dpi, and all mice died within 7 dpi. The body temperature of SARS-CoV-2-infected mice decreased in a similar trend to body weight, and analysis of lung tissues stained with hematoxylin and eosin revealed that SARS-CoV-2-infected K18-hACE2 mice developed progressive pneumonia (see Fig. 1b). Minimal perivascular immune cell infiltration was observed in the lung tissues of infected mice at 1 dpi. At 2 dpi, immune cells began to accumulate primarily in the perivascular area, occupying larger areas of the lung and extending into the alveolar spaces with perivascular and interstitial edema at 5 dpi. Inflammatory cells comprised primarily of a large number of monocytes and a small number of neutrophils. Furthermore, thickened alveolar septa, collapsed alveoli, and multifocal hemorrhages were observed in the parenchyma (see Figure 1b). To elucidate cellular and molecular changes associated with severe SARS-CoV-2 infection, we generated a time-lapse scRNA-seq lung atlas in K18-hACE2 mice (n = 2 or 4). A computational pipeline for quality control and batch correction was used to generate a time-resolved lung atlas of 55,340 quality-control-positive cells during SARS-CoV-2 infection (see Figures 1c and 1d). Cells were visualized using Uniform Manifold Approximation and Projection (UMAP), and a graph-based clustering algorithm was applied to group cells into 25 clusters. Based on the expression of canonical marker genes, 21 major cell types were defined and further sub-clustered into 36 cell subtypes. Significant changes in cellular composition were observed during the infection process (see Figures 1D and 1E).Non-immune cell compartments, including epithelial, endothelial, mesothelial, and fibroblast cells, were markedly reduced starting at 1 dpi (see Figures 1d and 1e). A significant expansion of monocytes and dendritic cells (DCs) was observed at 1 and 2 dpi, followed by a delayed expansion of macrophages and neutrophils at 5 dpi (see Figure 1e). Conversely, innate lymphoid cells showed a persistent decrease in frequency after infection, whereas other lymphocytes, including natural killer cells, proliferative T cells, and innate T cells, increased at 5 dpi (see Figure 1e). These results suggest that early accumulation of monocytes and DCs in the infected lung, followed by reciprocal expansion of macrophages and neutrophils, is a critical feature of lung disease in severe SARS-CoV-2 infection.

[0131] Next, we aimed to determine the differential enrichment of SARS-CoV-2 viral RNA across cell types and time points, considering peripheral viral RNA (see Figures 1f and 1g). As expected, significant enrichment of viral RNA was observed in epithelial cells at the onset of infection (adjusted P < 0.01). Macrophages and endothelial cells were also enriched in viral RNA (adjusted P < 0.01), a finding consistent with a recent scRNA-seq study in the lungs of COVID-19 patients. Both hACE2 and Krt18 were specifically expressed in epithelial and mesothelial cells. Furthermore, viral RNA levels in epithelial cells were significantly positively correlated with characteristic scores for apoptosis and inflammation. These results suggest that while epithelial cells are the primary target cell type for SARS-CoV-2 infection, viral RNA enrichment in other cell types may be a consequence of phagocytic engulfment. Together, these results demonstrate dynamic cellularity and potential cross-talk in lung tissues of K18-hACE2 TG mice upon SARS-CoV-2 infection.

[0132]

[0133] Cellular and molecular changes in the myeloid compartment

[0134] Given the dynamic changes in cell type composition and enrichment of viral RNA during the infection process, we sought to characterize the cellular and molecular changes in the myeloid compartment at higher resolution (see Fig. 2a). The bone marrow compartment contains plasmacytoid DCs (pDC; Siglech), conventional type 1 DCs (cDC1.Xcr1; Xcr1 and Clec9a), conventional type 2 DCs (cDC2.Itgax; Itgax and Itgam), a recently characterized subset of conventional DCs (cDC.Fscn1; Fscn1 and Ccr7), monocyte-derived DCs (MoDC; Ly6c2 and MHC class II), classical monocytes (Mon.Ly6c2; Ly6c2 and Vcan), non-classical monocytes (Mon.Itgal; Itgal and Fcgr4), Spp1+ macrophages (Mac.Spp1; Spp1 and Cd300lf), M2-like macrophages (Mac.Cd163; Cd163 and Lyve1), alveolar macrophages (Mac.Pparg; Pparg and Car4), Mast cells (Mast: Cpa3 and Ms4a2), and neutrophils (Ly6g and Retnlg).

[0135] Consistent with the marked increase in monocytes at 1 and 2 dpi shown in Figure 1e, we observed a marked increase in Mon.Ly6c2 classical monocytes (see Figure 2b). Similar to previous studies in K18-hACE2 TG mice and human patients, a rapid decrease in homeostatic alveolar macrophages (Mac.Pparg) and M2-like macrophages (Mac.Cd163) was observed in infected lung tissue after infection (see Figure 2b). The relative abundance of pDCs, cDCs.Fscn1, cDC1.Xcr1, and MoDCs increased at 1 and 2 dpi, but decreased at 5 dpi (see Figure 2b). These observations are consistent with the recently characterized negative correlation between pDC abundance in peripheral blood and COVID-19 severity. Interestingly, SARS-CoV-2 RNA was specifically enriched in Mac.Spp1 and Mac.Pparg subsets in various myeloid populations, indicating a unique cellular tropism of SARS-CoV-2 in the immune system (see Fig. 2c).

[0136] Next, we sought to identify SARS-CoV-2-induced changes in cellular pathways within the myeloid compartment. For each myeloid cell subset, we ranked all genes by their abundance at a given dpi compared to 0 dpi, then performed gene set enrichment analysis (GSEA) and gene set signature analysis, with the results presented in Figure 2D. Upregulated genes after infection were associated with type I and type II interferon (IFN) responses across most myeloid lineages and at most time points. The increase in the IFN response signature was accompanied by increases in genes associated with the proteasome, inflammatory response, tumor necrosis factor alpha (TNFα) signaling, complement activation, RIG-I-like receptor signaling, and antigen presentation in most myeloid subsets. These associations were more pronounced at 1 and 2 dpi but not at 5 dpi. We found that MYC signaling, oxidative phosphorylation, and DNA repair were upregulated at 5 dpi, whereas actin cytoskeleton regulation was downregulated. Interestingly, we observed a downregulation of signatures associated with inflammation and IFN responses at 5 dpi compared to 1 and 2 dpi (see Fig. 2d). Furthermore, SARS-CoV-2 infection significantly suppressed ribosome-related pathways in myeloid cells, suggesting that SARS-CoV-2 may possess potential immune evasion strategies similar to other viruses. The dramatic dynamics observed in the Mac.Pparg and Mac.Spp1 populations in the myeloid compartment during infection (see Fig. 2b) were further confirmed by staining lung tissue with SPP1 and F4 / 80 antibodies. We next aimed to identify key myeloid cell populations that promote lung fibrosis during the response to severe COVID-19. Compared to the Mac.Pparg population, the Mac.The Spp1 subpopulation was characterized by an enrichment in gene sets associated with CC chemokine receptor type 2 (CCR2) signaling (Ccl2, Ccl7, Ccl12, and Ccr2); myeloid leukocyte migration (C3ar1, Lgmn, Aif1, Csf1r, Cx3cr1, and Lgals3); and major histocompatibility complex (MHC) class II. Conversely, the Mac.Pparg subpopulation exhibited higher expression of inflammatory cytokines (Il-1a, Il-1b, Il-15, Il-16, Il-18, and Il-27) and upregulation of genes associated with fatty acid metabolism and phagocytosis. Consistent with this, Mac.Pparg showed increased phagocytosis in response to higher levels of viral transcripts. The role of CCR2 in recruiting circulating monocytes to sites of inflammation and the strong enrichment of SARS-CoV-2 RNA in the Mac.Spp1 subset suggest a potential role for the Mac.Spp1 subset in limiting viral spread via the CCR2 pathway. These results are consistent with previous reports on the role of CCR2 signaling in the increase of monocyte-derived cells that suppress viral replication and inflammation. Furthermore, similar to previous studies in bronchoalveolar lavage (BAL) fluid from COVID-19 patients, we found that the expression levels of Tgfbi, Spp1, and Lgmn were increased in Mac.Spp1 cells in proportion to the severity of COVID-19 disease. This suggests that the aberrant upregulation of Mac.Spp1 by SARS-CoV-2 infection induces fibrosis and damage in lung tissue to limit the effectiveness of the innate response and accelerate disease severity. To further elucidate the primary driver of lung inflammation in the bone marrow, we sought to estimate the amount of cytokine or IFN responses that peaked at 1 dpi and continued to decline until 5 dpi (see Figures 2f and 2g). At 1 and 2 dpi, the SARS-CoV2-induced cytokine storm was primarily driven by Mac.Pparg and Mon.We confirmed that Ly6c2 classical monocytes were dominant, and pDCs played a crucial role in inducing virus-induced IFN responses in lung tissue (see Figures 2f and 2g). Overall, these results indicate dynamics of functional changes in the myeloid compartment during the course of infection, characterized primarily by gene signatures for inflammatory and IFN responses, which peaked at 1 and 2 dpi in the lung but were completely resolved by 5 dpi.

[0137]

[0138] Cellular and molecular changes in the lymphoid compartment

[0139] To characterize the cellular and molecular changes in the lymphoid compartment, the lymphoid cell cluster was divided into 14 subsets based on the expression of canonical marker genes (see Figure 3a): 9 T cell subsets; 2 natural killer (NK) cell subsets; and 1 innate lymphoid cell (Il-7r and Kit), memory B cell (Cd19, Cd38, and H2-dma), and plasmablast (Sdc1 and Mki67) subset. Of the 9 T cell subsets, 3 subsets were assigned to CD4+ T cells, and 3 to CD8+ T cells were assigned to proliferating T cells (T. Prolif; Cd3d and Mki67), gd T cells (Cd3d, Zbtb16 and Trdc), and innate T cells, including invariant NK T and mucosal-associated invariant T cells (T. Innate; Cd3d and Zbtb16). Among CD4+ T cells, naive CD4+ T cells (T. CD4. Naive; Ccr7 and Shell), effector or effector memory CD4+ T cells (T. CD4. EM; Cd44), and regulatory T cells (Treg; Foxp3 and Il2ra) were distinguished. Three subsets of CD8+ T cells were annotated: naive CD8+ T cells (T.CD8.Naive; Ccr7 and Shell), cytotoxic CD8+ T cells (T.CD8.Cytotoxic; Gzma, Gzmb, and Prf1), and exhausted CD8+ T cells (T.CD8.Exhausted; Pdcd1, Havcr2, and Lag3). Two NK subsets, NK and NK.Cd226, are distinguished by the expression of Il7r and Cd226 (NK2.Cd226). Next, we aimed to determine SARS-CoV-2-induced cellular changes within the lymphoid compartment. The proportion of the T.CD4.Naive subset decreased over time, whereas the proportion of the T.CD4.EM subset increased, consistent with the previously described negative correlation between COVID-19 disease severity and naive T cell frequency in peripheral blood (see Figure 3b). The T.CD4.EM subset showed high expression of cytokines for T helper type 1 cells (Ifng and Tnf), suggesting that this subset is primarily composed of T helper type 1 cells. The T.CD8.Naive subset did not show significant changes in abundance over time, but the proportion of the T.CD8.Cytotoxic subset peaked at 1 dpi and then steadily decreased, and the T.CD8.Exhausted subset was identified at 5 dpi. T.The Prolif subset expanded after 2 dpi, consistent with previous reports of enriched proliferating T cells in the peripheral blood of patients with severe COVID-19. Furthermore, the NK.Cd226 subset, known to be a more activated NK subset compared to other NK subsets, showed an increase at 5 dpi. Given the increase at 5 dpi, we next analyzed the molecular characteristics of the T.CD8.Exhausted subset. Type I and type II IFN response signatures were increased in most lymphoid lineages at 1 and 2 dpi, with increased signatures of inflammatory response, TNFα signaling, and cytokine signaling (see Figure 3c). Similar to the myeloid lineage, the increased IFN signature decreased at 5 dpi in all lymphoid lineage subsets (see Figure 3c). This is consistent with previous reports showing a negative enrichment of type 1 IFN signatures in SARS-CoV-2-reactive CD8+ T cells from patients with severe disease. The T.CD8.Exhausted subpopulation exhibited both activated and exhausted phenotypes after infection (see Figure 3c), consistent with previous reports describing functional SARS-CoV-2-reactive CD8+ T cells expressing both cytotoxic and exhausted signatures. Single-cell trajectory analysis predicted that the T.CD8.Cytotoxic (expanded at 1 and 2 dpi) and T.CD8.Exhausted (expanded at 5 dpi) subsets differentiated along branching pathways starting from the T.CD8.Naive subset, suggesting that naive CD8+ T cells can differentiate into various subsets after infection in a time-dependent manner (see Figures 3d-g). Exhausted, but functional, CD8+ T cells were associated with impaired IFN responses at 5 dpi (see Figure 3h). This is consistent with recent reports showing a positive mechanistic link between IFN signaling and the exhaustion phenotype of CD8+ T cells.Overall, the results indicate a dynamic shift towards SARS-CoV-2 in a lymphoid context with an increase in activated T cells in the lungs of K18-hACE2 mice, which could potentially lead to lung damage.

[0140]

[0141] Damaged alveolar regeneration program in the epithelial compartment

[0142] Enrichment of viral RNA in epithelial cells (see Figure 1f) was confirmed by in situ hybridization, which revealed that alveolar cells exhibited a spatial distribution of SARS-CoV-2 virus in lung tissue (see Figure 4a). Because viral infection in alveolar cells has been shown to induce hypercytokine responses and abnormal alveolar cell differentiation in severe COVID-19, we next analyzed the transcriptional response to SARS-CoV-2 within the epithelial compartment, consisting of alveolar and airway epithelial cells. Among airway epithelial cells, ciliated cells (Foxj1), club cells (Scgb1a1), and neuroendocrine cells (Ascl1) were identified. Among alveolar epithelial cells, we identified alveolar type 1 pneumocytes (AT1; Ager, Cav1, and Pdpn), alveolar type 2 pneumocytes (AT2; Sftpa1 and Sftpc), activated AT2 cells (Lcn2 and Ptges), and damage-associated transient progenitor cells (DATP; Cldn4 and Krt8) (see Fig. 4b). The activated AT2 subpopulation is distinguished from the AT2 subpopulation by the downregulation of regulators essential for AT2 cell identity (Abca3, Cebpa, and Etv5) and the upregulation of damage-induced genes such as Lcn2, Cxcl17, Lrg1, Ptges, and Glrx. This suggests that AT2 cells lose their AT2 identity upon SARS-CoV-2 infection. AT2 cells, which produce large amounts of surfactant proteins to regulate surface tension in the alveolar epithelium, serve as alveolar stem cells with self-renewal capabilities and differentiate into AT1 cells for lung homeostasis and regeneration after injury. DATP, also known as pre-AT1 transitional cell state (PATS) or alveolar differentiation intermediate (ADI), has recently been identified as a cell state in which AT2 cells transition to AT1 cells in regenerating alveolar tissue, and this has been associated with impaired alveolar regeneration and pulmonary fibrosis.Given the DATP accumulation and pulmonary fibrosis observed in severe human COVID-19, we aimed to analyze the cellular dynamics of DATP during SARS-CoV-2 infection. While the DATP signature of alveolar epithelial cells increased over time, peaking at 5 dpi (see Figure 4c), we observed a progressive decline in AT2 signatures, including Etv5, essential for maintaining AT2 cell identity (see Figures 4d and 4g). AT1 cells were not readily apparent at 0 dpi (perhaps due to their thin and elongated morphology), but AT1 signatures and AT1 cell fraction gradually increased in later stages of infection (see Figures 4e and 4f). Interestingly, DATP and AT1 cells were not clearly distinguished by marker expression, suggesting an impaired lung degeneration program during infection (see Figure 4h). Next, we used Palantir and scVelo to track the time-resolved differentiation trajectory from AT2 cells to AT1 cells (see Figures 4i and 4j). At 0 dpi, a linear transition from AT2 cells to AT1 cells was observed via rare DATP (<6%) (pathway 1). This represents a trajectory by which AT2 cells spontaneously differentiate into AT1 cells in homeostasis.

[0143] [Revised 21.01.2025 under Rule 91] After infection, AT2 cells diverge from activated AT2 cells to generate either highly inflammatory activated AT2 cells (pathway 2) with a high IFN response signature at 1 and 2 dpi or AT1 cells (pathway 3) via DATP at 5 dpi (see Figures 4i and 4j). At 5 dpi, both expanded DATP and AT1 cells highly expressed DATP marker genes (Krt8, Cldn4, Cdkn1a, Sprr1a, and Tnip3) and exhibited increased features of hypoxia, p53 signaling, TNFα signaling, and apoptosis (see Figure 4h), which are associated with pulmonary fibrosis and severe COVID-19. Because hypoxic signaling controls AT2-DAPT conversion and inflammation impairs DAPT-AT1 differentiation, these data suggest that severe SARS-CoV-2 infection induces impaired alveolar regeneration characterized by persistence of DATP.

[0144]

[0145] Dynamic cell-to-cell communication network during SARS-CoV-2 infection

[0146] Finally, we aimed to systematically identify time-resolved cellular communication networks by investigating ligand-receptor interactions between cell types in lung tissue. We observed a significant increase in TNF signal intensity at 1 and 2 dpi, but a decrease at 5 dpi. This is consistent with a transient upregulation of the innate response of myeloid cells after SARS-CoV-2 infection. Notably, some signaling pathways known to induce pulmonary fibrosis (SPP1) or lung leak and damage (ANGPTL) after influenza infection were predicted to be upregulated at 5 dpi, whereas signaling pathways that maintain lung homeostasis (SEMA3) or suppress lung fibrosis (THY1) were downregulated after infection. Considering the altered cellular communication networks that induce dysregulated lung repair responses in the later stages of infection, we hypothesized that mechanical stress due to impaired alveolar regeneration induces lung fibrosis and dysfunction via TGFβ signaling. Within the alveolar epithelium, downstream features of TGFβ signaling (see Fig. 5a) and epithelial-mesenchymal transition (EMT) were increased at 5 dpi, consistent with increased TGFβ signaling from the medullary compartment to alveolar epithelial cells (see Fig. 5b). Since mechanical stress has been shown to induce EMT phenotypes and lung fibrosis via the midkine (MDK) Notch2-ACE signaling pathway and regulate TGFβ expression in lung tissue, enhanced MDK signaling from fibroblasts and mesothelial cells to all epithelial cell types, including AT2 and ciliated cells (at 2 dpi) or DATP and AT1 cells (at 5 dpi), suggests a possible role for MDK signaling in dysregulated lung repair responses. Interestingly, the TGFβ signaling network, which is predominantly pDC-dominated, significantly increased the expression of TGFβR during SARS-CoV-2 infection (see Figures 5c-5e) and was enriched together with the myeloid compartment as the infection progressed (see Figure 5d).Because TGFβ is known to impair pDC activity and IFN-I production, the impaired IFN production in pDCs observed at 5 dpi may be due to increased TGFβ signaling from myeloid cells to pDCs (see Figures 5d and 5e). To highlight the importance of this phenomenon, we performed an experiment in which lung lysates from SARS-CoV-2-infected mice were exposed to A549 cells cultured at a 2D air-liquid interface. This exposure was performed under two conditions: with and without concomitant administration of a TGFβ receptor inhibitor (SB525334). Remarkably consistent with insights derived from scRNAseq analysis, blocking TGFβ signaling significantly reduced the expression of fibrosis-related genes, particularly mmp1 and vimentin. These genes have previously been shown to be upregulated in response to SARS-CoV-2 lung lysate treatment. Taken together, these results suggest that the dysregulated lung repair response induced by SARS-CoV-2 infection may be due to disruption of cellular communication networks, exemplified by the aberrant TGFβ signaling pathway.

[0147]

[0148] Targeting SPP1 Inhibition to Alleviate SARS-CoV-2 Infection Pathogenesis

[0149] To verify whether SPP1 inhibition could be used as a therapeutic strategy against SARS-CoV-2 infection, we administered anti-SPP1 or isotype-matched anti-IgG treatment after SARS-CoV-2 infection and monitored disease progression in infected mice (see Fig. 6a). Notably, the group treated with anti-SPP1 showed a significantly reduced mortality rate in K18-hACE2 mice upon SARS-CoV-2 infection compared to the group treated with anti-IgG (survival rate - αIgG: 52% VS αSPP1: 86%) (see Fig. 6b). In other words, the survival rate was significantly improved, and SPP1 inhibition effectively improved inflammation and showed excellent effects on edema and capillary dilation (see Figs. 6c and 6d, top: 14 dpi, bottom: 42 dpi). Taken together, these results suggest that targeting SPP1 inhibition has potential as a therapeutic approach to alleviate the pathogenesis of SARS-CoV-2 infection, and is expected to contribute to the prevention or treatment of SARS-CoV-2-related diseases.

[0150]

[0151] Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible within a scope that does not depart from the technical spirit of the present invention described in the claims.

[0152] Sequence number 1: SPP1 amino acid sequence, Homo sapiens

[0153] MRIAVICFCLLGITCAIPVKQADSGSSEEKQTLPSKSNESHDHMDDMDDEDDDDHVDSQDSIDSNDSDDVDDTDDSHQSDESHHSDESDELVTDFPTDLPATEVFTPVVPTVDTYDGRGDSVVYGLRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQDLNAPSDWDSRGKDSYETSQLDDQSAETHSHKQSRLYKRKANDESNEHSDVIDSQELSKVSREFHSHEFHSHEDMLVVDPKSKEEDKHLKFRISHELDSASSEVN

Claims

1. A pharmaceutical composition for preventing or treating coronavirus infectious disease, comprising a SPP1 (Secreted Phosphoprotein 1) inhibitor as an active ingredient.

2. In paragraph 1, A pharmaceutical composition wherein the above SPP1 inhibitor is an agent that reduces the expression level of the SPP1 gene or an agent that inhibits the activity of a protein encoded by the SPP1 gene.

3. In paragraph 2, A pharmaceutical composition wherein the agent that reduces the expression level of the SPP1 gene is at least one selected from the group consisting of an antisense nucleotide that complementarily binds to the SPP1 gene, a short interfering RNA (siRNA), a short hairpin RNA, a microRNA (miRNA), a guide RNA (gRNA), and a ribozyme.

4. In paragraph 2, A pharmaceutical composition, wherein the agent that inhibits the activity of the protein encoded by the SPP1 gene is at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to the protein encoded by the SPP1 gene.

5. In paragraph 1, A pharmaceutical composition wherein the above coronavirus is a beta coronavirus.

6. In paragraph 5, A pharmaceutical composition wherein the above beta coronavirus is Severe Acute Respiratory Syndrome virus (SARS; SARS-CoV), Severe Acute Respiratory Syndrome virus-2 (SARS-CoV-2), Middle East Respiratory Syndrome virus (MERS; MERS-CoV), human coronavirus OC43 (HCoV-OC43), or human coronavirus HKU1 (HCoV-HKU1).

7. In paragraph 1, A pharmaceutical composition, wherein the coronavirus infectious disease is at least one selected from the group consisting of coronavirus enteritis, coronavirus diarrhea, coronavirus hepatitis, coronavirus encephalitis, coronavirus gastritis, coronavirus pulmonary inflammation, coronavirus pulmonary fibrosis, coronavirus pulmonary edema, coronavirus telangiectasia, severe acute respiratory syndrome coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome (MERS).

8. A food composition for preventing or improving coronavirus infection disease, comprising a SPP1 (Secreted Phosphoprotein 1) inhibitor as an active ingredient.

9. In paragraph 8, A food composition, wherein the above coronavirus infectious disease is at least one selected from the group consisting of coronavirus enteritis, coronavirus diarrhea, coronavirus hepatitis, coronavirus encephalitis, coronavirus gastritis, coronavirus pulmonary inflammation, coronavirus pulmonary fibrosis, coronavirus pulmonary edema, coronavirus telangiectasia, severe acute respiratory syndrome coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome (MERS).

10. A method for preventing or treating a coronavirus infectious disease, comprising administering a SPP1 (Secreted Phosphoprotein 1) inhibitor to a subject other than a human.

11. In Article 10, A method according to claim 1, wherein the coronavirus infectious disease is at least one selected from the group consisting of coronavirus enteritis, coronavirus diarrhea, coronavirus hepatitis, coronavirus encephalitis, coronavirus gastritis, coronavirus pulmonary inflammation, coronavirus pulmonary fibrosis, coronavirus pulmonary edema, coronavirus telangiectasia, severe acute respiratory syndrome coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome (MERS).

12. A step of contacting a candidate substance with a biological sample containing cells expressing SPP1 (Secreted Phosphoprotein 1); and A method for screening a composition for treating a coronavirus infectious disease, comprising: a step of measuring the expression level of SPP1 in the sample.

13. In paragraph 12, A method comprising a step of determining that the candidate substance is a composition for treating a coronavirus infectious disease if the expression level of SPP1 in the sample decreases.

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