US Patent Application for METHODS AND COMPOSITIONS FOR THE TREATMENT OF INFLAMMATORY CONDITIONS ASSOCIATED WITH INFECTIOUS DISEASES Patent Application (Application No. 20230159932 issued May 25, 2023) (2023)

This application claims the benefits of US Provisional Application No. 63/198,706, filed Nov. 6, 2020, and US Provisional Application Ser. 63/013,865, filed Apr. 22, 2020, applications which are incorporated herein by reference in their entirety.

I. BACKGROUND

There are currently no effective biological treatments for coronaviruses, especially for COVID-19. Commercially available products do not act simultaneously and synergistically to directly affect viral infection and/or replication, nor to regulate downstream inflammation and vascular cell-related pathologies in response to viral infection. Therefore, what is needed is a method and composition for the intravenous or pulmonary delivery of autogenous or allogeneic MSC-derived growth factors and exosomes, wherein the products are acellular and useful for the treatment of various viruses, while simultaneously avoiding the potential negative long-term-permanent effects associated with the introduction of cellular living MSCs into the recipient.

II. ABSTRACT

Disclosed are methods and compositions relating to a composition comprising an MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) and/or one or more biomolecules for use in the treatment of disease.

In one aspect, disclosed herein are compositions comprising a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) and one or more biomolecules (such as, for example, a peptide, polypeptide, protein, siRNA, shRNA, and/or microRNA (miRNA)) that selectively bind to one or more microbial immunogens or inhibit the ability of the microbe to colonize the host, or inhibit, reduce , reduce, ameliorate and/or prevent one or more secondary conditions caused by microbial infection (such as, for example, composed of peptides, polypeptides, proteins, siRNAs, shRNAs, microRNAs (miRNAs) that act simultaneously and synergistically to directly affect viral infection and/or replication and/or act simultaneously and synergistically to regulate downstream inflammation and vascular cell-related pathologies in response to microbial infection). For example, disclosed herein are compositions comprising a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) and one or more the biomolecules contain the protein ferritin, PAI-1, thrombomodulin and/or the miRNA is selected from the group of miRNAs containing hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d -3p, hsa-let-7e-5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a -3p, hsa -miR-106a-5p, hsa-miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a -5p, hsa -miR-125b-5p, hsa-miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa -miR-138 -5p, hsa-miR-139-5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p , hsa-miR -148a-3p, hsa-miR-152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa -miR-16 -5p, hsa-miR-17-5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR -197-3p , hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a -5p, hsa -mir-203a, hsa-miR-203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir -221, hsa -miR-221-3p, hsa-mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24 -1, hsa-mir-24-2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p, hsa-miR-26a-5p, hsa-miR-27a -3p, hsa -mir-27b, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa -miR-30b -5p, hsa-miR-30c-5p, hsa-mir-30d, hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p , hsa-miR -31-5p, hsa-miR-320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR -376a-3p , hsa-miR-376c-3p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1 , hsa-mir -486-2, hsa-miR-486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hsa-mir -92a-1 , hsa-mir-92a-2, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a -5p and hsa-miR-99b-5p.

Also disclosed herein are compositions of any preceding aspect, wherein one or more biomolecules inhibit the bradykinin pathway (for example, by inhibiting the translation of bradykinin 2), the angiotensin-converting enzyme 2 (ACE 2) receptor, inhibit the transmembrane protease, serine 2 (TMPRSS2) enzyme , inhibits kallikrein B1 and/or inhibits IL-1β, IL-6, TNF-α, GM-CSF or M-CSF.

In one aspect, the compositions of any preceding aspect are disclosed herein, wherein the MSC secretome further comprises prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 ( SDF -1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TNF-α receptor, insulin - such as growth factors, fibroblast growth factors (FGF) 1-23 (especially FGF1 and FGF2), bone morphogenetic proteins (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) macrophage- stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM -CSF estrogen, and/or thyroid hormones.

Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection (such as, for example, a viral, bacterial, fungal, or parasitic infection) or its symptoms (including, but not limited to, microbially induced cytokine storm, microbially initiated bradykinin storm and/or acute respiratory distress syndrome) in a subject comprising administering to the subject a composition of any of the preceding aspects.

In one aspect, disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection or symptoms thereof of any preceding aspect, wherein the microbial infection includes infection by a virus selected from the group consisting of Herpes Simplex virus -1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus (including but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV- HKU1, HCoV -NL63, SARS-CoV, SARS-CoV-2 or MERS-CoV), influenza A virus, influenza B virus, measles virus, polyomavirus, human papilloma virus, respiratory syncytial virus, adenovirus, Coxsackie virus, Chikungunya virus, Dengue virus, mumps virus, poliovirus, rabies virus, Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type-1, Hantavirus, rubella virus, simian virus immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2.

Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection or symptoms thereof of any of the foregoing aspects, wherein the microbial infection includes infection by a bacterium selected from the group consisting ofMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovissoj BCG, supstrovi BCG,Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marine, Mycobacterium ulcerans, Mycobacterium aviumparatuberculosis subspecies,Mycobacterium chimaera, Nocardia asteroides,anotherNocardiaspecies,Legionella pneumophila,anotherLegionellaspecies,Acetinobacter baumanii, Salmonella typhi, Salmonella enterica,anotherSalmonellaspecies,Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri,anothershigellaspecies,Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida,anotherPasteurellaspecies,Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus,anotherBrucelaspecies,Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpiianotherBordetellaspecies,Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, ricketsspecies,Erlichiusspecies,Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacterspecies,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,anotherPseudomonasspecies,Haemophilus influenzae, Haemophilus ducreyi,anotherHemophilusspecies,Clostridium tetanianotherClostridiumspecies,Yersinia enterolitica,and othersJersinijatype, imycoplasmaspecies. In one respect bacteria are notBacillus anthracis.

In one aspect, also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection or symptoms thereof of any of the preceding aspects, wherein the microbial infection comprises infection with a fungus selected from the group consisting ofCandida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicillium marneffi,iAlternaria alternates.

Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating and/or preventing a microbial infection or symptoms thereof of any of the foregoing aspects, wherein the microbial infection includes a parasitic infection with a parasite selected from the group consisting of parasitic organisms consisting ofToxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae,anotherplasmodiumspecies,Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidiumspp.,Trypanosoma brucei, Trypanosoma cruzi, Leishmania major,anotherLeishmaniaspecies,Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoebaspecies,Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni,anotherSchistosomaspecies,Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa,iEntamoeba histolytica.

IV. DETAILED DESCRIPTION

Before these compounds, compositions, articles, devices and/or methods are disclosed and described, it should be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to specific reagents unless otherwise specified. , as such can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from "about" one particular value and/or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and/or to another particular value. Similarly, when values ​​are expressed as approximations, the use of the antecedent "about" will imply that the particular value constitutes another realization. It will further be understood that the endpoints of each range are significant both relative to the other endpoint and independently of the other endpoint. It is also understood that there are a range of values ​​disclosed herein and that each value is also disclosed herein as being "about" that particular value in addition to the value itself. For example, if the value "10" is published, then "about 10" is also published. It is also understood that when a value is detected that is "less than or equal to" a value, "greater than or equal to" and possible ranges between values ​​are also detected, as appropriately understood by one skilled in the art. For example, if the value "10" is published, "less than or equal to 10" as well as "greater than or equal to 10" are also published. It is also understood that throughout the application, data is provided in a variety of different formats and that these data represent end points, start points, and ranges for any combination of data points. For example, if a certain data point "10" and a certain data point 15 are detected, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are detected as well as between 10 and 15. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are published, then 11, 12, 13 and 14 are also published.

The term "subject" is defined herein to include animals such as mammals, including but not limited to primates (eg, humans), cows, horses, pigs, sheep, goats, dogs, cats, rabbits, rats, mice, and As. In some embodiments, the subject is a human.

"Administration" to a subject includes any route of introduction or delivery of an agent to the subject. Administration may be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarteriolar, intraarticular, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, inhalation, via an implanted container, parenterally (eg subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional and intracranial injection or infusion techniques) and the like. "Simultaneous administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein means that the compounds are administered at the same point in time or essentially immediately after each other. In the latter case, the two compounds are given at times close enough that the observed results do not differ from those obtained when the compounds are given at the same time point. "Systemic administration" refers to the introduction or delivery of an agent to a subject by a route by which the agent is introduced or delivered to large areas of the subject's body (eg, greater than 50% of the body), for example through entry into the circulation or lymphatic systems. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject by a route that introduces or delivers the agent to an area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, topically applied agents are readily detectable in the local vicinity of the site of administration, but are undetectable or detectable in negligible amounts in distal parts of the subject's body. Management includes self-management and management by others.

"Biocompatible" generally refers to a material and any of its metabolites or breakdown products that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

"Contains" means that the compositions, methods, etc. include the specified elements, but do not exclude others. "Consisting principally of" when used to define compositions and methods, means including the specified elements but excluding other elements of any essential importance to the combination. Accordingly, a composition consisting mainly of elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consists of" means the exclusion of more than trace elements of other ingredients and significant steps of the method for providing the compositions of this invention. Embodiments defined by each of these transitional terms are within the scope of this invention.

A "control" is an alternate subject or sample used in an experiment for comparison purposes. Control can be "positive" or "negative".

An "effective amount" of an agent refers to a sufficient amount of the agent to achieve the desired effect. The amount of agent that is "effective" will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Therefore, it is not always possible to determine a quantified "effective amount". However, an appropriate "effective amount" in any given case can be determined by a person of ordinary skill in the art using routine experimentation. Also, as used herein, and unless otherwise noted, an "effective amount" of an agent may also refer to an amount that encompasses both therapeutically effective amounts and prophylactically effective amounts. The "effective amount" of an agent required to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to achieve an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced according to the requirements of the therapeutic situation.

"Decrease" can refer to any change that results in less gene expression, protein production, amount of symptoms, disease, composition, condition, or activity. A substance is also considered to reduce the genetic effect of a gene when the genetic effect of the gene product with the substance is less than the effect of the gene product without the substance. Also, for example, a reduction may be a change in the symptoms of a disorder such that the symptoms are less than previously observed. The reduction can be any single, medium or average reduction of the condition, symptoms, activity, composition in a statistically significant amount. So the decrease can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95 or 100% reduction as long as the reduction is statistically significant.

"Inhibit", "inhibit" and "inhibition" means to reduce an activity, response, condition, disease or other biological parameter. This may include, but is not limited to, complete ablation of an activity, response, condition or disease. This may also include, for example, a 10% reduction in activity, response, condition or disease compared to natural or control levels. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between compared to natural or control levels.

"Treatment", "treatment", "treatment" and their grammatical variations as used herein include the administration of a composition with the intention or purpose of partially or completely preventing, delaying, treating, healing, alleviating, alleviating, altering, repairing, ameliorating, stabilizing, alleviating and/or reducing the intensity or frequency of one or more diseases or conditions, symptoms of a disease, disorder, injury or condition, or the underlying cause of a disease or condition. Treatments according to the invention can be applied preventively, prophylactically, palliatively or curatively. Prophylactic treatments are administered to a subject before the onset (eg, before obvious signs of cancer), during early onset (eg, after initial signs and symptoms of cancer), or after established cancer development. Prophylactic administration can occur day(s) to a year before the onset of symptoms of infection.

The terms "prevent", "prevent", "prevent" and their grammatical variations as used herein refer to a method of partially or completely delaying or preventing the onset or recurrence of a disease and/or one or more associated symptoms or prohibiting a subject from acquiring or get the disease again or reduce the subject's risk of acquiring or re-acquiring the disease or one or more associated symptoms.

"Pharmaceutically acceptable" component may refer to a component that is not biologically or otherwise undesirable, ie. component can be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant adverse biological effects or interacting in a deleterious manner with any other component of the formulation in which it is contained. When used in relation to administration to humans, the term generally implies that the component has met the required standards of toxicology and manufacturing testing or is included in the Guide to Inactive Ingredients prepared by the US Food and Drug Administration.

"Pharmaceutically acceptable carrier" (sometimes referred to as "carrier") means a carrier or excipient useful in the preparation of a pharmaceutical or therapeutic composition that is generally safe and nontoxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" may include, but are not limited to, phosphate buffered saline, water, emulsions (such as oil/water or water/oil emulsion), and/or various types of wetting agents. As used herein, the term "carrier" includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as further described here.

"Pharmacologically active" (or simply "active"), as in "pharmacologically active" derivative or analog, can refer to a derivative or analog (eg, salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound and approximately the same degree.

"Therapeutic agent" refers to any composition that has a beneficial biological effect. Useful biological effects include therapeutic effects, e.g. treatment of disorders or other undesirable physiological conditions, and prophylactic effects, e.g. prevention of disorders or other undesirable physiological conditions (eg non-immunogenic cancer). The terms also include pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms "therapeutic agent" are used, or when a specific agent is specifically identified, it is understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs etc.

A "therapeutically effective amount" or "therapeutically effective dose" of a composition (eg, a composition containing an agent) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is the control of type I diabetes. In some embodiments, the desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, sex, and weight of the subject. The term may also refer to an amount of therapeutic agent, or rate of delivery of a therapeutic agent (eg, amount over time), that is effective to achieve a desired therapeutic effect, such as alleviation of pain (ie, nociception). The precise desired therapeutic effect will vary depending on the condition being treated, the tolerance of the subject, the agent and/or formulation of the agent being administered (eg, the strength of the therapeutic agent, the concentration of the agent in the formulation, and the like), and a number of other factors that are appreciated by those skilled in the art. common in this field of technology. In some cases, the desired biological or medical response is achieved after administration of multiple doses of the composition to a subject over a period of days, weeks or years.

In this specification and in the requirements that follow, reference will be made to a number of terms which will be defined to have the following meanings:

"Optional" or "optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which said event or circumstance occurs and instances in which it does not.

Various publications are cited throughout this application. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the related art. The disclosed references are also individually and specifically incorporated by reference herein for the material contained therein that is discussed in the sentence in which the reference is made.

B. Compositions

The components that will be used to prepare the compositions described are disclosed as well as the compositions themselves that will be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. are disclosed. of these materials, while specific reference to each different individual and collective combination and permutation of these compounds need not be explicitly disclosed, each is separately discussed and described herein. For example, if a particular MSC secretome (including but not limited to MSC exosomes (with or without growth factors) referred to herein as extracellular vesicle isolate product (EVIP)) is detected and discussed and a number of possible modifications are considered. molecules including the MSC secretome, and specifically contemplates any combination and permutation of the MSC secretome and modifications that are possible unless otherwise noted. Therefore, if a class of molecules A, B and C, as well as a class of molecules D, E and F and an example combination of molecules, A-D is disclosed, then even if each is not individually stated each is individually and collectively considered combinations of meaning, A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are considered discovered. Likewise, any subset or combination of these is also disclosed. So, for example, a subset of A-E, B-F, and C-E would be considered discovered. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Accordingly, if there are a number of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

Currently, COVID-19 is sweeping the world. It has been widely reported that children are less likely to become seriously ill and die from COVID-19. A recent study of 44,672 people with confirmed COVID-19 infection found that children under the age of 10 accounted for less than 1 percent of those cases and none of the 1,023 deaths. A direct explanation would be that children resist the infection at all, but this does not seem to be the case. Children are just as likely to get infected as adults. So what is child protection? Experts suspect that this may be due to the unique way in which children's immune systems react to these viruses.

A common complication of COVID-19, SARS and MERS in adults is when the immune response against the coronavirus becomes excessive and causes life-threatening lung damage. The resulting leakage of fluid and immune cells into the lungs can result in the life-threatening problems of acute respiratory distress syndrome (ARDS). Even if those immune responses try to help by attacking the virus, they can end up blocking the supply of oxygen to the lungs. Because children's immune systems are still developing, one suggestion is that they are protected from this type of dangerous immune response, called a cytokine storm, when they get COVID-19 or similar illnesses.

Injury to airway epithelial cells is also important in the pathogenesis of COVID-19. Damaged lung epithelial cells are a source of inflammatory mediators such as IL-6, TNF-α, GM-CSF and CXCL-8, which can act in both an autocrine and paracrine manner. IL-6 induces airway cell remodeling by regulating and stimulating myofibroblast differentiation, which is the main cause of fibrosis development during airway remodeling. The paracrine activity of these mediators is mediated, at least in part, by inflammatory exosomes.

Acute inflammation is a key pathological feature of the COVID-19 process. For example, cytokines produced by natural killer cells, mast cells, macrophages, and monocytes at the site of inflammation play a key role in the development of the COVID-19 cytokine storm that results in ARDS. Proinflammatory biomarkers, such as cytokines, have been found in chronic and acute lung disease states, suggesting a direct or facilitating role in the pathology. During the inflammatory reaction, more cytokines are produced. Cytokines contribute to inflammatory processes by activating specific signal transduction mechanisms, as well as by activating other types of cells. Cytokines are found extracellularly (in the blood) and in interstitial compartments, where they can activate cells in an autocrine/paracrine manner. Increased cytokine levels are hypothesized to influence and contribute to respiratory symptoms of COVID-19 by increasing nociceptor sensitization. When tissue is attacked or destroyed by pulmonary leukocytes during an inflammatory episode, several mediators such as interleukin-1 (IL-1), IL-8, IL-6, and tumor necrosis factor alpha (TNF-α) migrate to the site. Also included in these mediators are nerve growth factor and prostaglandins. All this results in pneumonia.

Mesenchymal stem cells (MSCs) have attracted much attention due to their ability to regulate inflammatory processes. In many types of lung trauma, inflammatory conditions at the site of injury interfere with the natural processes of recovery by local progenitor and mature cells. MSCs help through paracrine mechanisms and modulate the regenerative environment through anti-inflammatory and immunomodulatory mechanisms. In response to inflammatory molecules such as interleukin-1 (IL-1), IL-2, II-6, IL-12, tumor necrosis factor-a (TNF-α) and interferon-gamma (INF-γ), MSCs they secrete a variety of growth factors and anti-inflammatory proteins with complex feedback mechanisms among many types of immune cells. Key immunomodulatory cytokines include prostaglandin 2, TGF-β1, HGF, SDF-1, indoleamine 2,3-dioxygenase, IL-4, IL-10, IL-1 receptor antagonist, and soluble tumor necrosis factor receptor-a. MSCs prevent the proliferation and dysfunction of many inflammatory immune cells, including T cells, natural killer cells, B cells, monocytes, macrophages, and dendritic cells.

The primary trophic property of MSCs is the secretion of growth factors and exosomes to stimulate cell proliferation and angiogenesis. Exosomes express mitogenic proteins such as transforming growth factor alpha (TGF-α), TGF-β, hepatocyte growth factor (HGF), epithelial growth factor (EGF), basic fibroblast growth factor (FGF-2), and insulin-like factor growth factor -1 (IGF-1). They increase the division of fibroblasts, epithelial and endothelial cells. Vascular endothelial growth factor (VEGF), IGF-1, EGF, and angiopoietin-1 are released to recruit cells of the endothelial lineage and initiate vascularization.

MSCs help through paracrine mechanisms and modulate the regenerative environment through anti-inflammatory and immunomodulatory mechanisms. In response to inflammatory molecules such as interleukin-1 (IL-1), IL-6, IL-2, IL-12, tumor necrosis factor-α (TNF-α), and interferon-gamma (INF-γ), MSCs they secrete a variety of growth factors and anti-inflammatory proteins with complex feedback mechanisms among many types of immune cells. Key immunomodulatory cytokines include prostaglandin 2, TGF-β1, HGF, SDF-1, nitric oxide, indoleamine 2, 3-dioxygenase, IL-4, IL-10, IL-1 receptor antagonist, and soluble tumor necrosis factor receptor-α. MSCs inhibit the proliferation and function of many inflammatory immune cells, including T-cells, natural killer cells, B-cells, monocytes, macrophages, and dendritic cells. Although MSCs of all species are capable of regulating T-cell activity, the mechanisms are not identical among mammalian species.

A characteristic of chronically inflamed environments is a constant imbalance in the types of helper T-cells and macrophages. MSC exosomes indirectly promote the transition of TH1 to TH2 cells by decreasing INF-γ and increasing IL-4 and IL-10. It has been proven that the restored TH1/TH2 balance improves tissue regeneration in cartilage, muscle and other soft tissue injuries, alleviates the symptoms of autoimmune diseases and has an anti-diabetic effect. Similarly, reduction of INF-γ and secretion of IL-4 promotes a shift in macrophages from the M1 (pro-inflammatory, anti-angiogenic and tissue growth inhibiting) type to M2 (anti-inflammatory, pro-remodeling and tissue healing), an effect required for skeletal, muscle and neural healing and regeneration.

Here, a complex composition of secreted biomolecules (proteins, lipids and ribonucleic acids) and/or extracellular vesicles containing biomolecules, originating from cells of the mesenchymal lineage, which directly interact with microbial proteins (such as, for example, the coronavirus protein) and/or or nucleic acid content (such as, for example, microbial ribonucleic acid content) to disrupt the capacity of the microbe to establish an infection in a subject, infect cells and/or replicate within cells, and simultaneously interact with infected cells and the immune cells responsible for resolving hyperactive responses (such as, for example, microbially induced cytokine storm, microbially initiated bradykinin storm, and/or acute respiratory distress syndrome). In one aspect, disclosed herein are compositions comprising a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) and one or more biomolecules (such as, for example, a peptide, polypeptide, protein, siRNA, shRNA, and/or microRNA (miRNA)) that selectively bind to one or more microbial immunogens or inhibit the ability of the microbe to colonize the host, or inhibit, reduce , reduce, ameliorate and/or prevent one or more secondary conditions caused by microbial infection (such as, for example, composed of peptides, polypeptides, proteins, siRNAs, shRNAs, microRNAs (miRNAs) that act simultaneously and synergistically to directly affect viral infection and/or replication and/or act simultaneously and synergistically to regulate downstream inflammation and vascular cell-related pathologies in response to microbial infection).

In some embodiments, the composition comprises bone marrow-derived mesenchymal lineage cells that adhere to culture plastic and can differentiate in culture into multiple mature cell fates including but not limited to adipocyte, osteoblast, and chondrocyte fates and/or secreted extracellular vesicles containing a nucleus a protein composition that includes any combination of core composition proteins selected from the group consisting of: Ferritin, NUP85, LAMP2, GPR115, Serpin F1, OPN, PAI-1, DAPP1, Cathepsin B, Semaphorin 6C, PDGF R alpha, Sortilin, Serpin B6 , Dkk-3, Thrombomodulin, PF4, MIF, Periostin, Furin, TIMP-1, Decorin, PCK1, CD99, CD63, CD9, CD81, Transferrin, DcR3, Lumican, TIMP-2, SLITRK5, FAP, Artemin, DPPII, cIAP -1, Pentraxin 3, Visfatin, Neprilysin, Albumin, Galectin-1, UNC5H3, IL-20 R beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midkine, Calreticulin, Osteoactivin, Legumain, TAZ, Cathepsin L, RBP4, Serpin A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin 3, SIRP alpha and Syndecan-4, and at least one protein selected from the group consisting of: Ferritin, IGFBP-4 IL -1 R6 GSTM1 , NUP85, LAMP2, MeprinA, IL-1 F10, bIG-H3, GPR115, TGFb1, Ephrin-A4, CD109, Serpin F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN, PAI-1, DAPP1, GDF-9, cathepsin B, IGFBP-2, semaphorin 6C, IGF-2, PDGF R alpha, sortilin, serpin B6, Dkk-3, CNTF, TSP-1, GM-CSF Ra, thrombomodulin, endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Furin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP-4, Lumican, TIMP-2, TFPI , SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF -2R, ADAMTSL -1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRL5, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb, Pentraxin 3, Angiotensinogen, Follistatin, CF VII , Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin, Stabilin-2, IL- 17 RD, Albumin , Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 ligand, IL-20 R beta , Semaphorin 6A, TSK , Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO , PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, C1qTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc, LILRB4, BMP-2, MIA, CD34, CD63 , CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matriptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R, Nidogen-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, thyroid peroxidase, aminopeptidase P2, IL-1 RI, ADAM9, OSM R beta, thrombospondin-2, SMPD1, B2M, MFRP , LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF-23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL , Neurturin, Kremen-2, EMMPRIN, Activin RIB, Neuroligin 2, Epiregulin, CASA, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53 , EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A, CHST2, Pappalysin-2, Syndecan-3, Jagged 1, AKR1C4, Olfactomedin-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin alpha 5, VAMP-2 , FLRG, Ret Midkine , CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA125, HAO-1 , PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, Cathepsin V, Testican 2, Glypican 5, CD6 , Siglec-2, Legumain, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 beta, TAFA1, Podocalyxin, Ra1A, CRELD2, GRAP2, SP-D, BID, GFR alpha-2, Notch-3, VEGF R3, DLL4, TGFb2 , LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckin, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4, OSCAR, VEGF, IGSF3, Biglycan, Neudesin, ILT4 , uPAR, Ax1, WIF-1, IL-7 R alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Cochlin, Presenilin 1, NPTXR, SLAM , COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b, Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A , HGF R, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF-15, Epimorphin, GRK5, PD -1, Serpin A4 , ADAM23, NOV, Galectin-2, Neurexin 3 beta, TLR3, Sirtuin 2, Numb, IL-28 R alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM- A, Calreticulin-2 , ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin-3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I -TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-1ra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM -1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II, Desmoglein-3, Nectin-4, SCF, Serpin A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFA5, Galectin-9, vWF-A2, TACE, Activin RIIB, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF-11, Nrf2, TROP-2 , NUDT5, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gas6, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR, IL-22, PILR-alpha, NRG1-b1 , FABP4, RGM-A, RELT, TrkC, C5a, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galectin-3, CXCL16, JAM-B, DR6, Nogo receptor, TLR4, VEGF R2, Tie-2 , IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP-2, Desmoglein 2, Aggrecan , Syntaxin 4, VAMP -1, Nectin-2, FGF-21, Flt-3, GFAP, TIM-1, Inhibin A, Cadherin-4, P1GF-2, Neurogranin, HE4, IL-23 R, Galectin-7, GALNT3, GITR L, CD14, R-spondin 2, CK19, cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP-S, SDF-1a, PDGF -AB, GPVI, CD40, SCF R, Thrombospondin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL-1, ROBO3, RANTES, Endocan , Granulysin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin 3, ARSB, LIF R alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL-1 R4, CXCL14, IL -31, SIRP alpha , Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23, CD36, TNFb, Shh-N, Ficolin-1 , Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, Insulin, Syntaxin 6, GRO, Bc1-w, Lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE -1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.1, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-gamma R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin, HVEM, ENPP-7, Syndekan-4, IL -2 Rg, MICA, Dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA-total, IL-15, MAP1D, C1q, TNF4, Dtk, Endoglin, ENA -78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, Galectin-8, CA4, Cystatin EM, FUT8, B7-H3, GCP-2, CD40L, MDC, 4 -1BB, HO- 1, SOST, S100A13, kallikrein 7 and IL-13.

In some embodiments, the composition contains one or more biomolecules that can selectively bind to a microbial antigen (such as a viral, bacterial, fungal, or parasitic antigen), block its function, and/or enzymatically process the protein so that it is detected by the host's immune system and then activates the virus's immune response to disable the virus's ability to infect cells. For example, the biomolecule can bind to a viral antigen from a virus selected from the group consisting of Herpes Simplex virus-1 (such as, for example, glycoprotein D and/or glycoprotein G), Herpes Simplex virus-2 (such as, on for example, glycoprotein D and/or glycoprotein G), Varicella-Zoster virus (such as, for example, glycoprotein E), Epstein-Barr virus (such as, for example, EBV glycoprotein), cytomegalovirus (such as, on e.g., CMV glycoprotein), human herpes virus-6, Variola virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus (including but not limited to hepatitis B virus surface antigen), hepatitis C virus (such as, on example, hepatitis C E1, E2 or E3 proteins), hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus (including but not limited to spike or coat proteins from avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15 ), porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, SARS-CoV, SARS-CoV-2 (including but not limited to variant B1.351, variant B .1.1.7 , and P.1 variant), or MERS-CoV), influenza A virus (such as, for example, hemagglutinin (HA) protein including HA1 and HA2 protein and including trimeric HA), influenza B virus (such as which is, for example, hemagglutinin (HA) protein including HA1 and HA2 proteins and including trimeric HA), measles virus (such as, for example, hemagglutinin protein), polyomavirus, human papillomavirus, respiratory syncytial virus (such as, for example, RSV G protein) , Adenovirus, Coxsackie virus, Dengue virus (such as, for example, capsid protein, envelope protein and/or premembrane/membrane protein), mumps virus, poliovirus, rabies virus (including but not limited to rabies glycoprotein), Rous sarcoma virus, reovirus, yellow fever virus, Zika virus (such as capsid protein, envelope protein and/or premembrane/membrane protein), Ebola virus, Marburg virus, Lassa virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louisa virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A (including but not limited to viral protein 4 and viral protein 7), Rotavirus B (including but not limited to viral protein 4 and viral protein protein 7), rotavirus C (including but not limited to viral protein 4 and viral protein 7), Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, Hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type-1 (such as, for example, glycoprotein (gp), envelope protein (Env) or gag protein) and human immunodeficiency virus type-2. For example, the presence of a furin protein of the invention may allow enzymatic processing of the secreted spike protein of viral particles into an upright immunologically detectable conformation, enhancing the immune response to degrade extracellular viral particles. In addition, for example, the preparation may contain ferritin protein content that effectively increases the secretion of IL-10 by immune regulatory cells to inhibit the action of hyperactive immune cells, collectively referred to as cytokine storm, or PAI-1 protein that can block the production of plasmin to inhibit " bradykinin storms". The biomolecule of the preparation may also contain thrombomodulin, which can suppress blood micro-coagulation, reducing pathogenic coagulation and reducing thrombotic emboli; and other protein components that inhibit NETosis (neutrophil-induced nucleic acid and protein networks designed to trap pathogenic invading species, for example, viruses and bacteria within the vasculature)

Extracellular vesicles (EVs) are small membrane-bound spheres containing proteins and RNA (a subset of which are exosomes). Exosomes are lipid bilayer vesicles of small (eg 20-150 nm) diameter that are secreted by cells to enable paracrine communication. Other EV populations originate directly from the plasma membrane or are formed during apoptosis (apoptotic bodies). Recently, miRNA sequences homologous to the SARS-CoV-2 RNA sequence were compared and a list of miRNAs that appeared to bind to the viral sequence in the 3′ untranslated region, which has a low mutation rate, was identified. Therefore, miRNA should be effective in its action against multiple virus variants. Accordingly, in some embodiments, the biomolecule composition includes micro RNA content that can bind to microbial RNA sequences and block translation from or activate degradation of the microbial RNA sequence. The composition can therefore reduce the rate of microbial replication (including but not limited to the rate of viral replication) and reduce host cell death. For example, disclosed herein are compositions comprising a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) and one or more biomolecules containing miRNAs were selected from the group of miRNAs consisting of hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e - 5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa - miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa - miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139 - 5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR - 152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-17 - 5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-197-3p, hsa-miR-199a-3p , hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-miR-203a, hsa-miR - 203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa - mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24 - 2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p, hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR - 27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c - 5p, hsa-mir-30d, hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR - 320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-376c-3p , hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR - 486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2 , hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p and hsa-miR-99b -5p.

In some embodiments, the composition comprises a miRNA (such as hsa-miR-19a-3p, hsa-miR-19b-3p) that can be effective in inhibiting the translation of bradykinin receptor 2, which is critical for the activation of bradykinin signaling responsible for the severe vascular response to coronavirus infection.

In some embodiments, the composition comprises a microRNA that can inhibit the translation of kallikrein B1 (such as hsa-miR-24-3p) and other kallikrein proteins that are involved in the proteolytic digestion of bradykinin precursor protein to produce the bradykinin peptide.

In some embodiments, the biomolecule of the preparation includes microRNA content that can inhibit the translation of cellular proteins involved in enabling virus fusion to the cell membrane using the angiotensin-converting enzyme 2 (ACE 2) receptor protein or by blocking the activity of proteins activated through the process of virus binding to the ACE 2 receptor protein. For example, the transmembrane protease enzyme serine 2 (TMPRSS2) can be inhibited. TMPRSS2 is required to allow the SARS-CoV-2 spike protein to interact with the ACE 2 receptor and initiate membrane fusion. Exemplary microRNA content may include human miRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-214-3p and hsa-miR-27a-3p, all of which have TMPRSS2 mRNA binding sites.

In some embodiments, the biomolecule composition comprises a microRNA that can inhibit bradykinin pathway proteins. For example, microRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-let-7i , hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa -miR-20a-5p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-25-3p, hsa-miR -92a-3p, hsa-miR-92b-3p and hsa-miR-93-5p can bind and prevent the translation of hyaluronic synthase 2, thereby preventing the formation of hyaluronic acid complexes in the lungs. Hyaluronic acid complexes block the exchange of oxygen in the alveoli.

In some embodiments, the biomolecule composition comprises a microRNA content, a protein content, or a combination thereof that can inhibit cytokine storm. The composition can inhibit the cytokine storm by (i) inhibiting the translation of cytokine proteins by binding mRNA sequences to those proteins, (ii) sterically preventing ligand/receptor interactions, (iii) enzymatically modifying the ligand or receptor to inhibit their proinflammatory effects, or (iv) activating inhibitory proteins, lipids or RNA sequences that inhibit pro-inflammatory cytokines such as, but not limited to, IL-1beta, IL-6, TNF-alpha, GM-CSF, M-CSF. For example, the human miRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p and hsa-miR - 547-3p can bind IL-6 mRNA, while hsa-miR-130a-3p and hsa-miR-181a-5p can inhibit TNF-alpha translation.

It is understood and considered here that the MSC secretome contains exosomes and growth factors. Growth factors and exosomes can be allogeneic or autogenous. Growth factors and exosomes can be derived from any cell in the human body, such as ectodermal cells, endodermal cells, or mesodermal cells. For example, MSC secretomes may contain mesenchymal stem cell (MSC)-derived growth factors, MSC-derived exosomes, or MSC-derived growth factors and exosomes. In some embodiments, the method further includes adding at least one additive with exosomes and growth factors. In particular, MSCs under appropriate wound healing conditions can produce appropriate therapeutic agents, such as exosomes and growth factors, which can provide therapy for inflammatory lung diseases. In one aspect, compositions disclosed herein, wherein the MSC secretome composition further comprises prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1 ), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TNF-α receptor, insulin-like growth factors fibroblast growth factors (FGF) 1-23 (especially FGF1 and FGF2), bone morphogenetic proteins (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) macrophage-stimulating protein (MSP) , platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor (GM-CSF) estrogen and /or thyroid hormones.

As noted above, the composition contains proteins and microRNAs, some of which may be embedded in or surrounded by a lipid membrane to form vesicles ranging in size from about >20 nm to about 200 nm. The number of vesicles within the invention can range from about 1 million to about 100 billion vesicles per mL when suspended or about 10 million to about 1 trillion when formulated as a lyophilized powder.

1. Pharmaceutical carriers/delivery of pharmaceutical products

As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, together with the nucleic acid or vector, without causing any unwanted biological effects or interacting in a deleterious manner with any other components of the pharmaceutical preparation in which it is found. The carrier would naturally be chosen to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one skilled in the art.

The compositions can be administered orally, parenterally (eg, intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalation. As used herein, "topical intranasal administration" means delivery of the compositions to the nose and nasal passages through one or both nasal cavities and may include delivery by a spray mechanism or a droplet mechanism, or via nucleic acid or vector aerosolization. Administration of the compositions by inhaler may be through the nose or mouth by delivery by a nebulizer or droplet mechanism such as, for example, a metered dose inhaler, dry powder inhaler, nebulizer, vaporizer, or the like. Delivery can also be directly to any area of ​​the respiratory system (eg lungs) via intubation. The exact amount of compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector being used, the route of administration, and the like. Therefore, it is not possible to determine the exact amount for each composition. However, a suitable amount can be determined by a person of ordinary skill in the art using only routine experimentation in light of the teachings herein.

Parenteral administration of the preparation, if used, is generally characterized by injection. Injections can be prepared in the usual forms, either as liquid solutions or suspensions, solid forms suitable for suspension solution in liquid before injection, or as emulsions. A recently revised approach to parenteral administration involves the use of slow or sustained release systems so that a constant dose is maintained. See, e.g., U.S. Pat. 3,610,795, which is incorporated herein by reference.

Materials can be in solution, suspension (for example, embedded in microparticles, liposomes or cells). They can be targeted to a specific cell type via antibodies, receptors or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al.,Biokonjugat Chem.,2:447-451, (1991); Bagshawe, K. D.,Br. J. Rak,60:275-281, (1989); Bagshawe, et al.,Br. J. Rak,58:700-703, (1988); Center, et al.,Biokonjugat Chem.,4:3-9, (1993); Battelli, et al.,Cancer Immunol. Imunodrugo.,35:421-425, (1992); Pietersz and McKenzie,Immunologist. reviews,129:57-80, (1992); and Roffler, et al.,Biochem. Pharmacol,42:2062-2065, (1991)). Carriers such as stealth and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated DNA targeting through cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo . The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al.,cancer research,49:6214-6220, (1989); i Litzinger i Huang,Biochemical and Biophysical Journal,1104:179-187, (1992)). In general, receptors are involved in endocytosis pathways, either constitutive or ligand-induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through the acidified endosome where the receptors are sorted, and are then either recycled to the cell surface, stored inside the cell, or degraded in lysosomes. Internalization pathways serve diverse functions, such as nutrient uptake, clearance of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation at the receptor level. Many receptors follow more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valence, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis were reviewed (Brown and Greene,DNA and cell biology10:6, 399-409 (1991)).

a) Pharmaceutically acceptable carriers

The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described inRemington: The Science and Practice of Pharmacy(19th edition) ed. A. R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of a pharmaceutically acceptable carrier include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release compositions such as semi-permeable matrices of solid hydrophobic polymers containing the antibody, which are matrices in the form of molded articles, e.g. films, liposomes or microparticles. Those skilled in the art will appreciate that certain carriers may be preferred depending on, for example, the route of administration and the concentration of the formulation being administered.

Pharmaceutical carriers are known to those skilled in the art. These would most commonly be standard vehicles for human drug administration, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The preparations can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

The pharmaceutical preparation can be applied in several ways depending on whether local or systemic treatment is desired, and on the area being treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, inhalation or parenteral, for example intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarily or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic injectable esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixed oils. Intravenous vehicles include fluids and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases and the like.

Topical formulations may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oil bases, thickeners and the like may be necessary or desirable.

Preparations for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents or binders may be preferred.

Some of the compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as are formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

b) Therapeutic use

Effective doses and schedules for administration of the compositions can be determined empirically, and such determination is within the skill of the art. The dosage ranges for administration of the preparations are those large enough to produce the desired effect in which the symptoms of the disorder are affected. The dose must not be so high as to cause harmful side effects, such as unwanted cross-reactions, anaphylactic reactions and the like. In general, the dosage will vary depending on the patient's age, condition, sex, and degree of disease, route of administration, or whether other drugs are included in the regimen, and can be determined by one skilled in the art. The dose can be individually adjusted by the doctor in case of contraindications. Dosage may vary and may be administered in one or more doses per day, over one or several days. Guidelines for appropriate dosages for certain classes of pharmaceutical products can be found in the literature. For example, guidelines for selecting appropriate doses for antibodies can be found in the literature on the therapeutic use of antibodies, e.g.Handbook of Monoclonal Antibodies,Ferrone et al., ur., Noges Publications, Park Ridge, N.J., (1985) pogl. 22 i str. 303-357; Smith i dr.,Antibodies in human diagnostics and therapy,Haber et al., eds., Raven Press, New York (1977) p. 365-389. A typical daily dose of the antibody used alone can range from about 1 μg/kg to 100 mg/kg body weight or more per day, depending on the above factors.

C. Mesenchymal stem cells

As noted throughout, the therapeutic compositions disclosed herein may utilize MSC secretomes and/or growth factors derived from mesenchymal stem cells (MSCs). While allogeneic MSC IV infusion treatments are in high demand, there are numerous safety and regulatory issues related to allogeneic cell preparations. Inherent problems with IV infusions of live MSCs include entrapment of the cells in the lung, causing the cells to die within 24 hours. The cellular debris from this cell death is transported to the liver and spleen, where it is disposed of. Current autogenous bone marrow concentrate treatments yield only a few thousand MSCs. While allogeneic expanded MSC IV infusions can deliver hundreds of millions of live MSCs, they remain trapped in the lungs and die. The long-term effect of introducing foreign DNA into the recipient is not clear, and questions have been raised as to whether the introduction of large amounts of foreign DNA can be carcinogenic.

Existing autogenous and allogeneic MSCs contained in bone marrow, bone marrow concentrate, mesenchymal stem cells (MSCs) derived from synovium or stromal vascular fractions (SVF) derived from adipose tissue or various postnatal products from umbilical cord, placenta or amnion, expanded MSC cultures are currently used for the treatment of wounds, orthopedic pathology and spine pathology. To avoid recognition by the immune system, undifferentiated MSCs express low to intermediate levels of human leukocyte antigen (HLA) class I and low levels of HLA class II. This property gives donor MSCs the so-called 'stealth' ability to be undetected by the host's immune system in allogeneic therapies. However, class I antigen is present at measurable levels, and class II antigen expression can be induced by INF-γ. Several cases of allogeneic MSC rejection and chronic immune responses have been reported in animal studies and human clinical trials. This entire problem could be avoided by using only mesenchymal stem cell secretomes, including but not limited to growth factors, proteins, peptides, glycosaminoglycans, proteoglycans, chemokines, cytokines, extracts, extracellular vesicles and/or exosomes collected from conditioned growth media. Acellular versus cellular MSC product

In fact, prior to this discovery, no active MSC growth factor product had been developed that could be used for these applications. Thus, in one aspect, disclosed herein are MSC secretome compositions (including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or preparations containing extracellular vesicles) for use in treating, inhibiting, reducing, reducing, enhancing , and/or prevention of microbial infection, symptoms of said infection, or secondary conditions (such as, for example, microbial-induced cytokine storm, microbial-induced bradykinin storm, and/or microbial-induced acute respiratory distress syndrome), said treatment compositions containing (i) a powdered growth factor additive comprising a composition derived from mesenchymal stem cells (MSC) (including but not limited to MSC secretome composition) and (ii) a pharmaceutically acceptable carrier.

As noted above, MSCs are multipotent cells that have the ability to differentiate into a multitude of cell types including myocytes, chondrocytes, adipocytes, and osteoblasts. Typically, these cells can be found in the placenta, cord blood, adipose tissue, bone marrow, or amniotic fluid, including perivascular tissue. As used herein, "MSC" refers to non-terminally differentiated cells including but not limited to multipotent stem cells, multipotent stromal cells, stromal vascular cells, pericytes, perivascular cells, stromal cells, pluripotent cells, multipotent cells, adipose-derived fibroblasts tissues. similar cells, adipose tissue-derived stromal vascular fraction, adipose tissue-derived MSC, bone marrow-derived fibroblast-like cells, bone marrow-derived stromal vascular fraction, bone marrow-derived MSC, tissue-derived fibroblast-like cells, adult stem cells, adult stromal cells, keratinocytes and/or melanocytes.

It has long been known that MSCs, in addition to their differentiation potential, also have immunomodulating abilities that result in the expression of many different cytokines and growth factors. As used herein, "MSC preparation" or "MSC secretome composition" refers to a composition containing MSC growth factors, MSC exosomes, extracellular vesicles, extracellular vesicle isolate product (EVIP), or acellular MSC extracts and/or MSC lysates derived from human MSCs, fibroblast-like cells and non-human animal MSCs including but not limited to MSCs from horses, cows, pigs, sheep, non-human primates, dogs, cats, rabbits, rats and mice. In embodiments, the MSCs may be derived from the patient to whom the composition will be administered (autologous) or derived from another individual (allogeneic). MSCs can be expanded in culture to collect conditioned media or to increase the amount of cells for lysate or can be used fresh prior to incorporation into the composition of the present disclosure.

MSC secretome preparations (including but not limited to MSC growth factor, MSC exosome, MSC extracts and/or compositions containing extracellular vesicles) may contain about 0.00001 to about 20 wt. %, such as from about 0.01 to about 10 wt. % of mesenchymal stem cell (MSC) extract, MSC exosomes or MSC growth factor preparations. The MSC preparation may contain either MSC conditioned medium or MSC lysate from expanded cell culture MSCs. In some embodiments, the composition may further comprise from about 0.01 to about 10 wt. % of cell-free medium conditioned by the growth of MSCs or cells of the MSC lineage, wherein the cells are cultured under normal hyperoxic culture conditions or under artificial wound healing conditions.

As disclosed herein, MSCs used to produce the disclosed MSC additives (including secretome composition of growth factors either frozen or powdered) can be selectively stimulated to produce MSC growth factors, secretomes, cytokines, chemokines, mesenchymal stem proteins cells, peptides, glycosaminoglycans, extracellular matrix (ECM), proteoglycans, secretomes and exosomes. Growth factors and exosomes can be derived from any cell in the human body, such as ectodermal cells, endodermal cells, or mesodermal cells. As used herein, MSC growth factors include, but are not limited to, prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TNF-α receptor, insulin-like growth factors, growth fibroblast factors (FGF) 1-23 (especially FGF1 and FGF2), bone morphogenetic proteins (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) macrophage-stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), such as and hormones including estrogen and thyroid hormones.

As mentioned above, culture of MSCs can occur under normal hyperoxic culture conditions or under hypoxic wound healing conditions. Hyperoxic conditions may contain approximately 21% oxygen with or without serum supplementation, while hypoxic conditions may contain about 1% to about 5% oxygen with inflammatory cytokines, angiogenic factors, reduced serum, reduced glucose, or these elements in various combinations. The combined reduced nutrient and metabolite environment can induce cultured cells to produce wound healing and anti-inflammatory ECM proteins and growth factors to direct tissue healing. Direct tissue healing is likely in the form of new ECM proteins, such as collagen and glycosaminoglycans (GAGs), as well as growth factors and cytokines. In one aspect, the MSC composition (such as, for example, an MSC secretome composition) comprises MSC growth factors, MSC exosomes, and/or MSC cell extracts or MSC lysates derived from MSC- and cultured under standard hyperoxic culture conditions (for example, 21% oxygen) or MSCs cultured under artificial wound healing conditions (such as, for example, 0.1% to about 5% oxygen in the presence of inflammatory cytokines, angiogenic factors, and reduced glucose ).

As disclosed herein, artificial wound healing conditions simulate growth conditions in real wounds where there is a reduction in nutrient supply and a reduction in waste removal typically caused by disruption of local blood circulation. This creates a harsh environment for the cells until new blood vessels are formed and blood circulation is restored. Accordingly, artificial wound healing conditions used to culture MSCs may include one or more of the following growth conditions, reduced glucose availability, reduced oxygen tension, reduced pH, and elevated temperature.

In one aspect, the availability of glucose may be reduced relative to a normal control. The modified culture medium to reduce glucose but without damaging the cells can be between 0 and 50% glucose reduction, more preferably between about 5% and 40% glucose reduction. For example, the culture conditions of MSC artificial wound healing culture may include reducing glucose by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50% such as a reduction in glucose from about 5% to about 15%, from about 10% to about 20%, from about 15% to about 25%, from about 20% to about 30%, or from about 25% to about 35%.

In one aspect, the oxygen tension can be reduced to oxygen levels under hypoxic conditions. Normal atmospheric oxygen is approximately 21% and any reduction is considered hypoxic. Thus, in one aspect, MSCs can be cultured at between 0.0% and 20.9% oxygen, from about 0.1% to about 0.5% oxygen, from about 0.1% to about 2.0% , from about 0.1% to about 5.0% oxygen, from about 0.5% to 5.0%, from about 1.0% to about 10% oxygen, from about 5.0% to about 10.0% oxygen; and from about 10.0% to about 15.0% under conditions of artificial wound healing. Preferably, during culture of MSCs, the oxygen tension is between about 0.5% and 20.5% oxygen, such as, for example, 0, 0.05, 0.1, 0.2, 0.3, 0 ,4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.7, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6 , 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.5 , 4.6 , 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 , 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5 , 9.6 , 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5 , 16, 16.5, 17, 17.5, 18, 18.5, 19 , 19.5, 20 or 20.5% oxygen.

The pH may also decrease under conditions of artificial wound healing. Physiological pH is maintained very tightly and is usually very close to neutral pH=7.2±0.2 (7.0-7.4). However, in the early acidic environment it can have pH=6.2±0.2 (ie pH from 6.0 to about 6.4). Accordingly, under culture conditions for artificial wound healing, the pH may be from about 6.0 to about 7.4, for example, from 6.0 to about 6.4, from about 6.2 to about 6.4 , from about 6.2 to about 6.6, from about 6.4 to about 6.6, from about 6.4 to about 6.8, or from about 6.6 to about 7.0, such as 6 ,0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 , 7.3 or 7.4.

Under culture conditions of artificial wound healing, the ambient temperature of the culture can be raised to simulate an increase in temperature at the wound site. Physiological homeostasis temperature is maintained at 37°C (98.6°F). A slight increase or decrease can cause significant changes in cellular metabolism. Increasing the temperature above 37° C. to any temperature up to about 40° C. (104° F.) can create a "feverish" environment. Therefore, in one aspect, artificial wound healing culture conditions for MSCs may comprise from about 35°C to about 39°C, from about 35°C to about 36°C, from about 36°C to about 37°C, from about 37°C to about 38°C, from about 38°C to about 39°C, from about 39°C to about 40°C. In one aspect, the culture temperature for artificial wound healing can be 35.0, 35.1, 35.2, 35.3, 36.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36.0, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.6. 7, 36, 8, 36.9, 37.0, .37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38.0, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38. 8, 38.9, 39.0 , 39.1, 39.2, 39.3, 39.4, 39.5 , 39.6, 39.7, 39.8, 39.9 or 40.0°C.

In one aspect, MSC secretome compositions (including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) may further comprise a protective coating (such as, for example, a cryoprotective oligosaccharide and a solution protein) to reduce the breakdown of growth factors. It is understood and considered herein that the protective coating can be constructed as a polymer. "Polymer" refers to an organic compound of relatively high molecular weight, natural or synthetic, whose structure can be represented by a repeating small unit, a monomer. Non-limiting examples of polymers include polyethylene, rubber, cellulose. Synthetic polymers are usually formed by addition or condensation polymerization of monomers. The term "copolymer" refers to a polymer formed from two or more different repeating units (monomer residues). By way of example and without limitation, the copolymer may be an alternating copolymer, a random copolymer, a block copolymer, or a graft copolymer. It is also contemplated that, in certain aspects, the various block segments of a block copolymer may themselves comprise copolymers. The term "polymer" encompasses all forms of polymers including, but not limited to, natural polymers, synthetic polymers, homopolymers, heteropolymers or copolymers, addition polymers, etc. In one aspect, the gel matrix may comprise copolymers, block copolymers, diblock copolymers, and/or triblock copolymers.

In one aspect, the protective coating may comprise a biocompatible polymer. In one aspect, the biocompatible polymer may be cross-linked. Such polymers can also be used to slowly release an adipose tissue browning agent and/or a fat modulating agent into the tissue. As used herein, biocompatible polymers include but are not limited to polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG) and poly(ethylene oxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide); poly(hydroxyalkyl methacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol), polyhydroxy acids such as poly(lactic acid), poly(glycolic acid) and poly(lactic acid-co-glycolic acid); polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyether esters; polyacetals; polycyanoacrylates; polyacrylates; polymethyl methacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid), as well as their copolymers. Biocompatible polymers may also include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols (PVA), methacrylate PVA (m-PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes. , polyurethanes and their copolymers, alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitro cellulose, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly ( isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly (ethylene) oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, polyvinyl chloride polystyrene and polyvinylpyrrolidone, their derivatives, linear and branched copolymers and their block copolymers, and their mixtures. Examples of biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene amines), poly(caprolactones), poly(hydroxybutyrates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphospholiazenes, their derivatives, linear and branched copolymers and their block copolymers, and their mixtures.

In some embodiments, the protective coating includes a carbohydrate structure of monosaccharides as well as carbohydrate polymers such as disaccharides or polysaccharides including but not limited to non-reducing polys or disaccharides as well as any combination thereof. Examples of carbohydrates that can be used in the protective coating include glucose, aldoses (D-allose, D-altrose, D-mannose, etc.), glucopyranose, pentahydroxyhexanal, α-D-glucopyranosyl-D-glucose, α-D-Glucopyranosyl- dihydrate, β-D-glucopyranosyl unit polymer, β-D-fructofuranosyl α-D-glucopyranoside (anhydrous/dihydrate), β-D-galactopyranosyl-D-glucose, α-D-glucopyranosyl-α-D-glucopyranoside (anhydrous/ dihydrate), galactose, pentoses (ribose, xylose, lyxose), dextrose, dodecacarbon monodecahydrate, fructose, sucrose, lactose, maltose, trehalose, agarose, D-galactosyl-β-(1-4)-anhydro-L-galactosyl, cellulose , polymer units of β-D-glycopyranosyl and starch, as well as polyhydric alcohols, polyalcohols, alditols, erythritol, glycitols, glycerol, xylitol and sorbitol.

In some embodiments, the protective coating comprises biocompatible and/or biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid) and poly(lactic-co-glycolic acid). The particles may further comprise one of the following polyesters: homopolymers including glycolic acid units, referred to herein as "PGA", and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D, L -lactic acid, poly-L-lactide, poly-D-lactide and poly-D,L-lactide5 collectively referred to herein as "PLA", and caprolactone units, such as poly(e-caprolactone), collectively referred to here as "PCL"; and copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by a lactic acid:glycolic acid ratio, collectively referred to herein as "PLGA" ; and polyacrylates, and their derivatives. Examples of polymers also include polyethylene glycol (PEG) copolymers and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers." In certain embodiments, the PEG region can be covalently linked to the polymer to give "PEGylated polymers" using a cleavable linker. In one embodiment, the polymer contains at least 60, 65, 70, 75, 80, 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent acetal pendant groups.

The triblock copolymers disclosed herein contain a core polymer such as, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol, polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), poly(vinyl pyrrolidone-co-vinyl acetate), polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oils, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic) acid, poly(lactic-co-glycolic) acid (PLGA), cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like.

Examples of diblock copolymers that can be used in the protective coatings disclosed herein include a polymer such as, for example, polyethylene glycol (PEG), polyvinyl acetate, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), poly (vinyl pyrrolidone-co-vinyl acetate), polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil, polycaprolactam, polylactic acid, polyglycolic acid, poly(lactic-glycolic) acid, poly(lactic-co-glycolic) acid (PLGA).

In one embodiment, the protective coating comprises (i.e. encapsulated, encapsulated compositions may further comprise lecithin or hydrolysed lecithin as a carrier or encapsulating material. As used herein, lecithin and/or hydrolysed lecithin coatings include coatings that contain phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidylserine and phosphatidic acid Lecithin sources can be pnat or animal sources.

In one embodiment, any of the polymers, monosaccharides, disaccharides, or polysaccharides used to form the protective shell formed by placing the MSC additive in the encapsulating solution can be at an appropriate concentration to form the protective shell. For example, the polymers, monosaccharides, disaccharides, or polysaccharides can be at any concentration between 0.01 mM and 10.0 M, for example, from about 0.01 M to about 0.1 M, from about 0.1 mM to about 1.0 M, from about 1.0 M to about 10.0 M. Example concentrations include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3 , 0.4, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 110, 120, 130, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, , 500, 600, 700, 800, 900 mM, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10M.

In one aspect, MSC secretome compositions (including but not limited to MSC growth factor, MSC exosome, MSC extracts and/or compositions containing extracellular vesicles) disclosed herein may comprise any known ingredients typically found in the pharmaceutical fields. such as means to fight against free radicals; bactericides; funds for allocation; preservatives; alkalizing or acidifying agents; smells; surfactants; fillers; natural products or extracts of natural products, such as aloe or green tea extract; vitamins; or coloring materials. Other ingredients that may be combined with the powder may include antioxidants, which may be selected from a variety of antioxidants. Suitable antioxidants include vitamins, such as vitamin C (L-ascorbate, ascorbate-2 phosphate magnesium salt, ascorbyl palmitate, tetrahexyldecyl ascorbate), vitamin E (tocotrienol), vitamin A (retinol, retinal, retinoic acid, provitamin A carotenoids, such as which are beta-carotene), N-acetyl glucosamine or other glucosamine derivatives. Other ingredients may include at least one essential fatty acid, such as Ω-3, Ω-6 and Ω-9 polyunsaturated fatty acids, such as linoleic acid (LA), gamma-linoleic acid (GLA), alpha- linoleic acid (ALA), dihomo-y-linolenic acid (DGLA), arachidonic acid (ARA) and others. Fatty acids can come from a variety of sources, including evening primrose oil, black currant oil, borage oil, or GLA-modified safflower seed. Other ingredients may include platelet-rich fibrin matrix, at least one ingredient to support ECM production and hyaluronic acid production, such as N-acetyl glucosamine or other glucosamine derivatives, ultra-low molecular weight (ULMW) hyaluronic acid, chondroitin sulfate, or keratin sulfate.

Production of MSC secretome compositions may include culturing MSCs collected from a donor to create a culture medium under culture conditions selected from the group consisting of normal hyperoxic culture conditions and hypoxic wound healing conditions including reduced oxygen and nutrition; stimulating cultured cells to selectively secrete desired anti-inflammatory proteins, peptides, glycosaminoglycans, proteoglycan exosomes and secretomes by adjusting cell growth conditions; collecting, combining the conglomerate mixture with an encapsulation solution, and freezing the conglomerate mixture, wherein the conglomerate mixture contains exosomes, peptides, proteins, cytokines, growth factors, extracellular matrix (ECM), proteoglycans, glycosaminoglycans; and chemokines selected from the group consisting of human MSC, animal MSC, multipotential stromal cells, fibroblasts and fibroblast cells; combining the conglomerate mixture with an encapsulating solution, such as oligosaccharides, such as a trehalose solution or a protein solution, and freezing the mixture; and lyophilizing or freeze-drying the frozen mixture to form a dry powder. Alternatively, MSCs can be lysed to collect all MSCs from the culture process, creating an extracted lysate; concentrating the extracted lysate and combining the extracted lysate with an encapsulating solution, such as oligosaccharides such as trehalose solution or protein solution, and freezing the mixture; and lyophilizing or freeze-drying the frozen mixture to form a dry powder. The powder contains a highly concentrated collection of analgesic MSC secretomes and exosomes and extracellular matrix components that are specific for anti-inflammatory activity.

The method may also include filter-sterilizing, concentrating, freezing, or freeze-drying the MSC-conditioned culture medium. In addition, the MSC culture medium can be combined with a cryoprotectant before freezing.

There are different methods for MSC lysis. Lysing can be achieved by addition of a hypotonic solution or by repeated freezing and thawing procedures to disrupt cell membranes. Moreover, cells can be lysed while attached to the culture surface or in suspension. Cells can also be enzymatically released and/or lysed by mechanical homogenization.

Inducing MSCs to selectively secrete the desired anti-inflammatory proteins, peptides, glycosaminoglycans, proteoglycans, exosomes and secretomes can be achieved by adjusting cell growth conditions, such as cell confluence, culture medium supplements, nutritional supplements, oxygen levels, length of culture under these conditions, the number of cell passages or their combinations and the like.

D. Methods of treatment of microbial infections and/or their symptoms

In one aspect, it is understood and contemplated herein that the disclosed comprises a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts and/or extracellular vesicles containing compositions) and one or more biomolecules (such as, for example, a peptide, polypeptide, protein, siRNA, shRNA, and/or microRNA (miRNA)) that selectively bind to one or more microbial immunogens or inhibit the ability of the microbe to colonize the host or inhibit, reduce , reduce, ameliorate, and/or prevent one or more secondary conditions caused by microbial infection (such as, for example, composed of peptides, polypeptides, proteins, siRNAs, shRNAs, microRNAs (miRNAs) ) can act simultaneously and synergistically to directly affect to viral infection and/or replication and/or act simultaneously and synergistically to regulate downstream inflammation and vascular cell-related pathologies in response to microbial infection). Accordingly, disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection (such as, for example, a viral, bacterial, fungal, or parasitic infection) or its symptoms (including, but not limited to microbially induced cytokine storms, microbially initiated bradykinin storms and/or acute respiratory distress syndrome) in a subject comprising administering to the subject any of the compositions described herein. For example, disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection (such as, for example, a viral, bacterial, fungal, or parasitic infection) or its symptoms (including, but not limited to, microbial induced cytokine storm, microbially initiated bradykinin storm, and/or acute respiratory distress syndrome) in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts and/or preparations containing extracellular vesicles) and one or more biomolecules (such as, for example, a peptide, polypeptide, protein, siRNA, shRNA and/or microRNA (miRNA)) that selectively bind to one or more microbial immunogens, or inhibit the ability of the microbe to colonize the host, or inhibit, reduce, reduce, ameliorate and/or prevent one or more secondary conditions caused by microbial infection (such as, for example, contain a peptide, polypeptide , protein, siRNA, shRNA, microRNA (miRNA).

As noted above, a composition for use in the disclosed methods can comprise bone marrow-derived mesenchymal lineage cells that adhere to culture plastic and can differentiate in culture into multiple mature cells including but not limited to adipocyte, osteoblast, and chondrocyte fates and /or secreted extracellular vesicles containing a core protein composition that includes any combination of core protein composition selected from the group consisting of: Ferritin, NUP85, LAMP2, GPR115, Serpin F1, OPN, PAI-1, DAPP1, Cathepsin B, Semaphorin 6C, PDGF R alpha, Sortilin, Serpin B6, Dkk-3, Thrombomodulin, PF4, MIF, Periostin, Furin, TIMP-1, Decorin, PCK1, CD99, CD63, CD9, CD81, Transferrin, DcR3, Lumican, TIMP-2, SLITRK5 , FAP, Artemin, DPPII, cIAP-1, Pentraxin 3, Visfatin, Neprilysin, Albumin, Galectin-1, UNC5H3, IL-20 R beta, SREC-II, JAM-C, TNF RI, htPAPP-A, eNOS, MSP R, TPP1, LAMP1, B2M, NCAM-1, HIF-1 alpha, ST6GAL1, CD99-L2, Plexin A4, EMMPRIN, p53, Semaphorin 7A, NKp80, Cystatin B, Osteoadherin, Midkine, Calreticulin, Osteoactivin, Legumain, TAZ, Cathepsin L, RBP4, Serpin A4, JAM-A, MCSF, LIMPII, OPG, IL-22, Galectin-3, MOG, Trypsin 3, SIRP alpha and Syndecan-4, and at least one protein selected from the group consisting of: Ferritin, IGFBP-4 IL-1 R6 GSTM1, NUP85, LAMP2, MeprinA, IL-1 F10, bIG-H3, GPR115, TGFb1, Ephrin-A4, CD109, Serpin F1, IGFBP-6, HS3ST4, aminopeptidase LRAP, OPN . PAI-1, DAPP1, GDF-9, Cathepsin B, IGFBP-2, Semaphorin 6C, IGF-2, PDGF R alpha, Sortilin, Serpin B6, Dkk-3, CNTF, TSP-1, GM-CSF Ra, Thrombomodulin, Endoglycan, IGFBP-3, RGM-C, PF4, MIF, TGM4, Periostin, Furin, TIMP-1, PAPP-A, Decorin, PCK1, Arylsulfatase A, CD99, CA2, PRDX4, Transferrin, DcR3, GP73, LAIR2, ULBP -4, Lumican, TIMP-2, TFPI, SOX2, SLITRK5, FAP, Spinesin, ENPP-2, CD97, CTACK, Integrin alpha 1, EXTL3, IL-18 BPa, PD-L2, PSMA, IL-20 Ra, Glyoxalase II, Trypsin 1, IGF-2R, ADAMTSL-1, Erythropoietin, Plexin D1, DNMT3A, BCL-2, CL-P1, Ephrin-B3, FABP6, CHI3L1, FCRL5, TFF3, Artemin, DPPII, cIAP-1, PDGF Rb , Pentraxin 3, Angiotensinogen, Follistatin, CF VII, Persephin, TRAIL R1, THAP11, CD200, CLEC-2, AMIGO, IGFBP-5, PON1, SOX7, GALNT10, Visfatin, Progranulin, PCSK2, GKN1, IL-18, Neprilysin , Stabilin-2, IL-17 RD, Albumin, Follistatin-like 1, MMP-10, FKBP51, LRRC4, Pref-1, Galectin-1, Troponin C, UNC5H3, FLRT2, CD314, Semaphorin 6B, Netrin-4, CD27 Ligand, IL-20 R beta, Semaphorin 6A, TSK, Cytokeratin-8, CHST3, Mc1-1, DPPIV, SREC-II, Norrin, JAM-C, Bc1-10, Wnt-4, LSECtin, Kell, TNF RI, PTP1B, htPAPP-A, IDO, PDGF-CC, Galanin, Activin A, TLR2, SCCA2, FABP1, eNOS, SHP-1, ICOS, C1qTNF9, MMP-1, TC-PTP, IL-24, gp130, C-myc , LILRB4 , BMP-2, MIA, CD34, CD63, CD9, CD81, IFNab R2, Glypican 2, MSP R, DSCAM, Matriptase, KIR2DL3, CD30, Siglec-10, CLEC-1, TPP1, Ubiquitin+1, ANGPTL4, TWEAK R , Nidogen-1, CD2, Kallikrein 1, TSLP R, LAMP1, TROY, VCAM-1, Siglec-11, S100A1, PAR1, Thyroid Peroxidase, Aminopeptidase P2, IL-1 RI, ADAMS, OSM R beta, Thrombospondin- 2, SMPD1, B2M, MFRP, LRP-6, ST3GAL1, NCAM-1 (CD56), Granzyme B, Adiponectin, IL-22BP, TPST2, PD-ECGF, LH, LEDGF, Cyr61, ULBP-3, IFNb, THSD1, FGF- 23, LAMA4, Adipsin, AIF, SorCS2, SULT2A1, CD39L2, Insulin R, HIF-1 alpha, OX40 Ligand, Pax3, UCH-L3, cMASP3, Langerin, Desmin, SOX9, ST6GAL1, MEP1B, CD99-L2, Plexin A4, Semaphorin 4D, ROBO2, PDX-1, APRIL, Neurturin, Kremen-2, EMMPRIN, Activin RIB, Neuroligin 2, Epiregulin, CA5A, MMP-12, GALNT2, CEACAM-5, VEGF R1, DSPG3, SorCS1, Matrilin-2, sFRP-3, p53, EphB3, NCK1, Semaphorin 7A, NKp80, Prolactin, Cystatin B, Sirtuin 1, FGF-16, FGF R5, NQO-1, Semaphorin 6D, FGF-3, GATA-4, VAP-A , CHST2 , Pappalysin-2, Syndecan-3, Jagged 1, AKR1C4, Olfactomedin-2, Osteoadherin, NKp44, Thyroglobulin, IL-21R, Chemerin, EphA1, CD48, MICB, FGF-5, TRANCE, CES2, ULBP-1, Integrin alpha 5, VAMP-2, FLRG, Ret Midkine, CD73, TRACP, proGRP, Granzyme H, PRX2, p27, Siglec-6, Dectin-1, CD51, Notch-1, Calreticulin, DR3, DCTN1, CDC25B, Osteoactivin, ACE, CA125, HAO-1, ​​PSMA1, FCRLB, BMP-9, CRIM1, LIF, SPINK1, EphB6, RGM-B, HS3ST1, ROR1, CMG-2, 4-1BB ligand, L1CAM-2, p63, cathepsin V, Testican 2, Glypican 5, CD6, Siglec-2, Legumain, PRELP, CES1, TAZ, NSE, TECK, HTRA2, HIF-1 beta, TAFA1, Podocalyxin, Ra1A, CRELD2, GRAP2, SP-D, BID, GFR alpha- 2, Notch-3, VEGF R3, DLL4, TGFb2, LIGHT, XIAP, ST8SIA1, Cathepsin L, 6Ckine, MIS RII, Kallikrein 5, TGM3, FCAR, Contactin-2, CD83, IL-1 R3, SALM4, GBA3, ROBO4 , OSCAR, VEGF, IGSF3, Biglycan, Neudesin, ILT4, uPAR, Axl, WIF-1, IL-7 R alpha, GPR56, CEACAM-3, MCEMP1, FABP2, Plexin B3, MEPE, Activin RIIA, ANG-2, Cochlin , Presenilin 1, NPTXR, SLAM, COMT, SPHK1, RBP4, Nectin-1, GUSB, Nidogen-2, IL-17F, SR-AI, TAFA2, N-Cadherin, IL-17B, IL-17 RC, MIP-3b , Cystatin C, Cystatin D, AMSH, FcERI, CLEC10A, HGF R, ANG-1, Prolactin R, FGF-20, CD28, Nogo-A, HSD17B1, IL-19, Enteropeptidase, Cathepsin E, TSLP, TCN2, GDF- 15, Epimorphin, GRKS, PD-1, Serpin A4, ADAM23, NOV, Galectin-2, Neurexin 3 beta, TLR3, Sirtuin 2, Numb, IL-28 R alpha, IL-33, Lin28, FCRL1, KLF4, NKp30, Lymphotactin, Cystatin SN, JAM-A, Calreticulin-2, ErbB4, BMP-8, IL-27 Ra, Fas, IL-4 Ra, Kallikrein 14, Matrilin-3, Olig2, Kallikrein 12, CA13, IL-9, Nectin -3, MPIF-1, Cystatin S, ADA, IL-2 Rb, GFR alpha-1, Smad4, ICAM-1, MEF2C, TREM-1, L-selectin, Hepsin, CD42b, MCSF, RANK, CHST4, CA8, FCRL3, ASAH2, CF XIV, PYY, HGF, I-TAC, Semaphorin 4C, SorCS3, Tie-1, IL-31 RA, Arginase 1, POGLUT1, IL-1ra, Podoplanin, TIM-3, CREG, CD300f, uPA, EphA2, LRRTM4, LIMPII, Tenascin R, CPE, PECAM-1, DNAM-1, DKK-1, OPG, CPB1, TSH, MMP-2, Siglec-9, ICAM-3, Cystatin SA, Galectin-4, Pepsinogen II , Desmoglein-3, Nectin-4, SCF, Serpin A5, PTH, FGF-19, MSP, IL-28A, FGF-12, METAP2, ASAHL, EDIL3, NTAL, EGF R, TAFA5, Galectin-9, vWF-A2 , TACE, Activin RIIB, Cathepsin S, LDL R, BMPR-IA, OX40, IL-13 R2, B7-H4, MMP-13, ANGPTL7, TRAIL R4, IGSF4B, Sirtuin 5, PEAR1, SH2D1A, Cerberus 1, GDF- 11, Nrf2, TROP-2, NUDT5, ROR2, EphB4, Glypican 1, LAP(TGFb1), Gas6, Contactin-1, IL-27, UNC5H4, ICAM-2, MBL, HS3ST3B1, RCOR1, IL-10 Rb, XEDAR , IL-22, PILR-alpha, NRG1-b1, FABP4, RGM-A, RELT, TrkC, C5a, SREC-I, Nestin, TPO, ErbB3, Kirrel3, FLRT1, Galectin-3, CXCL16, JAM-B, DR6 , Nogo receptor, TLR4, VEGF R2, Tie-2, IL-15 R, Caspr2, LTbR, LAMP, ALCAM, GLP-1, NG2, IL-22 R alpha 1, AMIGO2, HCC-1, TFPI-2, ULBP -2, desmoglein 2, aggrecan, syntaxin 4, VAMP-1, nectin-2, FGF-21, Flt-3, GFAP, TIM-1, inhibin A, cadherin-4, P1GF-2, neurogranin, HE4, IL- 23 R, Galectin-7, GALNT3, GITR L, CD14, R-spondin 2, CK19, Cardiotrophin-1, TREML1, HAPLN1, CD27, ANG-4, Siglec-7, CD155, VEGF-C, TNF RII, PGRP- S, SDF-1a, PDGF-AB, GPVI, CD40, SCF R, Thrombospondin-5, IL-1 RII, Neuropilin-2, Cadherin-13, E-Selectin, GITR, WISP-1, Renin, AgRP, MDL- 1, ROBO3, RANTES, Endocan, Granulysin, hCGb, Mesothelin, TLR1, TRAIL, MOG, DDR1, NGF R, TRAIL R3, Trypsin 3, ARSB, LIF R alpha, BAFF R, CD157, Granzyme A, 2B4, ESAM, IL -1 R4, CXCL14, IL-31, SIRP alpha, Uromodulin, CTRC, CEACAM-1, TARC, MIP-3a, SDF-1b, NKp46, MCP-3, IL-32 alpha, TGFb3 FOLR2, CD58, IL-23 , CD36, TNFb, Shh-N, Ficolin-1, Reg4, ILT2, Mer, TREM-2, Flt-3L, CDS, IL-6, CD229, insulin, syntaxin 6, GRO, Bc1-w, lipocalin-2, PDGF-AA, IL-2 Ra, Angiogenin, LYVE-1, CD4, RAGE, CDNF, Brevican, NAP-2, PU.1, EDAR, ADAMTS13, Kynureninase, PTH1R, IFN-gamma R1, CrkL, B7-1, PARC, Draxin, VE-Cadherin, Procalcitonin, SOX15, Kallikrein 11, BCMA, Dectin-2, EpCAM, HCC-4, TGFa, IP-10, BLAME, CILP-1, PIGF, LOX-1, MCP-2, Resistin , HVEM, ENPP-7, Syndecan-4, IL-2 Rg, MICA, Dopa decarboxylase, NPDC-1, MCP-4, EG-VEGF, Glycoprotein V, Semaphorin 4G, IL-12p40, PSA-total, IL-15 , MAP1D, C1q, TNF4, Dtk, Endoglin, ENA-78, Reg3A, MIP-1b, FGF-17, IL-6R, IL-8, Galectin-8, CA4, Cystatin EM, FUT8, B7-H3, GCP- 2, CD40L, MDC, 4-1BB, HO-1, SOST, S100A13, kallikrein 7 and IL-13.

In some embodiments, a composition for use in the disclosed methods comprises one or more biomolecules that can selectively bind to a microbial antigen (such as a viral, bacterial, fungal, or parasitic antigen), block its function, and/or enzymatically process the protein so that it is immunologically the host system can detect to then activate the immune response of the virus to disable the virus's ability to infect cells. For example, the biomolecule can bind to a viral antigen from a virus selected from the group consisting of Herpes Simplex virus-1 (such as, for example, glycoprotein D and/or glycoprotein G), Herpes Simplex virus-2 (such as, on for example, glycoprotein D and/or glycoprotein G), Varicella-Zoster virus (such as, for example, glycoprotein E), Epstein-Barr virus (such as, for example, EBV glycoprotein), cytomegalovirus (such as, on e.g., CMV glycoprotein), human herpes virus-6, Variola virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus (including but not limited to hepatitis B virus surface antigen), hepatitis C virus (such as, on example, hepatitis C E1, E2 or E3 proteins), hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus (including but not limited to spike or coat proteins from avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15 ), porcine epidemic diarrhea virus (PM V), HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, SARS-CoV, SARS-CoV-2 (including but not limited to variant B1.351, B .1.1.7 variant and P.1 variant), or MERS-CoV), influenza A virus (such as, for example, hemagglutinin (HA) protein, including HA1 and HA2 protein and including trimeric HA), influenza B virus ( such as, for example for example hemagglutinin (HA) protein, including HA1 and HA2 proteins and including trimeric HA), measles virus (such as, for example, hemagglutinin protein), polyomavirus, human papillomavirus, respiratory syncytial virus (such as, for example , RSV G protein ), Adenovirus, Coxsackie virus, Dengue virus (such as, for example, capsid protein, envelope protein and/or premembrane/membrane protein), mumps virus, poliovirus, rabies virus (including but not limited to rabies glycoprotein), Rous sarcoma virus, reovirus, yellow fever virus, Zika virus (such as capsid protein, envelope protein and/or premembrane/membrane protein), Ebola virus, Marburg virus, Lassa virus, Eastern equine encephalitis, Japanese encephalitis virus, St. Louis encephalitis, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A (including but not limited to viral protein 4 and viral protein 7), Rotavirus B (including but not limited to viral protein 4 and viral protein 7), rotavirus C (including but not limited to viral protein 4 and viral protein 7), Sindbis virus, simian immunodeficiency virus,

Human T-cell leukemia virus type 1, Hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1 (such as glycoprotein (gp), envelope protein (Env), or gag protein) and human immunodeficiency virus type 1 2. For example, the presence of the furin protein of the invention may allow enzymatic processing of the secreted viral particle spike protein into an upright immunologically detectable conformation, enhancing the immune response to degrade extracellular viral particles. Additionally, for example, the preparation may contain ferritin protein content that effectively increases the secretion of IL-10 by immune regulatory cells to inhibit the action of hyperactive immune cells, collectively referred to as cytokine storm, or PAI-1 protein that can block the production of plasmin to inhibit " bradykinin storms". The biomolecule of the preparation may also contain thrombomodulin, which can suppress blood micro-coagulation, reducing pathogenic coagulation and reducing thrombotic emboli; and other protein components that inhibit NETosis (neutrophil-induced nucleic acid and protein networks designed to capture pathogenic invading species, for example, viruses and bacteria within the vasculature)

In some embodiments, the biomolecule composition for use in the disclosed methods includes micro RNA content that can bind to microbial RNA sequences and block translation from or activate degradation of the microbial RNA sequence. The composition can therefore reduce the rate of microbial replication (including but not limited to the rate of viral replication) and reduce host cell death. For example, disclosed herein are compositions comprising a therapeutically effective amount of MSC secretome (such as, for example, including but not limited to MSC growth factor, MSC exosome, MSC extracts, and/or compositions containing extracellular vesicles) and one or more miRNA-containing biomolecules are selected from the group of miRNAs consisting of hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-3p, hsa-let-7e - 5p, hsa-let-7g-5p, hsa-let-7i, hsa-let-7i-5p, hsa-miR-100-5p, hsa-miR-103a-3p, hsa-miR-106a-5p, hsa - miR-106b-5p, hsa-mir-10b, hsa-miR-10b-5p, hsa-mir-1246, hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-125b-5p, hsa - miR-130a-3p, hsa-mir-130b, hsa-miR-130b-3p, hsa-miR-132-3p, hsa-miR-136-5p, hsa-miR-138-5p, hsa-miR-139 - 5p, hsa-mir-140, hsa-miR-140-3p, hsa-miR-145-5p, hsa-mir-146a, hsa-miR-146a-5p, hsa-miR-148a-3p, hsa-miR - 152-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-mir-16-1, hsa-mir-16-2, hsa-miR-16-5p, hsa-miR-17 - 5p, hsa-miR-181a-5p, hsa-miR-191-5p, hsa-miR-193a-5p, hsa-miR-193b-3p, hsa-miR-197-3p, hsa-miR-199a-3p , hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-miR-203a, hsa-miR - 203a-3p, hsa-miR-214-3p, hsa-mir-21, hsa-miR-21-3p, hsa-miR-21-5p, hsa-mir-221, hsa-miR-221-3p, hsa - mir-222, hsa-miR-222-3p, hsa-miR-22-3p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24 - 2, hsa-miR-24-3p, hsa-mir-25, hsa-miR-25-3p, hsa-miR-26a-5p, hsa-miR-27a-3p, hsa-mir-27b, hsa-miR - 27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-30a-5p, hsa-miR-30a-5p, hsa-miR-30b-5p, hsa-miR-30c - 5p, hsa-mir-30d, hsa-miR-30d-5p, hsa-mir-30e, hsa-miR-30e-5p, hsa-miR-31-3p, hsa-miR-31-5p, hsa-miR - 320a, hsa-miR-342-3p, hsa-miR-345-5p, hsa-miR-34a-5p, hsa-miR-361-5p, hsa-miR-376a-3p, hsa-miR-376c-3p , hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424-5p, hsa-miR-484, hsa-mir-486-1, hsa-mir-486-2, hsa-miR - 486-5p, hsa-miR-570-3p, hsa-miR-574-3p, hsa-miR-663a, hsa-miR-874-3p, hsa-mir-92a-1, hsa-mir-92a-2 , hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-mir-93, hsa-miR-93-5p, hsa-miR-940, hsa-miR-99a-5p and hsa-miR-99b -5 p. For example, a composition containing a miRNA (such as hsa-miR-19a-3p, hsa-miR-19b-3p) that may be effective in inhibiting the translation of bradykinin receptor 2, which is critical for the activation of bradykinin signaling responsible for severe vascular responses to coronavirus infection. Similarly, wherein the composition comprises a microRNA capable of inhibiting the translation of kallikrein B1 (such as hsa-miR-24-3p) and other kallikrein proteins involved in the proteolytic digestion of bradykinin precursor proteins to produce bradykinin peptide. Also, for example, where the biomolecule of the preparation contains microRNA content that can inhibit the translation of cellular proteins that are involved in enabling the fusion of the virus to the cell membrane using the angiotensin-converting enzyme 2 (ACE 2) receptor protein or by blocking the activity of activated proteins through the process of binding the virus to the protein ACE 2 receptors. For example, the transmembrane protease, enzyme serine 2 (TMPRSS2) can be inhibited. TMPRSS2 is required to allow the SARS-CoV-2 spike protein to interact with the ACE 2 receptor and initiate membrane fusion. Exemplary microRNA content may include human miRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-214-3p and hsa-miR-27a-3p, all of which have TMPRSS2 mRNA binding sites. Additionally, wherein the biomolecule of the preparation contains a microRNA that can inhibit proteins of the bradykinin pathway. For example, microRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-let-7i , hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa -miR-20a-5p, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-mir-24-1, hsa-mir-24-2, hsa-miR-25-3p, hsa-miR -92a-3p, hsa-miR-92b-3p and hsa-miR-93-5p can bind and prevent the translation of hyaluronic synthase 2, thereby preventing the formation of hyaluronic acid complexes in the lungs. Hyaluronic acid complexes block the exchange of oxygen in the alveoli. In some embodiments of the methods disclosed herein, the biomolecule composition comprises a microRNA content, a protein content, or a combination thereof that can inhibit cytokine storm. The composition can inhibit the cytokine storm by (i) inhibiting the translation of cytokine proteins by binding mRNA sequences to those proteins, (ii) sterically preventing ligand/receptor interactions, (iii) enzymatically modifying the ligand or receptor to inhibit their proinflammatory effects, or (iv) activating inhibitory proteins, lipids or RNA sequences that inhibit pro-inflammatory cytokines such as, but not limited to, IL-1beta, IL-6, TNF-alpha, GM-CSF, M-CSF. For example, the human miRNA sequences hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7g-5p, hsa-let-7i-5p and hsa-miR - 547-3p can bind IL-6 mRNA, while hsa-miR-130a-3p and hsa-miR-181a-5p can inhibit TNF-alpha translation.

It is understood and herein contemplated that the MSC secretome used in the compositions of the disclosed methods may contain exosomes and growth factors. Growth factors and exosomes can be allogeneic or autogenous. Growth factors and exosomes can be derived from any cell in the human body, such as ectodermal cells, endodermal cells, or mesodermal cells. For example, MSC secretomes may contain mesenchymal stem cell (MSC)-derived growth factors, MSC-derived exosomes, or MSC-derived growth factors and exosomes. In some embodiments, the method further includes adding at least one additive with exosomes and growth factors. In particular, MSCs under appropriate wound healing conditions can produce appropriate therapeutic agents, such as exosomes and growth factors, which can provide therapy for inflammatory lung diseases. In one aspect, the compositions of any preceding aspect are disclosed herein, wherein the MSC secretome composition further comprises prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), nitric oxide, indoleamine 2,3-dioxygenase, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), IL-1 receptor antagonist and soluble TNF-α receptor, insulin -as growth factors, fibroblast growth factors (FGF) 1-23 (especially FGF1 and FGF2), bone morphogenetic proteins (BMP) 1-15, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) macrophage -stimulating protein (MSP), platelet-derived growth factor (PLGF), vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF), insulin, granulocyte-colony-stimulating factor (G-CSF), macrophage-granulocyte-colony-stimulating factor ( GM-CSF estrogen and/or thyroid hormones.

As mentioned above, a microbial infection can include a viral, bacterial, fungal or parasitic infection. In one aspect, disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection or symptoms thereof of any preceding aspect, wherein the microbial infection includes infection by a virus selected from the group consisting of Herpes Simplex virus -1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus (including but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV- HKU1, HCoV -NL63, SARS-CoV, SARS-CoV-2 or MERS-CoV), influenza A virus, influenza B virus, measles virus, polyomavirus, human papilloma virus, respiratory syncytial virus, adenovirus, Coxsackie virus, Chikungunya virus, Dengue virus, mumps virus, poliovirus, rabies virus, Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type-1, Hantavirus, rubella virus, simian virus immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2.

Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection or symptoms thereof of any of the foregoing aspects, wherein the microbial infection includes infection by a bacterium selected from the group consisting ofMycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovissoj BCG, supstrovi BCG,Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marine, Mycobacterium ulcerans, Mycobacterium aviumparatuberculosis subspecies,Mycobacterium chimaera, Nocardia asteroides,anotherNocardiaspecies,Legionella pneumophila,anotherLegionellaspecies,Acetinobacter baumanii, Salmonella typhi, Salmonella enterica,anotherSalmonellaspecies,Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri,anothershigellaspecies,Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida,anotherPasteurellaspecies,Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus,anotherBrucelaspecies,Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpiianotherBordetellaspecies,Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, ricketsspecies,Erlichiusspecies,Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacterspecies,Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa,anotherPseudomonasspecies,Haemophilus influenzae, Haemophilus ducreyi,anotherHemophilusspecies,Clostridium tetanianotherClostridiumspecies,Yersinia enterolitica,and othersJersinijatype, imycoplasmaspecies. In one respect bacteria are notBacillus anthracis.

In one aspect, also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing a microbial infection or symptoms thereof of any of the preceding aspects, wherein the microbial infection comprises infection with a fungus selected from the group consisting ofCandida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicillium marneffi,iAlternaria alternates.

Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating and/or preventing a microbial infection or symptoms thereof of any of the foregoing aspects, wherein the microbial infection includes a parasitic infection with a parasite selected from the group consisting of parasitic organisms consisting ofToxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae,anotherplasmodiumspecies,Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidiumspp.,Trypanosoma brucei, Trypanosoma cruzi, Leishmania major,anotherLeishmaniaspecies,Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoebaspecies,Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni,anotherSchistosomaspecies,Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa,iEntamoeba histolytica.

E. EXAMPLES

The following examples are presented to provide those of ordinary skill in the art with a full disclosure and description of how the compounds, compositions, articles, devices, and/or methods claimed herein were made and evaluated, and are intended to be exemplary only and do not intent to limit disclosure. Efforts have been made to ensure accuracy in terms of numbers (eg quantities, temperature, etc.), but some errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, temperature is in °C or at ambient temperature, and pressure is at or near atmospheric.

Experimental treatments showed that this publication's treatment method greatly improved health in critical cases of COVID-19, resulting in all tested patients avoiding ventilator use or being removed from ventilators within 72 hours.

SUCH1A-1Cshow representative transmission electron microscopy (TEM) images showing the size distribution of EVs present within a sample of the invention from the batch of cyclic guanosine monophosphate (cGMP) manufactured #PV-441-2002C.SUCH1A,1Bend1Chave scales of 200 nm, 100 nm, and 100 nm, respectively. Figures 1, 2 and 3 are detailed views of the structures shown inPICTURE1C. The Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) is an imaging system used to capture imaging data on nanoparticles and convert the information into particle size, concentration and identity. One such commercial system is the NANOVIEW ExoView™ system.

SUCH2A-2Dshows CD81, CD63, and CD9 that label captured exosomes.PICTURE2Ais a chart illustrating the results of lyophilized test lot 4411901C.PICTURE2Bis a diagram illustrating the results of a series of tests of preexcipient 441901D.PICTURE2Cis a diagram illustrating the results of a series of tests of preexcipient 441902A.PICTURE2Dis a diagram illustrating the results of a series of tests of preexcipient 441902B. All samples were stained with fluorescently labeled monoclonal antibodies. CD63 antibody (Ab) is indicated in red, CD81 Ab is indicated in green, and CD9 Ab is indicated in blue.PICTURE2includes results for the following proteins. CD9 refers to a protein expressed in exosomes and EV plasma membranes, a member of the tetraspanin protein family, associated with integrins. CD9 regulates sperm-egg interactions, platelet aggregation and activation, and cell adhesion. In myocytes, CD9 associates with CD81 and inhibits myotube formation during muscle regeneration. In monocytes/macrophages, CD9 associates with CD81 and integrins and prevents the formation of giant cells and osteoclasts. CD63 refers to a protein expressed in exosomes, a member of the tetraspanin protein family. CD63 functions as a surface receptor for TIMP-1, activates cell signaling cascades, promotes cell survival, and activates the AKT and FAK/PTK2 pathways. CD81 refers to a protein expressed in exosomes, a member of the tetraspanin protein family. In myocytes, CD81 associates with CD9 and inhibits myotube formation during muscle regeneration. In monocytes/macrophages, CD81 associates with CD9 and integrins and prevents the formation of giant cells and osteoclasts. CD81 is expressed in B-cells and is involved in the trafficking of the CD19 receptor.

SUCH3A-3Dare a set of photomicrographs of CD63 captured exosomes.PICTURE3Aillustrates lyophilized test lot 4411901C.PICTURE3Billustrated by the precursor test lot 441901D.PICTURE3Cillustrated by the precursor test lot 441902A.PICTURE3Dillustrated by the precursor test lot 441902B. The image resolution limit for all images is 20 nm. All samples were stained with CD63 (red), CD81 Ab (green) and CD9 Ab (blue) antibodies. Nanoparticle Tracking Analysis (NTA) is a method that enables the visualization and analysis of particles in suspension.

PICTURE4reproduces the list of microRNA content of the MSC secretome (also referred to under the trade name EXOFLO™) identified as Table 1 in Park et al. Table 1 is highlighted to identify sequences found in EXOFLO™ that can bind directly to the SARS-CoV-2 RNA virus sequence. The footnotes of Table 1 include the following notes. 1. 3' UTR binding predictions were performed with the PITA tool. 2. To predict the binding of miRNAs to the 3′ UTR and region, whole genome analysis using miRDB was also performed. 3. Expression (%) indicates the level of each miRNA relative to total miRNA levels, calculated miRNA CPM/total CPM *100. 4. The location where each miRNA is expected to bind the SARS-CoV-2 genome. 5. Seed match length. 6. Seed mismatch length. 7. Number of oscillations caused by pairing of bases G and U. 8. Thermodynamic energy required for binding. A lower energy indicates a stronger binding prediction. The higher the score, the more likely there is a strong connection. 9. Prediction result from the miRDB method. According to miRDB, a predicted target with a score >80 is most likely to be tightly bound. 10. The initial location of the miRNA seed location corresponding to the RNA genome of SARS-CoV-2. 11. Rank of expression level within extracellular vesicles (EV).

PICTURE5is a schematic representation of the multiple mechanisms of action contemplated herein by which the invention may alleviate coronavirus infection and subsequent symptoms.

PICTURE6is a graphical representation of the inhibition of viral toxicity (infectivity and replication) as a function of extracellular vesicle concentration, showing the dose-response relationship of the inventive composition on in vitro live SARS CoV-2 virus in a "cyto-protective" assay.PICTURE6includes dashed lines to indicate linear regression and exponential regression of the data.

PICTURE7is a compilation of graphs showing the effect of the invention when administered to patients with severe ARDS associated with COVID-19. Biomarkers in a clinical study are indicators of the mechanism of action to reduce the hyperinflammatory state (cytokine storm).

PICTURE8is a graph showing one mechanism of action is inhibition of a major inflammatory cytokine, IL-1β Severity of COVID-19 is associated with peripheral blood levels of IL-1β. Herein, the capacity of the disclosed MSC secretome compositions (also referred to under the trade name EXOFLO™) to consistently regulate inflammation via inhibition of the pro-inflammatory cytokine IL-1β in human peripheral blood mononuclear cells stimulated with LPS (lipopolysaccharides or endotoxin), an in vitro model that is used to simulate the onset of a hyperimmune response, also known as a "cytokine storm".

PICTURE9illustrates that one mechanism of action is to regulate the hyperactive neutrophil immune response by reducing neutrophil extracellular trap formation (Netosis). The ability of MSC secretomes (also referred to under the trade name EXOFLO™) to inhibit virally or bacterially induced NETosis by inhibiting neutrophil NET formation and reducing the incidence of NET-associated microthrombi formation, for example, associated with SARS-CoV -2 formation, or high levels of endotoxin associated with bacterial infection. Therefore, the reduction in NET formation is associated with a reduction in disseminated intravascular coagulation (microclot formation) associated with COVID-19.