Methods for identifying small RNA biomarkers and evaluating the function of circulating extracellular vesicles, including exosomes

Abstract

Methods for identifying small RNA biomarkers and for assessing the function of circulating extracellular vesicles, including exosomes, are provided. [Solution] A purification method for capture of extracellular vesicles by an Antibody of CHoice and Enzymatic Release (EV-CATCHER), designed for high-throughput analysis of low-abundance small RNA cargo by next-generation sequencing, and the use of the method in preparing purified populations of biological particles or cells from biological samples for in vitro assessment of the risk of developing severe viral infections and treatment of severe viral infections, as well as the use of the method for enhancing the therapeutic effect of convalescent plasma therapy in patients at risk of severe coronavirus infection and for treating patients with severe coronavirus infection are described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 130,545, entitled "Method for Small-RNA Biomarker Identification and Functional Evaluation of Circulating Extracellular Vesicles Comprising Exosomes," filed December 24, 2020, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The filename of said ASCII copy, created on December 22, 2021, is 129642-00420_SL.txt and the file size is 21,261 bytes.

[0003] The present disclosure relates to methods for isolating and purifying circulating extracellular vesicles. [Background technology]

[0004] Nucleic acids released from cells during infection, inflammation, cancer, and other physiological and pathological processes circulate in human blood and may be powerful biomarkers of disease [1,2]. For example, detection of circulating cell-free genomic tumor DNA mutations is promising for evaluating the efficacy of cancer treatment and the presence of residual disease [3-6]. While large single-stranded RNA transcripts are degraded in circulation, small RNAs, especially microRNAs (miRNAs; ~22 nt in length), remain intact and can be detected and measured to reflect pathological processes. miRNAs are master transcriptional regulators that regulate the activity of specific mRNA targets and play important roles in a wide range of normal and pathological processes [7-12]. Although great progress has been made in detecting circulating miRNAs, the low proportion of these molecules among the large pool of circulating miRNAs obtained from diverse cells and tissues requires higher detection sensitivity and specificity, and discovery of disease-associated miRNA biomarkers remains a challenge. To address this issue, studies are now focused on the analysis of miRNA cargo encapsulated in extracellular vesicles (EVs), rather than total RNA purified from blood, serum, or plasma [13-15]. EVs are actively released by most types of cells into the microenvironment and circulatory system and are known to be involved in intercellular communication via uptake of target cells, and vesicle-transported miRNAs are involved in the reprogramming of recipient cells [16-20].

[0005] Extracellular vesicles (EVs) with lipid bilayers are distinguished by both their unique size range (30–150 nm in diameter) and biogenesis

[21] . CD9, CD63, CD81, CD37, and CD82 are actively secreted and specifically enriched in membrane-bound tetraspanins, making them targets for antibody purification [21, 22]. During the process of secretion, EVs also acquire surface molecules (proteins, lipids, etc.) from their cell of origin, making antibody-based purification more selective [23–25]. Although significant progress has been made in the last few years in the development of microfluidic-based systems for EV purification, few have reached the commercial market, and laboratory-based techniques for the purification and characterization of EVs from biofluids remain the gold standard. To date, four main approaches have been developed to purify circulating EVs, most of which offer the selection of large amounts of EVs for averaged biomarker characterization [26–28]. The three classical approaches to purify extracellular vesicles are based on ultracentrifugation, precipitation, and size exclusion [26-29]. Although these methods yield pure extracellular vesicle preparations (with caveats for precipitation-based methods), all confound subsequent analysis by contaminating subpopulations of extracellular vesicles released into the circulation from various tissues and cell types. A fourth method is to immunopurify extracellular vesicles by selecting for surface biomarkers of extracellular vesicles using magnetic beads coated with monoclonal antibodies [25, 30, 31]. Streptavidin magnetic beads may allow for customizable biotinylated antibody-based selection, but optimizing this customization remains challenging and highly dependent on the quality (surface polymer) and design (streptavidin coating) of the magnetic beads. In general, most commercially available antibody-based purification assays are static systems that rely solely on the recognition of known extracellular vesicle surface markers (e.g., CD63, CD81, CD9, etc.) and do not allow for customized selection and evaluation of different cell-specific extracellular vesicle subpopulations.Although these established assay methods allow for robust selection of extracellular vesicles, customizable antibody selection assays that can select cell-specific surface markers from biofluids are more promising for identifying circulating disease biomarkers

[32] . Finally, few antibody-based methods have been constructed that allow for the intact release of selected extracellular vesicles for downstream in vitro analysis. A recent immunoaffinity-based extracellular vesicle isolation assay (exo-PLA) has resolved many of these issues by using an enzymatically degradable DNA linker between the extracellular vesicle-binding antibody and the magnetic beads, allowing for the release of intact extracellular vesicles

[31] . However, our initial evaluation indicated that the magnetic beads used in this approach may nonspecifically bind small RNAs to an extent that significantly limits the detection of low-abundance miRNA cargo.

[0006] Extracellular vesicles are not only a valuable source of biomarkers but also play important roles in multiple biological processes, especially in the immune system [33-38]. Recent studies suggest that extracellular vesicles may be involved in antigen presentation to the immune system [37, 38] and mediate immune defense during viral infections [33, 34]. The mechanistic role that extracellular vesicles play in immunity to well-studied viruses (HIV, EBV, Ebola) has been previously evaluated [39-41]. However, few studies have evaluated their potential impact on SARS-CoV-2 infection. Our healthcare network (Hackensack Meridian Health) is located in the epicenter of the SARS-CoV-2 pandemic in the United States, with nearly half of the Covid-19 patients in New Jersey admitted to our hospital. This allowed us to obtain serum samples from Covid-19 patients, which we could use for proof-of-principle testing of applying our novel extracellular vesicle purification assay method to clinical specimens. Summary of the Invention

[0007] The described invention provides a method for purifying exosomes and / or extracellular vesicles, the Extracellular Vesicle Capture by AniTbody of CHoice and Enzymatic Release (EV-CATCHER) assay. We describe the step-by-step design and evaluation of this assay for the selection and release of customizable circulating extracellular vesicles. We combined the EV-CATCHER assay with highly sensitive small-RNA cDNA library preparation protocols [14, 42] to purify circulating extracellular vesicles from serum of Covid-19 patients for high-throughput small-RNA sequencing and demonstrated its applicability by identifying differentially expressed miRNAs associated with disease severity among hospitalized patients. Furthermore, we assessed the integrity of EV-CATCHER-purified and released extracellular vesicles and confirmed the neutralizing activity of convalescent extracellular vesicles from serum of Covid-19 convalescents that was unexpectedly identified using ultracentrifuge-purified extracellular vesicles.

[0008] According to one aspect, the present disclosure provides a method for preparing a population of purified biological particles from a biological sample from a subject and a method for evaluating the cargo of the purified population of biological particles, the method comprising: a) preparing the purified population of biological particles by (1) obtaining a biological sample containing biological particles; (2) contacting the biological sample from the subject containing biological particles with a binding agent for one or more biological particle surface agents that binds to nucleic acids, wherein the binding agent is immobilized to a surface of a solid support; b) isolating the biological particles bound by the binding agent from the biological sample; c) releasing the biological particles bound to the binding agent; d) eluting the biological particles from the binding agent to form a population of free purified biological particles; and e) evaluating the cargo and surface molecules, including proteins, DNA, RNA or proteins, of the purified population of biological particles.

[0009] According to some embodiments, step (e) evaluating the cargo and surface molecules further comprises one or more of: (i) identifying proteins specific to the surface of the biological particles by mass spectrometry; or (ii) identifying protein cargo by mass spectrometry; or (iii) identifying DNA molecules by sequencing or quantitative PCR; or (iv) extracting RNA from said population of purified biological particles and identifying and quantifying expression of small non-coding RNAs, including microRNAs (miRNAs), encapsulated by the population of purified biological particles.

[0010] According to some embodiments, the method includes an initial ultrafiltration or ultracentrifugation step to provide a starting pooled heterogeneous population of biological particles. According to some embodiments, the biological sample includes a bodily fluid. According to some embodiments, the bodily fluid includes whole blood, serum, plasma, cerebrospinal fluid (CSF), lymph, urine, stool, sweat, tears, nipple aspirate, or semen, or a secreted biological fluid. According to some embodiments, the bodily fluid is a circulating or secreted bodily fluid. According to some embodiments, the circulating fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF), or lymph.

[0011] According to some embodiments, the binding agent that binds to one or more biological particle surface antigens is an antibody, an antibody binding fragment, or an aptamer. According to some embodiments, the aptamer is a nucleic acid or a polypeptide. According to some embodiments, the biological particle surface antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, heterologous cell surface polypeptides, and / or other cell surface markers carried by the biological particle. According to some embodiments, the nucleic acid comprises DNA, RNA, or a combination thereof. According to some embodiments, the nucleic acid comprises DNA. According to some embodiments, the DNA comprises one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil. According to some embodiments, the DNA comprises a restriction enzyme recognition site. According to some embodiments, the nucleic acid comprises a non-naturally occurring nucleotide. According to some embodiments, the nucleic acid further comprises a binding site on the first end of the nucleic acid and a binding site on the second end of the nucleic acid, and the binding site on the first end of the nucleic acid and the binding site on the second end of the nucleic acid are different. According to some embodiments, the binding site on the first end of the nucleic acid is an avidin, streptavidin or carboxyl binding site. According to some embodiments, the binding site is biotin. According to some embodiments, the binding site on the second end of the nucleic acid is an amine moiety. According to some embodiments, the amine moiety is an azide. According to some embodiments, the binding agent for one or more biological particle surface antigens comprises a dibenzocyclooctyne (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester) SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), Sulfo-SMCC, or derivatives thereof.In some embodiments, the solid support is a well plate, a polymer, or a surface. In some embodiments, releasing the isolated biological particles includes (i) enzymatically degrading the nucleic acid, or (ii) displacing the first strand of the nucleic acid linked to the antibody from the second strand of the nucleic acid linked to the support by strand displacement using a nucleic acid complementary to the first and second strands of the nucleic acid and an enzyme having strand displacement activity and releasing the antibody from the support, or (iii) isolating the annealed DNA strands and releasing the antibody from the platform by polymerase chain reaction using an oligonucleotide complementary to the region of the DNA bound to the antibody, without damaging the DNA strand bound to the antibody. In some embodiments, the enzymatic cleavage is with uracil glycosylase. In some embodiments, the enzymatic cleavage is with a restriction enzyme. In some embodiments, the enzyme with strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. In some embodiments, the method includes identifying one or more small uncoated RNAs, including miRNAs, encapsulated in one or more biological particles by next generation sequencing. According to some embodiments, the isolated biological particles are derived from a healthy subject or a subject suffering from a disease. According to some embodiments, the disease includes viral infection, cancer, placentation abnormalities, exercise-induced muscle damage, heart failure, Alzheimer's disease, liver cirrhosis, viral and bacterial infection, kidney disease, bone remodeling after injury, wound healing or COPD and asthma. According to some embodiments, the viral infection is a severe coronavirus infection. According to some embodiments, the severe coronavirus infection is caused by SARS-CoV-1, MERS, or SARS-CoV-2. According to some embodiments, the severe coronavirus infection is caused by SARS-CoV-2.According to some embodiments, the isolated and quantified miRNAs from subjects suffering from severe coronavirus infection with SARS-CoV-2 include one or more of hsa-miR-146a, hsa-miR-126-3p, hsa-miR-15a, hsa-miR-424, hsa-miR-151-3p, hsa-miR-126-5p, hsa-miR-627-5p, hsa-miR-145, hsa-miR-205, hsa-miR-200c, hsa-miR-550-5p, and hsa-miR-629. According to some embodiments, the miRNA markers of severe SARSCoV-2 disease include hsa-miR-146a, hsa-miR126-3p, or both.

[0012] According to another aspect, the present disclosure provides a method for in vitro assessment of a risk of a subject developing a severe coronavirus infection, and a method for treating a severe coronavirus infection, comprising: a. (1) obtaining a biological sample from a subject comprising biological particles; (2) preparing a population of purified biological particles by contacting the biological sample from the subject comprising biological particles with a binding agent for one or more biological particle surface agents, the binding agent binding to nucleic acids, the nucleic acids being immobilized on a surface of a solid support; b. isolating the biological particles bound by the binding agent from the biological sample; c. releasing the biological particles bound to the binding agent; d. eluting the biological particles from the binding agent to form a population of free purified biological particles; e. (i) extracting RNA from the population of purified biological particles; and (ii) extracting RNA from one or more biological particles encapsulated by the purified population of exosomes. (iii) determining a cargo profile of the purified biological particles by assessing the cargo of the population of purified biological particles by comparing the cargo profile of the purified biological particles to the cargo profiles of (1) control subjects not infected with coronavirus, (2) control subjects infected with coronavirus and who developed mild disease, and (3) control subjects infected with coronavirus and who developed severe disease; (f) determining the patient's risk for severe viral infection if the small noncoding RNA or protein cargo profile of the purified biological particles is about 1.5-fold lower or 1.5-fold higher than the cargo profile from a control subject infected with coronavirus who developed severe disease; and (g) administering an appropriate therapy to patients at risk for severe viral infection.

[0013] According to some embodiments, the method includes an initial ultrafiltration or ultracentrifugation step to provide a starting pooled heterogeneous population of biological particles. According to some embodiments, the biological sample comprises a bodily fluid. According to some embodiments, the bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymph, urine, stool, sweat, tears, nipple aspirate, or semen, or a secreted biological fluid. According to some embodiments, the bodily fluid is a circulating or secreted bodily fluid. According to some embodiments, the circulating fluid is whole blood, serum, plasma, CSF, or lymph. According to some embodiments, the severe coronavirus infection is due to SARS-CoV-1, MERS, or SARS-CoV-2. According to some embodiments, the severe coronavirus infection is due to SARS-CoV-2. According to some embodiments, the miRNAs of patients infected with SARS-Cov-2, when compared to controls, include one or more of hsa-miR-146a, hsa-miR-126-3p, hsa-miR-15a, hsa-miR-424, hsa-miR-151-3p, hsa-miR-126-5p, hsa-miR-627-5p, hsa-miR-145, hsa-miR-205, hsa-miR-200c, hsa-miR-550-5p, and hsa-miR-629. According to some embodiments, the miRNA markers of severe disease due to SARS-CoV-2, when compared to controls, include hsa-miR-146a, hsa-miR126-3p, or both. According to some embodiments, the binding agent for one or more biological particle surface antigens is an antibody, antibody fragment, or aptamer. According to some embodiments, the exosome surface antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers carried by the biological particle. According to some embodiments, the nucleic acid comprises DNA comprising one or more ribonucleic acid nucleotides, wherein the ribonucleic acid nucleotide is uracil.In some embodiments, releasing the isolated biological particles includes (i) enzymatically cleaving the nucleic acid, or (ii) strand displacement with an enzyme having strand displacement activity with a nucleic acid complementary to the first or second strand of the nucleic acid to displace the first strand of the nucleic acid linked to the antibody from the second strand of the nucleic acid linked to the support, thereby releasing the antibody from the support, or (iii) separating the annealed DNA strands by polymerase chain reaction with an oligonucleotide complementary to a region of the DNA bound to the antibody, to release the antibody from the support without damaging the DNA strand bound to the antibody. In some embodiments, the enzymatic cleavage is with uracil glycosylase. In some embodiments, the enzymatic cleavage is with a restriction enzyme. In some embodiments, the enzyme having strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. In some embodiments, identification and quantification of small non-coding RNAs, including one or more microRNAs (miRNAs), encapsulated by the population of purified biological particles is performed by next generation sequencing. According to some embodiments, appropriate therapy for patients at risk for severe viral infection includes one or more of supportive care, antiviral agents, and immunomodulatory agents. According to some embodiments, (a) the supportive care consists of intravenous fluids, supplemental oxygen, or inhalation of steam via a nebulizer; or (b) the antiviral agent inhibits viral entry, reduces viral load, or both; or (c) the antiviral agent is selected from acyclovir, gancidol, foscarnet, ribavirin, amantadine, azidodeoxythymidine (zidovudine), nevirapine, tetrahydroimidazobenzodiazepinone (TIBO) compounds, efavirenz, remdesivir, lopinavir / ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine; or (d) the immunomodulatory agent is a glucocorticoid, or a recombinant interferon; or (e) the immunomodulatory agent is a glucocorticoid, and the glucocorticoid is a corticosteroid.According to some embodiments, the glucocorticoid comprises prednisone, dexamethasone, azathioprine, mycophenolic acid, mycophenolate mofetil, or a combination thereof.

[0014] According to another aspect, the disclosure provides a method for enhancing the therapeutic efficacy of convalescent plasma therapy for treating patients at risk for severe coronavirus or other viral infection, the method comprising: a) (1) obtaining convalescent serum from a convalescent subject comprising high IgG titers against coronavirus; (2) preparing the purified population of biological particles by contacting the biological sample from the subject comprising biological particles with a binding agent for one or more biological particle surface agents, the binding agent binding to nucleic acid, the nucleic acid being immobilized on a surface of a solid support; b) isolating the biological particles bound by the binding agent from the biological sample; c) releasing the biological particles bound to the binding agent; d) eluting the biological particles from the binding agent to form a population of free purified biological particles; e) measuring in vitro the neutralization titer of the population of purified biological particles against coronavirus; and f) administering the convalescent serum comprising high titers of neutralizing biological particles and high titers of neutralizing IgG to the subject.

[0015] According to some embodiments, the method includes an initial ultrafiltration or ultracentrifugation step to provide a starting pooled heterogeneous population of biological particles. According to some embodiments, the biological sample includes a bodily fluid. According to some embodiments, the bodily fluid includes whole blood, serum, plasma, cerebrospinal fluid (CSF), lymph, urine, stool, sweat, tears, nipple aspirate, or semen, or secreted biological fluids. According to some embodiments, the bodily fluid is a circulating bodily fluid. According to some embodiments, the circulating bodily fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF), or lymph.

[0016] According to some embodiments, the binding agent that binds to one or more biological particle surface antigens is an antibody, antibody binding fragment, or aptamer. According to some embodiments, the biological particle surface antigens include one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, heterologous cell surface polypeptides, and / or other cell surface markers carried by the biological particle. According to some embodiments, the nucleic acid includes DNA comprising ribonucleic acid nucleotides, the ribonucleic acid nucleotides being uracil, or the DNA comprising a restriction enzyme recognition site. According to some embodiments, releasing the isolated biological particles includes (i) enzymatically cleaving the nucleic acid, or (ii) strand displacement with an enzyme having strand displacement activity with a nucleic acid complementary to the first or second strand of the nucleic acid to displace a first strand of the nucleic acid linked to the antibody from a second strand of the nucleic acid linked to the support and release the antibody from the support, or (iii) separating the annealed DNA strands to release the antibody from the support without damaging the DNA strand linked to the antibody by polymerase chain reaction with an oligonucleotide complementary to a region of the DNA bound to the antibody. According to some embodiments, the enzymatic cleavage is with uracil glycosylase. According to some embodiments, the enzymatic cleavage is with a restriction enzyme. According to some embodiments, the enzyme having strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. According to some embodiments, the severe coronavirus infection is caused by SARS-CoV-1, MERS, or SARS-CoV-2. According to some embodiments, the severe coronavirus infection is caused by SARS-CoV-2. According to some embodiments, the population of neutralizing purified biological particles derived from convalescent serum with high IgG titers contains the ACE2-receptor.According to some embodiments, measuring the neutralization titer of the purified biological particle population against SARS-CoV-2 virus further comprises incubating mammalian cells infected with coronavirus in vitro with a dilution series of isolated purified biological particles from convalescent serum with high IgG titer and measuring viral particle production compared to a negative control (infected cells without purified biological particles from convalescent serum). According to some embodiments, viral particle production is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to the negative control. According to some embodiments, convalescent serum containing neutralizing purified biological particles may enhance the efficacy of convalescent plasma therapy by at least about 2-fold compared to treatment with convalescent plasma therapy alone, or (b) the neutralizing purified biological particles enable presentation of viral antigens to the immune system.

[0017] According to another aspect, the disclosure provides a method of treating a subject having a severe coronavirus infection, comprising: a) (1) obtaining a biological sample comprising biological particles; (2) contacting the biological sample from the subject comprising biological particles with a binding agent for one or more biological particle surface agents, the binding agent binding to nucleic acids, the nucleic acids being immobilized on a surface of a solid support; (3) isolating the biological particles bound by the binding agent from the biological sample; (4) releasing the biological particles bound to the binding agent (2); (5) eluting the biological particles from the binding agent to form a population of free purified biological particles; and (6) determining the neutralization titer of a population of biological particles purified from the subject's biological sample by measuring in vitro the neutralization titer of the population of purified biological particles; and b) determining that the neutralization titer of the population of purified biological particles purified from the subject is in vitro. and (c) preparing a population of purified biological particles from the convalescent plasma of the convalescent subject by: (i) obtaining convalescent serum from the convalescent subject comprising a high anti-coronavirus IgG titer; (ii) contacting the convalescent serum with a binding agent directed to one or more biological particle surface antigens; (iii) isolating the biological particles bound by the binding agent from the biological sample; (iv) releasing the biological particles bound to the binding agent; and (v) eluting the bound biological particles from the binding agent to form a population of free purified biological particles; d) measuring in vitro the neutralizing titer of the population of purified biological particles from the convalescent serum against the SARS-CoV-2 virus; and (e) administering to the subject the convalescent serum with a high IgG titer containing the neutralizing purified biological particles alone or the neutralizing purified biological particles.

[0018] According to some embodiments, the method includes an initial ultrafiltration or ultracentrifugation step to provide a starting pooled heterogeneous population of biological particles. According to some embodiments, the biological sample comprises a bodily fluid. According to some embodiments, the bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymph, urine, stool, sweat, tears, nipple aspirate, or semen, or a secreted biological fluid. According to some embodiments, the bodily fluid is a circulating or secreted bodily fluid. According to some embodiments, the circulating fluid is whole blood, serum, plasma, CSF, or lymph.

[0019] According to some embodiments, the binding agent that binds to one or more biological particle surface antigens is an antibody, an antibody binding fragment, or an aptamer. According to some embodiments, the aptamer is a nucleic acid or a polypeptide. According to some embodiments, the biological particle surface antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, heterologous cell surface polypeptides, and / or other cell surface markers inherited by the biological particle. According to some embodiments, releasing the isolated biological particles includes (i) enzymatically cleaving the nucleic acid, or (ii) strand displacement with an enzyme having strand displacement activity with a nucleic acid complementary to the first or second strand of the nucleic acid to displace the first strand of the nucleic acid linked to the antibody from the second strand of the nucleic acid linked to the support, thereby releasing the antibody from the support, or (iii) separating the annealed DNA strands by polymerase chain reaction with an oligonucleotide complementary to a region of the DNA bound to the antibody, to release the antibody from the support without damaging the DNA strand bound to the antibody. According to some embodiments, the nucleic acid comprises DNA comprising one or more ribonucleic acid nucleotides, the ribonucleic acid nucleotides being uracil, a restriction site sequence or a duplex of RNA and DNA molecules, and the enzymatic cleavage is performed by uracil glycosyltransferase, DNase or RNase. According to some embodiments, the enzymatic cleavage is with a restriction enzyme. According to some embodiments, the enzyme having strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. According to some embodiments, the severe coronavirus infection is caused by SARS-CoV-1, MERS, or SARS-CoV-2. According to some embodiments, the severe coronavirus infection is caused by SARS-CoV-2.

[0020] According to some embodiments, measuring the neutralization titer of (i) the purified biological particle population from the subject and (ii) the purified biological particle population from convalescent serum with high IgG titer against coronavirus comprises incubating mammalian cells infected in vitro with coronavirus with a dilution series of (i) the isolated purified biological particles from the subject and (ii) the isolated purified biological particles from convalescent serum with high IgG titer, and comparing the viral particle production compared to a negative control (infected cells alone). According to some embodiments, the neutralizing population of purified biological particles comprises the ACE2-receptor.

[0021] According to another aspect, the present disclosure provides a method of preparing a population of purified cells from a biological sample from a subject, the method comprising:

[0022] a) preparing a purified cell population by:

[0023] (1) obtaining a biological sample from a subject that contains cells;

[0024] (2) contacting the biological sample from the subject containing biological particles with a binding agent for one or more cell surface antigens, the binding agent binding to nucleic acid, the nucleic acid being immobilized on the surface of a solid support.

[0025] b) isolating the cells bound by the binding agent from the biological sample;

[0026] c) releasing the cells bound to the binding agent;

[0027] d) eluting the bound cells from the binding agent to form a purified cell population.

[0028] According to some embodiments, the method includes an initial ultrafiltration or ultracentrifugation step to provide a starting pooled heterogeneous population of cells.

[0029] According to some embodiments, the biological sample is prepared in vivo or in vitro.

[0030] According to some embodiments, the biological sample comprises a bodily fluid. According to some embodiments, the bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymph, urine, stool, sweat, tears, nipple aspirate, or semen, or a secreted biological fluid. According to some embodiments, the bodily fluid is a circulating or secreted bodily fluid. According to some embodiments, the circulating fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF), or lymph.

[0031] According to some embodiments, the one or more cell surface antigens comprise CD4, CD8, CD9, CD46, CD63, CD81, CD37, CD82, CD138, CD151, ALix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, a heterologous cell surface polypeptide.

[0032] According to some embodiments, the heterologous cell surface polypeptide is expressed by a chimeric antigen receptor T cell (CAR-T cell).

[0033] According to some embodiments, the binding agent that binds to one or more cell surface antigens is an antibody, an antibody-binding fragment or an aptamer. According to some embodiments, the aptamer is a nucleic acid or a polypeptide.

[0034] According to some embodiments, the nucleic acid comprises DNA, RNA, or a combination thereof. According to some embodiments, the nucleic acid comprises DNA. According to some embodiments, the DNA comprises one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil. According to some embodiments, the DNA comprises a restriction enzyme recognition site. According to some embodiments, the nucleic acid is a DNA / RNA duplex degradable by an endonuclease or RNAse (RNase-H). According to some embodiments, the nucleic acid comprises non-natural nucleotides.

[0035] According to some embodiments, the nucleic acid further comprises a binding site on a first end of the nucleic acid and a binding site on a second end of the nucleic acid, wherein the binding site on the first end of the nucleic acid and the binding site on the second end of the nucleic acid are different.

[0036] According to some embodiments, the binding site on the first end of the nucleic acid is an avidin, streptavidin or carboxyl binding site. According to some embodiments, the binding site is biotin. According to some embodiments, the binding site on the second end of the nucleic acid is an amine moiety. According to some embodiments, the amine moiety is an azide.

[0037] According to some embodiments, the binding agent to one or more cell surface antigens comprises a dibenzocyclooctyne (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester) SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), Sulfo-SMCC, or derivatives thereof.

[0038] According to some embodiments, the solid support is a well plate, a polymer, or a surface.

[0039] According to some embodiments, releasing the isolated cells comprises:

[0040] (i) enzymatic cleavage of nucleic acids; or

[0041] (ii) displacing the first strand of the nucleic acid linked to the antibody from the second strand of the nucleic acid linked to the support by strand displacement using a nucleic acid complementary to the first strand or the second strand of the nucleic acid and an enzyme having strand displacement activity, thereby liberating the antibody from the support; or

[0042] (iii) separating the annealed DNA strands by polymerase chain reaction using an oligonucleotide complementary to the region of DNA bound to the antibody, thereby releasing the antibody from the support without damaging the DNA strand bound to the antibody.

[0043] In some embodiments, the enzymatic cleavage is with uracil glycosylase. In some embodiments, the enzymatic cleavage is with a restriction enzyme. In some embodiments, the enzymatic cleavage is with an endonuclease or an RNase. In some embodiments, the enzyme with strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase.

[0044] According to some embodiments, the population of cells is derived from non-eukaryotic and eukaryotic species, or healthy normal or diseased tissue, or mouse orthotopic / xenograft / PDX organs, or blood, or tissue culture.

[0045] According to some embodiments, the eukaryotic cell is human, mouse, rat, dog, non-human primate, or cat.

[0046] In some embodiments, the population of purified cells is derived from a healthy subject or a subject suffering from a disease, in some embodiments, the disease consists of cancer, viral infection, placental abnormality, inflammation and other pathologies.

[0047] According to some embodiments, the cell is an endothelial cell, an epithelial cell, a T cell, a fibroblast cell, an adipocyte cell, a neuronal cell, a tumor cell, a blood cell, or a cardiac cell.

[0048] According to some embodiments, the cell population is derived from (a) non-eukaryotic and eukaryotic species, or (b) healthy normal or diseased tissue, or (c) mouse orthotopic / xenograft / PDX organs, or (d) blood, or (e) tissue culture.

[0049] According to some embodiments, the viral infection is a severe coronavirus infection. According to some embodiments, the severe coronavirus infection is due to SARS-CoV-1, MERS, or SARS-CoV-2. According to some embodiments, the severe coronavirus infection is due to SARS-CoV-2. [Brief description of the drawings]

[0050] [Figure 1] Schematic diagram of the EV-CATCHER assay (Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release), a customizable, low-background, high-affinity assay for specific immunocapture and release of circulating extracellular vesicles prior to small-RNA sequencing and identification of miRNA biomarkers. [Diagram 2]A is a schematic of the EV-CATCHER assay designed to purify extracellular vesicles from biofluids, which relies on the binding of degradable dsDNA-linkers (annealing of uracillated 5'-azide oligonucleotides with complementary uracillated 3'-biotin oligonucleotides) to DBCO-activated antibodies and a streptavidin-coated platform. B shows acrylamide gel migration of single-stranded oligonucleotides and double-stranded (ds) hybridized DNA linkers. Single-stranded (ss) biotin-labeled DNA (lane 2), ssDNA azide (lane 3), dsDNA linker (lane 4), and UNG-digested dsDNA linker (lane 5) were separated on a 15% non-denaturing PAGE gel. C shows activation of monoclonal human anti-CD63 antibody (Ab) and dsDNA-linker binding. Representative image of a Coomassie stained non-denaturing PAGE gel (12%) with migration of protein ladder (lane a), 1 μg native anti-CD63 Ab (lane b), dsDNA-linker bound to 1 μg DBCO-activated anti-CD63 antibody (using increasing amounts of dsDNA-linker) (lane c-e), and dsDNA-linker-Ab digested with UNG (500 ng-1 μg) (lane f). Experiments were performed in duplicate and imaged on a ChemiDoc MP imager. SEQ ID NOs: 1 and 2, respectively, are disclosed in order of appearance. [Figure 3A] Assay method to evaluate nonspecific small RNA binding to streptavidin-coated magnetic beads and wells. Schematic of experimental procedure for sample collection (top). A 1xPBS solution containing 1 ng of ath-miR-159a RNA oligonucleotide was incubated with wells or beads, and four different samples were collected, including wash #1, wash #2, wash #3, and final elution. The two graphs on the left show RT-qPCR quantifications obtained with control ath-miR-159a RNA (1 ng; black bars), washes #1, #2, #3 (gray bars), and final elution (white bars) for wells (top left graph) and beads (bottom left graph), without RNase-A treatment. The two graphs on the right show RT-qPCR quantifications obtained with samples collected in the same way, where wells and beads were first treated with RNase-A. [Figure 3B] Assay to evaluate extracellular vesicle non-specific binding of MCF-7. The experimental collection procedure is shown in the schematic (top). The two graphs represent RT-qPCR quantification of hsa-miR-21 (left) and hsa-miR-200c (right) using total RNA extracted from total extracellular vesicle control (10 μg, black bars), extracellular vesicles + RNase-A (dark grey bars), extracellular vesicles + RNase-A after incubation with Dynabeads™ (magnetic beads) and removal (light grey bars), and extracellular vesicles + RNase-A after incubation with wells and removal (white bars). All qPCR experiments were performed in triplicate for each sample. Data are technical replicates (subtraction of ath-miR-159a and 10 μg MCF-7 control) and are expressed as ΔΔCt. [Figure 4A] Evaluation of extracellular vesicle surface markers by Western blot using anti-Alix, anti-CD63, anti-CD9, and anti-CD81 antibodies. EV-CATCHER purification of CD63+ extracellular vesicles from MCF-7 tissue culture medium with decreasing total protein input before purification. For the left and center Western blots, 3 μg (lane 1), 2.75 μg (lane 2), 2.5 μg (lane 3), 2.25 μg (lane 4), 2 μg (lane 5), 1.75 μg (lane 6), and 1.5 μg (lane 7) of total protein were used before EV-CATCHER purification. [Figure 4B] Evaluation of extracellular vesicle surface markers by Western blot using anti-Alix, anti-CD63, anti-CD9, and anti-CD81 antibodies. EV-CATCHER purification of CD63+ extracellular vesicles from human plasma with reduced total protein input before purification. For the left and center Western blots, 3 μg (lane 1), 2.75 μg (lane 2), 2.5 μg (lane 3), 2.25 μg (lane 4), 2 μg (lane 5), 1.75 μg (lane 6), and 1.5 μg (lane 7) of total protein were used before EV-CATCHER purification. [Figure 4C]Evaluation of extracellular vesicle surface markers by Western blot using anti-Alix, anti-CD63, anti-CD9, and anti-CD81 antibodies. EV-CATCHER was also used to purify CD63+ extracellular vesicles from serum samples and was validated with four different surface protein antibodies. [Figure 4D] Transmission electron microscopy (TEM) images of MCF-7 extracellular vesicle stock (left, MCF-7 stock) and CD63 extracellular vesicles purified from MCF-7 extracellular vesicle stock (MCF-7), human plasma (Plasma), and human serum (Serum). The direct magnification of the TEM images is 20,000x, and the scale bar is 200 nm. [Figure 4E] Representative size distributions characterized by nanoparticle tracking (Spectradyne nCS1 equipped with a TS400 microfluidic cartridge) for CD63 extracellular vesicles purified from MCF-7 cell culture medium (left graph), human plasma (middle graph), and human serum (right graph). Peak filtering was performed for diameters <65 nm and transit times >80 μs. Concentrations of particles detected between 65 and 150 nm are represented. [Figure 5A] Next generation sequencing and heatmap representation of the top expressed circulating miRNAs in human serum. The heatmap includes the top 49 expressed miRNAs and their detectability in decreasing amounts of total RNA extracted from human serum (6 ng, 3 ng, and 1.5 ng, replicated twice). [Figure 5B] Heatmap representation showing the difference in miRNA expression between total RNA extracted from mouse RAW S264.7 extracellular vesicles (first two columns), commercial whole human plasma (columns 3 and 4), and extracellular vesicles obtained by ultracentrifugation from the same commercial whole human plasma (columns 5 and 6). Duplicate libraries were obtained using new (Repeats#1) and old (Repeats#2) 3' barcode adapters. [Figure 5C]EV-CATCHER specific capture of mouse extracellular vesicles spiked into human plasma. Heatmaps depicting differences in miRNA expression between mouse RAWS264.7 EVs and human plasma, including the top 58 differentially expressed miRNAs between human plasma and mouse RAWS264.7 EVs (columns 1 and 2) and the top 50 mouse specific transcripts (columns 5 and 6). Mouse RAWS EVs (1 μg) spiked into human plasma (100 μl) were purified from plasma using the EV-CATCHER assay bearing a mouse specific anti-CD63 antibody (columns 2 and 3). Duplicate libraries were obtained using new (Repeats#1) and old (Repeats#2) 3' barcode adapters. Data were analyzed using dedicated Bioconductor packages in the R platform and heatmaps were generated using the 'NMF' package (heatmap function). [Figure 6A] Individual boxplot analysis by next-generation small RNA sequencing with 10 differentially expressed miRNAs (green boxes, hsa-miR-146a, hsa-miR-126-3p, hsa-miR-424, hsa-miR-151-3p, hsa-miR-126-5p, hsa-miR-627-5p, hsa-miR-145, hsa-miR-205, and hsa-miR-200c) identified from EV-CATCHER-purified extracellular vesicles and 2 differentially expressed miRNAs (orange boxes, hsa-miR-550-5p, hsa-miR-629*) identified from whole serum of mild and severe Covid-19 hospitalized patients. Extracellular vesicles purified from serum were obtained by EV-CATCHER using a combination of anti-CD63, anti-CD9, and anti-CD81 antibodies. [Figure 6B] Box plot miRNA read representation of the top 10 differentially expressed miRNAs identified from serum exosomes and / or extracellular vesicles of mild (n=13) and severe (n=17) hospitalized Covid-19 patients compared to total miRNA reads. [Figure 6C]Box plot representation of miRNA integration signatures (including the top 10 miRNAs identified in serum exosomes and / or extracellular vesicles) between two small RNA libraries prepared using RNA extracted from exosomes and / or extracellular vesicles purified from mild and severe patients (library #3 included 7 mild and 8 severe cases, library #4 included 6 mild and 9 severe cases). [Figure 6D] Box plot representation of integrated miRNA signatures (10 miRNAs) between RNA extracted from EV-CATCHER purified extracellular vesicles and RNA extracted from whole serum of mild and severe patients. [Figure 6E] Box plots show RT-qPCR validation results for the top four differentially expressed miRNAs identified by small RNA sequencing between mild and severe hospitalized Covid-19 patients using RNA extracted from EV-CATCHER-purified extracellular vesicles. Data are presented as ΔΔCt, with technical triplicates performed for each sample. Differences in expression were assessed using DESeq2 (R / Bioconductor package) for sequencing results and t-tests for miRNA scores and qPCR results. [Figure 7A] Six convalescent serum samples were quantified for the presence of immunoglobulin (IgG) directed against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Three high IgG titer serum samples (left) and three IgG titer below the level of quantification (BLQ) were quantified by ELISA. High IgG titers were estimated by remaining activity at a 1:10,000 dilution, and the BLQ IgG serum sample contained no detectable IgG against the SARS-CoV-2 spike protein. [Figure 7B] Western blot analysis of CD63-purified extracellular vesicles from high IgG (lanes 1-3) and BLQ IgG (lanes 4-6) serum samples using the EV-CATCHER assay with anti-Alix, anti-CD63, anti-CD9, and anti-CD81 antibodies (Abcam). [Figure 7C]Representative transmission electron microscopy (TEM) images of high IgG (CDI-001, FIG. 7B-lane 1) and BLQ IgG (CDI-004, FIG. 7B-lane 6) isolated from convalescent serum using the EV-CATCHER assay are shown. Direct magnification is 20,000x, scale bar is 200 nm. [Figure 7D] Figure 1 shows nanoparticle size distribution assessed using EV-CATCHER purified extracellular vesicles from six donors using a Spectradyne nCS1 instrument equipped with a TS400 microfluidic cartridge. [Figure 7E] FIG. 1 is a schematic diagram of the experimental procedure used to obtain highly pure convalescent serum extracellular vesicles by ultracentrifugation. [Figure 7F] In vitro assessment of SARS-CoV-2 infection using Vero E6 cells treated with whole serum or purified extracellular vesicles. Healthy Vero E6 cell control (bar 1, no virus) was incubated for 72 h with mNG SARS-CoV-2 (bar 2, virus), ultracentrifuged extracellular vesicles with high IgG (bar 3) and BLQ IgG (bar 4) (extracellular vesicle control), SARS-CoV-2 with high IgG whole serum (bars 5, 7, and 9) or BLQ IgG whole serum (bars 11, 13, and 15), mNG SARS-CoV-2 with CD63+ extracellular vesicles of high IgG serum (bars 6, 8, and 10) or CD63+ extracellular vesicles of BLQ IgG serum (bars 12, 14, and 16), UNG digestion buffer (bar 17), and UNG digestion buffer with mNG SARS-CoV-2 (bar 18). Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc correction. All results are presented as mean ± SEM (n = 3), **** = p < 0.0001 vs virus. [Figure 7G]Fluorescence imaging of Vero E6 cells infected with mNeonGreen SARS-CoV-2 reporter virus. Hoechst 33342 and mNeonGreen were visualized using fluorescent Celigo Cell Imaging. Representative fluorescence images are of healthy Vero E6 cells (control without virus), mNG SARS-CoV-2 with high IgG whole serum (high IgG serum), mNG SARS-CoV-2 with CD63+ extracellular vesicles purified from high IgG serum (extracellular vesicles (high IgG serum), patient no. CDI-001), mNG SARS-CoV-2 (virus control), mNG SARS-CoV-2 with BLQ IgG whole serum (BLQ IgG serum, patient no. CDI-004), and mNG SARS-CoV-2 with CD63+ extracellular vesicles purified from BLQ serum (extracellular vesicles (BLQ serum)). Scale bars in images were 500 μM. [Figure 8] Heatmap representation of the top 117 expressed circulating small-RNAs in human serum identified by small-RNA next generation sequencing (columns 1 and 2). Small RNA sequencing was performed on RNA extracted from Dynabeads™ magnetic beads incubated in whole serum (columns 3 and 4), magnetic beads incubated in whole serum and treated with UNG (columns 5 and 6), magnetic beads covalently coupled to CD63 antibody incubated in whole serum (columns 7 and 8), and extracellular vesicles obtained by ultracentrifugation (columns 9 and 10). All experiments were repeated twice. [Figure 9] A and B show SDS PAGE analysis of exosome extracts from COVID-19 convalescent sera (left, high IgG titer convalescent serum, RBD IgG>10,000; right, BLQ convalescent serum) stained for total protein and ACE-2. B shows a graph of ACE-2 protein expression normalized to total protein in convalescent sera (left, high IgG titer convalescent serum, RBD IgG>10,000; right, BLQ convalescent serum), demonstrating a significant increase in ACE-2 in convalescent plasma from individuals with high IgG titers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] definition As used in this 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 "peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0052] Acute respiratory distress syndrome (ARDS) is characterized by noncardiogenic pulmonary edema, pneumonia, hypoxemia, and decreased lung compliance. The pathogenesis of ARDS remains unclear, and there is no gold standard diagnostic test. The Berlin definition of ARDS (Ranieri, VM et al. J. Am. Med. Assoc. (2012) 307(23):2526-33) provides the following criteria and definition:

[0053] [Table 1]

[0054] The term "angiotensin-converting enzyme 2" or "ACE2" as used herein refers to a type 1 integral membrane glycoprotein [Tikellils, C. and Thomas MC. Intl J. Peptides (2012) 256294, citing Tipnis, SR et al. J. Biol. Chem. (2000) 275(43):33238-43] that is expressed and active in most tissues and is a component of the renin-angiotensin system (RAS), which controls blood pressure by regulating body water. ACE2 is most highly expressed in the kidney, endothelium, lung, and heart [Donoghue, M. et al. Cir. Res. (2000) 87(5):E1-E9, cited above, Tipnis, SR et al. J. Biol. Chem. (2000) 275(43):33238-43].

[0055] The term "administering" as used herein, when used in conjunction with a therapeutic agent, means administering or applying the therapeutic agent directly to a target organ, tissue or cell, or administering the therapeutic agent to a subject, whereby the therapeutic agent positively affects the organ, tissue, cell or subject to which it is targeted. Thus, as used herein, the term "administering", when used in conjunction with compositions, for example, including exosomes and / or extracellular vesicles, can include, but is not limited to, providing the composition in or on the target organ, tissue or cell; or providing the composition to a patient so that the therapeutic agent reaches the target organ, tissue or cell. "Administration" can be accomplished parenterally, for example, by intravenous administration or injection techniques, inhalation or pneumoperitoneum, or by such methods in combination with other known techniques.

[0056] The term "Alix" as used herein refers to an accessory protein of the endosomal sorting complex required for transport (ESCRT). Evidence indicates that Alix is ​​involved in packaging of miRNAs during extracellular vesicle (EV) biogenesis [Iavello, A. et al. Int. J. Mol. Med. (2016) 37(4): 958-66].

[0057] As used herein, the term "angiotensin-converting enzyme 2 receptor" or "ACE2 receptor" refers to the protein that serves as a gateway for SARSCoV-2 to infect a wide range of human cells.

[0058] As used herein, the terms "animal," "patient," and "subject" include, but are not limited to, humans and non-human vertebrates, such as wild, domestic, and farm animals. The terms "animal," "patient," and "subject" may refer to mammals, including, but not limited to, humans.

[0059] The term "antibody" as used herein refers to a polypeptide or group of polypeptides consisting of at least one binding domain formed from the folding of a polypeptide chain having a three dimensional binding space with an internal surface shape and charge distribution complementary to an antigenic determinant characteristic of an antigen.

[0060] The basic structural unit of a whole antibody molecule consists of four polypeptide chains: two identical light (L) chains (containing about 220 amino acids each) and two identical heavy (H) chains (usually containing about 440 amino acids each). The two heavy and two light chains are linked by a combination of non-covalent and covalent (disulfide) bonds. The molecule consists of two identical parts, each with an identical antigen-binding site consisting of the N-terminal region of the light chain and the N-terminal region of the heavy chain. Usually, both the light and heavy chains cooperate to form the antigen-binding surface. Human antibodies exhibit two types of light chains, kappa and lambda, although individual immunoglobulin molecules usually contain only one of them.

[0061] Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, CDR-grafted, multispecific, bispecific, catalytic, chimeric, humanized, fully human, anti-idiotypic, or labelable antibodies in soluble or bound form, as well as fragments, variants, and derivatives thereof, which may be used alone or in combination with other amino acid sequences provided by known techniques. Monoclonal antibodies (mAbs) can be produced by fusing mouse spleen cells from immunized donors with mouse myeloma cell lines to obtain established mouse hybridoma clones that grow in selective media. Hybridoma cells are immortalized hybrid cells resulting from the in vitro fusion of antibody-secreting B cells with myeloma cells. In vitro immunization refers to the primary activation of antigen-specific B cells in culture and is another established means of producing mouse monoclonal antibodies. Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes can also be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer (single-chain Fv or scFv) can be generated by randomly combining heavy and light chain V genes using PCR. The combinatorial library can then be cloned and expressed on the surface of filamentous bacteriophage by fusion with a minor coat protein at the phage tip. The guided selection method is based on shuffling human and murine immunoglobulin V genes.The method involves (i) shuffling a repertoire of human lambda light chains with heavy chain variable region (VH) domains of mouse monoclonal antibodies reactive with the antigen of interest, (ii) selecting half-human Fabs with the antigen, (iii) using the selected lambda light chain genes as "docking domains" for a library of human heavy chains in a second round of shuffling to isolate cloned Fab fragments with human light chain genes, (v) transfecting mouse myeloma cells with a mammalian cell expression vector containing the genes by electroporation; and (vi) expressing the V genes of the Fabs reactive with the antigen as complete IgG1, lambda antibody molecules in mouse myeloma. The antibodies may be from any species. The term antibody also includes binding fragments of the antibodies of the invention. Examples of fragments include Fv, Fab, Fab', single chain antibodies (svFC), dimeric variable regions (Diabodies), and disulfide-stabilized variable regions (dsFv). Structural and functional domains can be identified by comparing the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated herein by reference in its entirety.

[0062] As used herein, the term "antigen" refers to any substance that elicits an immune response.

[0063] As used herein, the term "antigen-binding site" refers to the site at the end of each arm of an antibody that physically contacts and non-covalently binds to an antigen. The antigen specificity of an antigen-binding site is determined by its shape and the amino acids present.

[0064] The term "antigenic determinant" or "epitope" as used herein refers to that portion of an antigen molecule that contacts the antigen binding site of a given antibody or antigen receptor.

[0065] The term "aptamer" as used herein refers to a single-stranded synthetic oligonucleotide that folds into a three-dimensional shape that can non-covalently bind to a target molecule with high affinity and specificity. Rather than the primary sequence, the binding of an aptamer is determined by its tertiary structure. Target recognition and binding involve three-dimensional shape-dependent interactions, as well as hydrophobic interactions, base stacking, and intercalation.

[0066] The term "binding" and other grammatical forms as used herein refer to a persistent attraction between chemical entities. Binding specificity includes both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinity from low to high, and design elements may suppress undesirable interactions. Post-translational modifications may also alter the chemical nature and structure of the interaction. "Promiscuous binding" may involve a degree of structural plasticity, such that different subsets of residues may be important for binding to different partners. "Relative binding specificity" is the property of a molecule in a biochemical system that interacts differently with its targets or partners, thereby conferring different effects on them depending on the identity of the individual targets or partners.

[0067] As used herein, the term "binding agent" refers to a substance that is capable of binding to other substances, such as chemicals or antigens.

[0068] As used herein, the terms "biodegradable" and "biodegradable" refer to materials that erode into soluble species or break down under physiological conditions into smaller units or chemical species that are themselves non-toxic (biocompatible) to a subject and can be metabolized, eliminated, or excreted by the subject.

[0069] As used herein, the term "biological particle" refers to a minute part, fragment, or mass (particle) derived from an organism. Biological particles include, but are not limited to, exosomes, extracellular vesicles, viral particles, bacterial particles, or other secreted particles that consist of a surface membrane.

[0070] The term "biomarker" (or "biosignature") as used herein refers to a peptide, protein, nucleic acid, antibody, gene, metabolite, or other substance used as an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as a cellular or molecular indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention. The term "indicator" as used herein refers to any substance, number, or ratio derived from a set of observations that may reveal relative changes as a function of time, or a signal, sign, mark, note, or symptom that is visible or that is evidence of its presence or existence. Once a proposed biomarker is confirmed to be valid, it can be used to diagnose disease risk, the presence of disease in an individual, or adjust the treatment of the disease in an individual (e.g., select a drug treatment or administration regimen).

[0071] The term "cargo" as used herein refers to a cargo or something that is carried. In the context of exosomes and / or extracellular vesicles, the term cargo refers to a substance that is encapsulated in exosomes and / or extracellular vesicles. The compound or substance may be, for example, a nucleic acid (e.g., nucleotide, DNA, RNA), a polypeptide, a lipid, a protein, or a metabolite, or other substance that can be encapsulated in exosomes and / or extracellular vesicles. In the context of exosomes and / or extracellular vesicles, the term "cargo profile" as used herein refers to the measurement of the abundance of a cargo component (e.g., a nucleic acid (e.g., nucleotide, DNA, RNA), a polypeptide, a lipid, a protein, or a metabolite) that characterizes a population of exosomes and / or extracellular vesicles.

[0072] As used herein, the term "CD9" refers to a member of the tetraspanin protein family whose crystal structure exhibits an inverted cone-like molecular shape, which induces membrane curvature in crystalline lipid layers. (Umeda, R. et al. Nature Communic. (2020) 11: article 1606).

[0073] As used herein, the term "CD37" refers to a member of the tetraspanin protein family that is expressed exclusively on immune cells. (Zuidscherwoude, M. et al. Scientific Reports (2015) 5:12201).

[0074] The term "CD63" as used herein refers to a member of the tetraspanin protein family, whose C-terminal domain interacts with several subunits of the adaptor protein (AP) complex, linking the trafficking of this tetraspanin to the clathrin-dependent pathway (Andreu, Z. & Yanez-Mo, M., citing Rous, BA et al. Mol. Biol. Cell (2002) 13(3):1071-82). Among intracellular interacting proteins, CD63 has been shown to directly bind to the double PDZ domain-containing protein syntenin-1 (ibid., citing Latysheva, N. et al. Mol. Cell Biol. (2006) 26(20):7707-18). For syntenin-1, a key role in exosome biogenesis has been reported (ibid., Baietti, MF et al. Nat. Cell Biol. (2012) 14(7):677-85).

[0075] The term "CD81" as used herein refers to a member of the tetraspanin protein family that exhibits a crystal structure in which four transmembrane (TM) helices are arranged in an inverted teepee fashion, forming a central pocket in the intramembrane region and likely binding cholesterol in the central cavity. (Zimmerman, B. et al. Cell (2016) 167: 1041-51). During development, CD81 controls the trafficking of CD19, an essential costimulatory molecule for lymphocyte B cells and a well-known partner of CD81, along the secretory pathway. (Shoham, T. et al. J. Imunol. (2003) 171: 4062-72). CD9 and CD81 have been shown to control several cell-cell fusion processes. (Charrin, S. et al. J. Cell Science (2014) 127: 3641-48).

[0076] As used herein, the term "CD82" refers to a member of the tetraspanin protein family that is involved in the regulation of protein sorting into EVs and antigen presentation by antigen-presenting cells. (Andreu, Z. and Yanez-Mo, M. Front. Immunol. (2014) doi.org / 10.3389 / fimmu.2014.00442).

[0077] The term "click chemistry" as used herein refers to a chemical synthesis method to produce compounds using reagents that can be combined using efficient reagent conditions, can be carried out in a benign solvent or solvents, and can be removed or extracted by convenient methods such as evaporation, extraction, distillation, etc. Several types of reactions that meet these criteria have been identified, including nucleophilic ring-opening reactions of epoxides and aziridines, non-aldol type carbonyl reactions such as hydrazone and heterocycle formation, addition reactions to carbon-carbon multiple bonds such as oxidative formation of epoxides and Michael addition, and cycloaddition reactions. A representative example of click chemistry is the reaction of formula I below, which couples azides and alkynes to form triazoles. Copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) is 10 times faster than uncatalyzed 1,3-dipolar cycloaddition reaction.7 ~10 8 It is characterized by an overwhelming acceleration of the reaction rate by 100x. It is successful over a wide temperature range, is not affected by aqueous conditions and has a pH range of 4-12, and tolerates a wide range of functional groups. The pure product can be isolated by simple filtration or extraction without the need for chromatography or recrystallization. [ka]

[0078] A representative example of copper-free click chemistry is the attachment of dibenzocyclo-octyl (DBCO) tagged DNA molecules to azide-functionalized surfaces via a copper-free cycloaddition reaction, as shown in Scheme II.

[0079] [ka] [Eeftens, JM, et al. BMC Biophys. (2015)8:9].

[0080] As used herein, the term "clickable functional group" refers to a functional group that can be used in click chemistry to form a product. According to some embodiments, the clickable functional group is an azide or an alkyne.

[0081] As used herein, the term "conjugate" refers to a compound formed by the bonding or linking of two or more chemical compounds.

[0082] As used herein, the term "contact" and its various grammatical forms refer to a state of being in contact or in close proximity or local proximity.

[0083] As used herein, the term "convalescent serum" refers to blood serum obtained from an individual who has recovered from an infectious disease and which contains antibodies against the infectious agent that caused the disease. The terms "convalescent serum" and "convalescent plasma" can be used interchangeably.

[0084] The term "coronavirus" or "CoV" (CoVs, plural) as used herein refers to a large family of single-stranded RNA viruses that can infect a wide variety of animals, causing respiratory, enteric, hepatic and neurological diseases [Yin, Y., Wunderink, RG, Respirology (2018) 23(2):130-37, citing Weiss, SR, Leibowitz, IL, Coronavirus pathogenesis. Adv. Virus Res. (2011) 81:85-164]. Human coronaviruses were once considered relatively harmless respiratory pathogens in the past, but are now attracting worldwide attention as important pathogens in respiratory infections. CoVs, the largest known RNA viruses, are further divided into four genera: α-, β-, γ-, and δ-groups, with the β-group further composed of subgroups A, B, C, and D. [Xia, S. et al. Sci. Adv. (2019) 5:eaav4580].

[0085] CoVs are enveloped with non-segmented, positive-sense, single-stranded RNA of 26,000 to 37,000 bases, which is the largest genome among RNA viruses [Yang, Y. et al., J. Autoimmunity (2020) doi.org / 10.1016 / j.jaut.2020.102434 citing Weiss, SR et al. Microbiol. Mol. Biol. Rev. (2005) 69(4):635-64]. Viral RNA encodes structural proteins and genes interspersed among the structural genes, some of which play important roles in viral pathogenesis [Yang, Y. et al., J. Autoimmunity (2020) doi.org / 10.1016 / j.jaut.2020.102434, Zhao, L. et al. Cell Host Microbe (2012) 11(6):607-16, citing Fehr, AR, Perlman, S. Methods Mol. Biol. (2015) 1282:1-23]. The spike protein (S), which is responsible for receptor binding and subsequent viral entry into the host cell, is composed of S1 and S2 subunits. The membrane protein (M) and the envelope protein (E) play important roles in virus assembly; the E protein is required for virulence [DeDiego,ML,et al.J.Virol.(2007)81(4):1701-13 cited above, Nieto-Torres,JL et al.PLoS Pathog.(2014)10(5):e1004077]. The nucleocapsid (N) protein has two domains, both of which can bind to the viral RNA genome by different mechanisms and are required for RNA synthesis and packaging of the encapsidated genome into virions.[Citing Fehr, AR, Perlman, S. Methods Mol. Biol. (2015) 1282:1-23 Yang, Y. et al., J. Autoimmunity (2020) doi.org / 10.1016 / j.jaut.2020.102434., Song, Z. et al. al.Viruses(2019)11(1):59, Chang,CK et al.J.Biomed.Sci.(2006)13(1):59-72, Hurst,KR,et al.,J.Virol.(2009)83(14):7221-34]. The N protein is also an interferon antagonist and a virally encoded suppressor of RNA interference (RNAi) (VSR), benefiting viral replication [references cited above: Cui, L. et al. J. Virol. (2015) 89(17):9029-43].

[0086] By December 2019, six coronaviruses had been identified as infecting humans and causing disease. Among them, infections caused by H-CoV-229E and HCoV-NL63 of the α group, and HCoV-OC43 and HCoV-HKU1 of the β subgroup A are often mild, mostly causing cold-like symptoms [Xu, X. et al. J. Nuclear Medicine & Molec. Imaging (2020) doi.org / 10.1007 / s00259-020-04735-9 citing Su, S. et al. Trends Microbiol. (2016) 24: 490-502]. The other two, severe acute respiratory syndrome coronavirus (SARS-CoV) of beta subgroup B and Middle East respiratory syndrome coronavirus (MERS-CoV) of beta subgroup C, have different pathogenicity and cause fatal diseases [Ibid., citing Cui, J. et al. Nat. Rev. Microbiol. (2019) 17:181-92]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the seventh member of coronaviruses that infect humans [Zhu, N. et al. N. Engl. J. Med. (2020) 382:727-33]. CoV entry into host cells depends on binding of the viral spike (S) protein to a cellular receptor and priming of the S protein by host cell proteases, which involves cleavage of the S protein at the S1 / S2 and S2' sites, resulting in fusion of the viral and cellular membranes, a process driven by the S2 subunit. [Hoffmann, M. et al. Cell (2020) 181 (2): 271-80]. Angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4) are known as host receptors for SARS-CoV and MERS-CoV, respectively [Yang, Y. et al., J. Autoimmunity (2020) doi.org / 10.1016 / j.jaut.2020.102434 citing Kuhn, JH, et al. Cell Mol. Life Sci. (2004) 61 (21): 2738-43; Raj, VS, et al. Nature (2013) 495 (7440): 251-54].SARSCoV-2 uses the SARS-CoV receptor ACE2 for entry into host cells and the serine protease TMPRSS2 for priming the S protein. [Hoffman,M.et al.Cell(2020)181(2):271-80]. One mechanism by which SARS-CoV-2 enters is that the spike protein on the surface of SARS-CoV-2 binds to the ACE2 receptor and is cleaved at two cleavage sites ("priming"), causing a conformational change that allows viral-host membrane fusion. [Shrimp,JH et al.ACS Pharmacol.Trans.Sci.(2020)3(5):997-1007].

[0087] As used herein, the term "derived from" refers to any method for receiving, obtaining, or modifying something from an origin.

[0088] As used herein, the term "DNA library" refers to a collection of DNA fragments that have been cloned into vectors so that a researcher can identify and isolate DNA fragments of interest for further study.

[0089] The term "domain" as used herein refers to either a region of a protein having a characteristic tertiary structure and function, and to a three-dimensional subunit of a protein that constitutes the tertiary structure formed by folding of a linear peptide chain.

[0090] The term "encapsulated" as used herein refers to being enclosed in a capsule (meaning a membranous envelope that surrounds a portion).

[0091] As used herein, the term "endosomal sorting complexes required for transport" (ESCRT) refers to components involved in the biogenesis of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs). ESCRTs are composed of about 20 proteins that assemble into four complexes (ESCRT-0, -I, -II, -III) and associated proteins (VPS4, VTA1, ALIX) that are conserved from yeast to mammals (Colombo, M. et al. J. Cell Science (2013) 126:5553-65, citing Henne, WM, et al. (2011). Dev. Cell 21, 77-91; Henne et al., 2011; Roxrud, I. et al. (2010). ESCRT & Co. Biol. Cell 102, 293-318). The ESCRT-0 complex recognizes and sequesters ubiquitinated proteins in the endosomal membrane. On the other hand, ESCRT-I and -II complexes appear to be responsible for membrane deformation into buds with sequestered cargo, while ESCRT-III components subsequently promote vesicle detachment (Ibid., Hurley, JHand Hanson, PI (2010). Nat. Rev. Mol. Cell Biol. 11, 556-566; citing Wollert, T. et al. Nature (2009) 458: 172-77). ESCRT-0 consists of the hepatocyte growth factor-regulated tyrosine kinase dubstrat (HRS) protein, which recognizes monoubiquitinated cargo proteins and forms a complex with signaling adaptor molecule (STAM), Eps15, and clathrin. HRS recruits TSG101 of the ESCRT-I complex, while ESCRT-I is involved in the recruitment of ESCRT-II or ESCRT-III through the ESCRT accessory protein ALIX. Finally, dissociation and recycling of the ESCRT machinery requires interaction with the ATPase associated with various cellular activities (AAA-ATPase) Vps4, which releases ESCRT-III from the MVB membrane for further selection events.It is unclear whether ESCRT-II has a direct role in ILV biogenesis or whether its function is restricted to specific cargoes (Bowers, K. et al. (2006) J. Biol. Chem. 281, 5094-5105; above citing Malerod, L. et al. Traffic 8, 1617-1629).

[0092] Simultaneous depletion of ESCRT subunits belonging to the four ESCRT complexes does not completely impair the formation of MVBs, indicating that other mechanisms may be at work in the formation of ILVs and thus exosomes and / or extracellular vesicles (see above, Stuffers, S. et al. (2009) Traffic 10, 925-937). One of these pathways requires type II sphingomyelinase, which hydrolyzes sphingomyelin to ceramide (see above, Trajkovic, K. et al. (2008) Science 319, 1244-1247). Although depletion of different ESCRT components does not lead to a clear reduction in MVB formation or secretion of exosome- and / or extracellular vesicle-associated proteolipid protein (PLP), silencing neutral sphingomyelinase expression by siRNA or inhibiting its activity by the drug GW4869 reduces exosome and / or extracellular vesicle formation and release. However, it remains to be determined whether such a dependency on ceramide can be generalized to other cell types and additional cargoes that produce exosomes or extracellular vesicles. Depletion of type II sphingomyelinase in melanoma cells does not impair MVB biogenesis (reference cited above in van Niel, G. et al. (2011) Dev. Cell 21, 708-721), nor does it inhibit exosome and / or extracellular vesicle secretion, but in these cells the tetraspanin CD63 is required for ESCRT-independent sorting of the luminal domain of the melanosomal protein PMEL (van Niel et al., (2011) Dev. Cell 21, 708-721). Furthermore, it has been proposed that tetraspanin-rich domains function as sorting machinery enabling the formation of exosomes and extracellular vesicles (Perez-Hernandez, D. et al. J. Biol. Chem. (2013) 288, 11649-11661).

[0093] Despite evidence for ESCRT-independent mechanisms of exosome and / or extracellular vesicle formation, proteomic analysis of purified exosomes and / or extracellular vesicles from various cell types has identified ESCRT components (TSG101, ALIX) and ubiquitinated proteins (ibid., Buschow, S. et al., (2005) Blood Cells Mol. Dis. 35, 398-403; Thery, C. et al., (2006). Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22). It has also been reported that HRS, an ESCRT-0 component, is required for the formation and / or secretion of exosomes and / or extracellular vesicles by dendritic cells (DCs), which may affect their antigen-presenting ability (ibid., citing Tamai, K. et al., (2010) Biochem. Biophys. Res. Commun. 399, 384-390). The transferrin receptor (TfR) of reticulocytes, which are generally destined for exosome and / or extracellular vesicle secretion, is not ubiquitinated but interacts with ALIX for MVB sorting (ibid., citing Geminard, C. et al., (2004). Traffic 5, 181-193). It has also been shown that ALIX is involved in the biogenesis of exosomes and / or extracellular vesicles and exosomal sorting of syndecans (a major family of cell surface heparan sulfate proteoglycans (HSPGs) composed of sulfated glycosaminoglycans (GAGs), heparan sulfate (HS) or both HS and chondroitin sulfate (CS)) covalently linked to a core protein - Park, PW. Methods Cell Biol. (2018) 143: 317-33), through its interaction with syntenin (Colombo, M. et al. J. Cell Science (2013) 126: 5553-65 citing Baietti, MF et al., (2012). Nat. Cell Biol. 14, 677-685). Silencing genes for two components of ESCRT-0 (HRS, STAM1), one of ESCRT-I (TSG101), and a slow-acting component (VPS4B) induced consistent changes in exosome and / or extracellular vesicle secretion.[Colombo, M. et al. J. Cell Sci. (2013) 126:5553-65].

[0094] The term "exosomes and / or extracellular vesicles" as used herein refers to extracellular bilayer-bound vesicles of endosomal origin in the size range of approximately 40-160 nm in diameter (average ∼100 nm) that are produced by all cells and are actively secreted.

[0095] Biogenesis: Exosomes and / or extracellular vesicles are generated in a process that involves a double invasion of the plasma membrane and the formation of intracellular multivesicular bodies (MVBs) that contain intracellular vesicles (ILVs). The ILVs are eventually secreted as exosomes and / or extracellular vesicles with diameters of 40-160 nm by fusion of the MVBs to the plasma membrane and exocytosis. The initial invasion of the plasma membrane forms a cup-like structure that contains cell surface proteins and soluble proteins associated with the extracellular environment. This leads to the de novo formation of early sorting endosomes (ESEs), which in some cases may directly bind to pre-existing ESEs. The trans-Golgi network and endoplasmic reticulum may also contribute to the formation and content of ESEs (Kalluri, R., LeBleu, VS. Science (2020) 367(6478):eaau6977 citing Kalluri, RJ Clin. Invest. (2016) 126:1208-15; van Neil, G. et al. Nat. Rev. Mol. Cell Biol. (2018) 19:213-28; McAndrews, KM, Kalluri, R. Mol. Cancer (2019) 18:52; Mathieu, M. et al. Nat. Cell Biol. (2019) 21:9-17; Willms, E. et al. Front. Immunol. (2018) 9:738; Hessvik, NP, Llorente, A. Cell Mol Life Sci. (2018) 75: 193-208). ESEs mature into late sorting endosomes (LSEs) and eventually generate MVBs, also called multivesicular endosomes. MVBs are formed by invading the endosome limiting membrane (i.e., double invasion of the plasma membrane). During this process, MVBs are formed that contain multiple ILVs (future exosomes and / or extracellular vesicles). MVBs can fuse with lysosomes or autophagosomes for degradation, or fuse with the plasma membrane to release the contained ILVs as exosomes and / or extracellular vesicles [references cited above citing Kahler, C., Kalluri, RJ Mol. Med. (2013) 91: 431037].

[0096] Heterogeneity: The heterogeneity of extracellular vesicles is thought to reflect their particle size, contents, functional effects on recipient cells, and cellular origin. They acquire surface proteins from their cell of origin during secretion. They naturally transport mRNA, miRNA, and proteins between cells.

[0097] Biomarkers: There is general agreement that the membrane is specifically enriched for the tetraspanins CD9, CD37, CD63, CD81, and CD82.

[0098] Role: Extracellular vesicles are mediators of short- and long-range intercellular communication in health and disease, influencing multiple aspects of cell biology.

[0099] The term "extracellular vesicles (EVs)" as used herein refers to nano-sized membrane-bound vesicles released from cells that can transport cargo, including DNA, RNA and proteins, between cells as a form of intercellular communication. Various EV types, including microvesicles (MVs), exosomes, oncosomes and apoptotic bodies, have been characterized based on their biogenesis or release pathways. Microvesicles originate directly from the plasma membrane, are 100 nanometers (nm) to 1 micrometer (μm) in size, and contain cytoplasmic cargo (Zaborowski, MP et al. BioScience (2015) 65(8):783-97, citing Heijnen, HF et al. Blood (1999) 94:3791-99). Exosomes, another EV subtype, are formed by fusion between multivesicular bodies and the plasma membrane, which release smaller vesicles (exosomes) with diameters in the range of 40-160 nm (El Andaloussi, S. et al. Nature Reviews Drug Discovery (2013) 12:347-57; Cocucci, E. and Meldolesi J. Trends in Cell Biology (2015) 25:364-72 cited above). Dying cells release vesicular apoptotic bodies (50 nm-2 μm) that may be more abundant than exosomes or MVs under certain conditions and may differ in content between biological fluids (Thery, C. et al. J. Immunology (2001) 1666:7309-18; El Andaloussi, S. et al. Nature Reviews Drug Discovery (2013) 12:347-57 cited above). Membrane protrusions can also generate larger EVs called oncosomes (1-10 μm), which are mainly produced by malignant cells that are distinct from their non-transformed counterparts (Di Vizio, D. et al. Am. J. Pathol. (2012) 181:1573-84; Morello, M. et al. Cell Cycle (2013) 12:3526-36 cited above).

[0100] The term "inhibitor" as used herein refers to a molecule that reduces the amount or rate of a process, stops a process completely, or reduces, limits or prevents its action or function. An enzyme inhibitor is a molecule that binds to an enzyme, thereby reducing the enzyme's activity. Inhibitors can be evaluated by their specificity and potency. Inhibition can include a decrease or reduction of the amount, rate, action function or process of a substance by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99%.

[0101] The term "isolated" is used herein to refer to (1) a material, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, that is substantially or essentially free of components that are normally associated or interacting with it in its natural environment. The terms "substantially free" or "essentially free" are used herein to refer to more than about 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 100% free. An isolated material optionally contains materials that are not found with the material in its natural environment; or (2) if the material is in its natural environment, the material is synthetically (non-naturally) altered into a composition by deliberate human intervention and / or is placed in a location in a cell (e.g., the genome, or an organelle within a cell) that is not naturally present in the material found in that environment. Modifications to obtain synthetic materials can be performed on materials in their natural state or removed from them.

[0102] The term "Fab fragment" as used herein refers to an antibody fragment produced by cleavage of IgG with the enzyme papain, lacking the Fc region and consisting of a single antigen-binding arm of the antibody. It contains a complete light chain and the amino-terminal variable region and first constant region of the heavy chain linked by an interchain disulfide bond.

[0103] As used herein, "F(ab') 2 The term fragment refers to an antibody fragment produced by cleavage of IgG with pepsin, lacking the Fc region and consisting of two joined antigen-binding arms (Fab fragments).

[0104] The term "fragment" or "peptide fragment" as used herein refers to a small portion derived from, cleaved or truncated from a larger peptide, polypeptide or protein, which retains the desired biological activity of the larger peptide, polypeptide or protein. Antibody binding fragments (e.g., Fab, Fab', F(ab') 2 Antibodies that are fragments of human antibodies (e.g., Fv and single-chain (sc) antibodies) can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0105] The term "heterogeneous" as used herein refers to being composed of unrelated or dissimilar elements or parts; varied; made of a variety of different kinds; differing or totally different in structure, quality, etc.

[0106] As used herein, the term "high throughput screening" or "HTS" refers to the use of automated equipment to rapidly test thousands to millions of samples for biological activity at the level of model organisms, cells, pathways, or molecules.

[0107] As used herein, the term "homogeneous" refers to having the same characteristics, structure, quality, etc.; being essentially similar; having the same properties; being composed of similar or identical elements or parts; being uniform.

[0108] As used herein, the terms "immune response" and "immune-mediated" are used interchangeably to refer to any functional manifestation of a subject's immune system against either foreign or self antigens, regardless of whether the outcome of these responses is beneficial or harmful to the subject.

[0109] As used herein, the term "immunomodulatory" refers to a substance or agent that can directly or indirectly increase or decrease the immune response, for example, by inducing the expression of chemokines, cytokines, and other mediators of the immune response.

[0110] As used herein, the term "inhalation" refers to the act of breathing in the medicated vapor.

[0111] The term "inhalation delivery device" as used herein refers to a device / machine or component used to generate small droplets or aerosols from liquid or dry powder aerosol formulations, for example for oral administration to achieve intrapulmonary administration of a drug contained in a liquid, suspension, powder, etc.

[0112] As used herein, the term "couple" means to join, connect, or join one thing to another. Each of these terms is used interchangeably.

[0113] The term "long non-coding RNA" ("lncRNA") as used herein refers to a class of transcribed RNA molecules that are longer than 200 nucleotides but do not code for proteins. LncRNAs can fold into complex structures and interact with proteins, DNA, and other RNAs to regulate activity, DNA targets or partners in multiprotein complexes. Crosstalk between lncRNAs and miRNAs creates a complex network that exerts post-transcriptional regulation of gene expression. For example, lncRNAs can harbor miRNA binding sites and act as molecular decoys or sponges that sequester miRNAs from other transcripts. Competition between lncRNAs and miRNAs for binding to target mRNAs has been reported, which leads to derepression of gene expression (Zampetaki, A. et al. Front. Physiol. (2018) doi.org / 10.3389 / fphys.2018.01201, citing Yoon, JH et al. Semin. Cell Dev. Bio. (2014) 34:9-14; Ballantyne, MD et al. Clin. Pharmacol. Ther. (2016) 99:494-501). Finally, lncRNAs contain embedded miRNA sequences and serve as a source of miRNAs (Piccoli, MT et al. Cir. Res. (2017) 121:575-83, supra).

[0114] As used herein, the term "messenger RNA" ("mRNA") refers to coding RNA that functions in protein translation.

[0115] As used herein, the term "microRNA" (or "miRNA") refers to a class of small, 18-28 nucleotide long, non-coding RNA molecules whose primary role is in the post-transcriptional regulation of protein expression.

[0116] As used herein, the term "neutralization," and other grammatical forms thereof, is used to refer to the inhibition of viral infectivity, for example, by inhibiting viral particles from binding to and entering a host cell, thereby effectively inhibiting the viral replication cycle. As used herein, the term "neutralization titer" refers to the minimum amount or concentration of a substance from a biological sample required to cause neutralization.

[0117] As used herein, the terms "next generation sequencing", "NGS", "massively parallel sequencing" or "deep sequencing" refer to high-throughput methods used to determine the nucleotide sequence of an individual's entire genome at once in an automated process. First, a DNA library is prepared from a patient sample by fragmentation, purification, and amplification of the DNA sample. Individual fragments are then physically isolated by binding to a solid surface or small beads. The sequence of each of these fragments is elucidated simultaneously by techniques such as sequencing by synthesis. The resulting sequence data is aligned by computer to a "standard reference" genome.

[0118] The term "non-coding RNA" ("ncRNA") as used herein refers to functional RNA molecules that are transcribed from DNA but are not translated into proteins. They are classified into housekeeping and regulatory non-coding RNAs. Housekeeping ncRNAs include ribosomal RNAs (rRNAs, which are the RNA components of ribosomes), transfer RNAs (tRNAs, which function as adapters to match amino acids to mRNAs), small nuclear RNAs (snRNAs, which function in RNA processing, such as mRNA splicing), and small nucleolar RNAs (snoRNAs, which function in inducing chemical modifications of other RNAs). Regulatory non-coding RNAs are divided into short ncRNAs (less than 200nt) and long ncRNAs (greater than 200nt). Short non-coding RNAs less than 200nt include microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-binding RNAs (piRNAs), as well as long non-coding RNAs (greater than 200nt). [Losko, M. et al. Mediators of Inflammation (2016)1-12.10.1155 / 2016 / 5365209].

[0119] The term "nucleic acid" as used herein refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and, unless otherwise limited, includes known analogues which have the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

[0120] As used herein, the term "nucleotide" refers to a molecule consisting of a nitrogenous base (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), a phosphate group, and a sugar (deoxyribose in DNA; ribose in RNA).

[0121] As used herein, the term "particle" refers to extremely small entities, such as nanoparticles.

[0122] The term "PDZ domain" as used herein refers to an abundant protein interaction motif that often recognizes a short amino acid motif on the C-terminal side of target proteins. They have been implicated in regulating multiple biological processes, such as trafficking, ion channel signaling, and other signaling systems. (See Lee, HJ & Zheng, JJ. Cell Communication & Signaling (2010) 8:article 8).

[0123] The term "peptidomimetic" as used herein refers to small protein-like chains or non-peptidic structural elements that can mimic (meaning mimic) or antagonize (meaning neutralize or counteract) the biological action(s) of a natural parent peptide.

[0124] The term "placental alkaline phosphatase" or "PLAP" refers to an enzyme normally produced by primordial germ cells and syncytiotrophoblasts, and detection of its expression is diagnostic of germ cell tumors. PLAP immunoreactivity is found in normal human adult and fetal muscle tissue. This immunoreactivity appears to be related to the degree of myogenic differentiation in soft tissue tumors, being more frequently expressed in cells with skeletal muscle differentiation and least frequently expressed in cells with myofibroblastic characteristics. (Goldsmith, JD, et al. Am J. Surgical Pathol (2002) 26(12):1627-33).

[0125] As used herein, the term "plasma" refers to the fluid (non-cellular) portion of circulating blood and the fluid portion of lymph.

[0126] The term "polymer" refers to a large molecule or macromolecule composed of many repeating small units. The term "monomer" refers to a molecule that can be chemically bonded to other molecules to form polymers. As used herein, the term "copolymer" refers to a polymer derived from more than one type of monomer.

[0127] The terms "polypeptide" and "protein" are used herein in their broadest sense to refer to sequences of small amino acids, amino acid analogs, or peptidomimetics. The small units are linked by peptide bonds, unless otherwise noted. These terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid. The terms also encompass modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. As is well known and as noted above, it will be understood that polypeptides may not be entirely linear. For example, a polypeptide may be branched as a result of ubiquitination, or it may be cyclic, with or without branching, generally as a result of post-translational events, whether by natural processing or by events brought about by human manipulation that do not occur in nature. Cyclic, branched, and branched cyclic polypeptides may be synthesized by entirely synthetic methods.

[0128] The term "purified" as used herein and its various grammatical forms refer to a process of separating or freeing from foreign, extraneous, or objectionable elements. A composition is substantially pure in that it is nevertheless substantially separated from substances that may be associated with it during biological systems or synthesis. As used herein, the term "substantially pure" refers to a purity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as determined by analytical protocols. Such protocols include, but are not limited to, for example, flow cytometry, electrophoresis, small RNA sequencing, quantitative PCR, nanoparticle tracking, electron microscopy, mass spectrometry, Western blotting, ELISA, and various metabolic assays.

[0129] The term "quantitative PCR" (or "qPCR"), also called "real-time PCR" or "quantitative real-time PCR", refers to a polymerase chain reaction-based technique that couples the amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction.

[0130] The term "receptor binding domain" or "RBD" as used herein refers to a domain on a viral protein that binds the viral protein to a host cell receptor, thereby gaining entry and infection into the host cell. The RBD of SARS-CoV-2 corresponds to residues 331-524 of the S protein. [Tai, W. et al. Cellular & Molecular Immunol. (2020) 17: 613-20].

[0131] The renin-angiotensin system (RAS) is a central regulator of renal and cardiovascular functions. Classically, it consists of angiotensin-converting enzyme (ACE), its product, angiotensin (Ang) II, and the receptors of Ang II, angiotensin type 1 (AT1) and angiotensin type 2 (AT2) receptors. The RAS further contains a monocarboxypeptidase, ACE2, which generates Ang-(1-7) from Ang II. Angiotensin-(1–7) is an endogenous ligand for the G protein-coupled receptor Mas, and thus Mas mediates the biological actions of Ang-(1–7) [Singh, N. et al. Am J. Physiol. Heart Circ. Hysiol. (2015) 309(10):H1697-H1707, citing Santos, RA et al. Proc. Nat. Acad. Sci. USA (2003) 100:8258-63]. Ang II produces hypertensive, pro-oxidative, hypertrophic and pro-fibrotic effects in the cardiovascular system. Ang-(1-7) induces a counterregulatory effect on the ACE / AngII pathway by reducing its vasodilatory, antihypertensive, antihypertrophic, antifibrotic and antithrombotic effects [Ferreira, AJ, et al. Hypertension (2010) 55:207-13; Jusuf, D. et al. Eur. J. Pharmacol. (2008) 585:303-12 cited above].

[0132] The term "restriction enzyme" as used herein refers to an enzyme produced by bacteria that recognizes a short sequence in a nucleic acid molecule and cleaves the nucleic acid molecule at or near that sequence, resulting in a double-stranded or single-stranded break. The term "restriction enzyme recognition site" or "restriction site" as used herein is a short, specific sequence of DNA base pairs recognized by each restriction enzyme.

[0133] As used herein, the term "serum" refers to the fluid portion of blood obtained after removal of the fibrin clot and blood cells.

[0134] As used herein, the term "static system" refers to a system without feedback, where the output at any time depends only on the input at that time. It is distinguished from a "dynamic system", where the output at any time depends on the past and future values ​​of the data.

[0135] The term "strand displacement" as used herein refers to a reaction in DNA homologous recombination and DNA mismatch repair. It is a reaction in which one of the strands in a double-stranded DNA is replaced by another nearly identical strand. DNA strand displacement involves three single strands, named the "invading", "existing" and "substrate" strands. Basically, it is an exchange reaction between the invading strand and the existing strand on the substrate strand. A double-stranded nucleic acid molecule is partially or completely separated into two separate single-stranded nucleic acid strands by an enzyme with strand displacement activity (e.g., relaxation activity), such as a topoisomerase or helicase, and an invading strand that is complementary to the existing strand in the double-stranded nucleic acid. The enzyme relaxes the double-stranded nucleic acid to allow the complementary invading strand to hybridize to one of the separated strands of the double-stranded nucleic acid, thereby preventing the existing double-stranded nucleic acid strand and its matching substrate strand from reannealing to form the original double-stranded DNA molecule. The displaced pre-existing single-stranded DNA then undergoes branch migration until the pre-existing strand is completely replaced by the invading strand. (Broadwater, Jr., DWB, Kim, HD, Biophys. J. (2016) 110(7):1476-84). The described and claimed assay involves DNA displacement, where a small oligonucleotide hybridizes and overhangs the end of the double strand of the double stranded DNA in the DNA strand that is bound to the antibody, and a DNA polymerase reaction separates the initial DNA strands (the strand linked to the antibody and the strand linked to the scaffold via biotin), so that a unique antibody with a unique DNA strand can be specifically pulled apart from a group of different antibodies targeting different surface markers of exosomes.

[0136] The term "tetraspanin" as used herein refers to membrane-spanning proteins with a conserved structure that primarily functions as a membrane protein organizer. Members of the tetraspanin family of proteins have four transmembrane domains that contribute to create a small (EC1) and a large (EC2) extracellular loop [Termini, CM, Gillette, JM, Front. Cell Dev. Biol. (2017) 5:34, citing Abe, M. et al. Cancer Lett. (2008) 266:163-70]. The large extracellular loop contains a conserved Cys-Cys-Gly amino acid motif (CCG motif), as well as two other conserved cysteine ​​residues. Many members of the tetraspanin family also contain post-translational modifications.

[0137] The term "therapeutic agent" as used herein refers to a drug, molecule, nucleic acid, protein, metabolite, cell, composition or other substance that provides a therapeutic effect. The term "active" as used herein refers to the ingredient, component or constituent of the described composition of the invention that is responsible for the intended therapeutic effect. The terms "therapeutic agent" and "active agent" are used interchangeably herein.

[0138] The term "therapeutic component" as used herein refers to a therapeutically effective dosage (i.e., dose and frequency of administration) that eliminates, reduces or prevents the progression of a particular disease manifestation in a certain percentage of the population. One example of a commonly used therapeutic component is the ED50, which represents the dose at a particular dosage that is therapeutically effective against a particular disease manifestation in 50% of the population.

[0139] The terms "therapeutic amount", "effective amount" or "therapeutically effective amount" of an active agent are used interchangeably to refer to an amount sufficient to provide the intended therapeutic benefit. However, dosage levels are based on a variety of factors, including the age, weight, sex, medical condition, severity of the condition, route of administration, and the specific active agent employed. Thus, dosing regimens can vary widely, but can be routinely determined by a physician using standard methods. In addition, the terms "therapeutic amount" and "therapeutically effective amount" include prophylactic or preventative amounts of the compositions of the invention described. In the preventative or preventative uses of the invention described, the pharmaceutical composition or medicament is administered to a patient susceptible to or at risk of a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of a disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes that appear during the progression of the disease, disorder, or condition. It is generally preferred to use a maximum dose, i.e., the highest safe dose according to some medical judgment. The terms "dose" and "dosage" are used interchangeably herein.

[0140] The term "therapeutic effect" as used herein refers to the outcome of a treatment, which outcome is deemed desirable and beneficial. Therapeutic effect may include, directly or indirectly, the prevention, reduction or elimination of disease symptoms. Therapeutic effect may also include, directly or indirectly, the prevention, reduction or elimination of the progression of disease symptoms.

[0141] For any therapeutic agent described herein, the therapeutically effective amount can be initially pre-determined in in vitro tests and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. It is within the ability of a person skilled in the art to adjust the dose to provide maximum efficacy based on the above and other well-known methods.

[0142] General principles for determining therapeutic efficacy can be found in Chapter 1 of The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001) by Goodman and Gilman, incorporated herein by reference, and are summarized below.

[0143] Pharmacokinetic principles provide a basis for modifying dosing regimens to obtain the desired degree of therapeutic efficacy while minimizing unacceptable adverse effects. In situations where the plasma concentration of a drug can be measured and related to a therapeutic window, further guidance for dosage modifications can be obtained.

[0144] In most clinical settings, drugs are administered in a series of repeated doses or as continuous infusions to maintain a steady-state concentration of the drug in relation to the therapeutic window. To maintain a selected steady-state or target concentration ("maintenance dose"), the rate of drug administration is adjusted so that the rate of input is equal to the rate of loss. If the clinician selects the desired concentration of the drug in the plasma and knows the clearance and bioavailability of that drug in a particular patient, the appropriate dose and dosing interval can be calculated. However, live cell therapy breaks this concept because it divides and, in the case of autologous cell therapy, may even acquire a permanent presence in the body. Thus, what is initially administered may have little correlation over time with what is present in the recipient.

[0145] As used herein, the term "TCID50" or "50% tissue culture infectious dose" refers to the concentration at which 50% of cells become infected when a dilute solution of the virus fluid is plated on a surface on which the cells are cultured.

[0146] As used herein, the term "T cell immunoglobulin mucin domain-containing 4" or "Tim-4" refers to a phosphatidylserine receptor that is selectively expressed on antigen-presenting cells but not on T cells and may regulate T cell proliferation. (Liu, W. et al. Front. Immunol. (2020) doi.org / 10.3389 / fimmu.2020.00537).

[0147] The term "titer" as used herein is a measure of the concentration and strength of a substance in a biological sample, such as the concentration of antibodies in blood. Titer is estimated by the highest dilution that still allows a detectable effect. For example, if 1:100 gives a positive test result and higher dilutions are negative, the titer is 100.

[0148] As used herein, the terms "treat", "treated" or "treating" refer to both therapeutic procedures and / or prophylactic or preventative measures aimed at preventing or slowing down an undesirable physiological condition, disorder or disease, or obtaining a beneficial or desired clinical outcome. For purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delay in onset or slowing of progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and detectable or undetectable (partial or complete) remission or improvement or amelioration of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without unacceptable side effects. Treatment also includes prolonging survival compared to expected survival in the absence of treatment.

[0149] As used herein, the term "viral load" refers to a measurement of the amount of virus, usually expressed in virus particles per milliliter, in an organism, typically in the bloodstream.

[0150] method According to one aspect, the present disclosure provides an in vitro method for assessing a subject's risk of developing and treating a severe viral infection, the method comprising:

[0151] a) preparing a population of purified biological particles comprising a cargo, said preparing comprising:

[0152] (1) obtaining a biological sample containing biological particles from a subject;

[0153] (2) contacting a biological sample containing biological particles from a subject with a binding agent for one or more surface antigens of the biological particles, whereby the binding agent binds to the nucleic acid and the nucleic acid is immobilized on the solid support;

[0154] b) isolating the biological particles bound by the binding agent from the biological sample; and

[0155] c) releasing the biological particles bound to the binding agent; and

[0156] (d) eluting the bound biological particles from the binding agent to form a population of free purified biological particles;

[0157] (e) assessing the cargo of the population of purified biological particles; and

[0158] (f) determining a patient's risk of severe viral infection based on the cargo profile of the population of purified biological particles; and

[0159] (g) providing appropriate treatment to patients at risk of severe viral infections.

[0160] According to some embodiments, the cargo comprises one or more of encapsulated miRNA and / or small non-coding RNA, encapsulated polypeptide, encapsulated DNA, surface polypeptide, or surface lipid. According to some embodiments, evaluating the cargo comprises identifying and quantifying the cargo, for example, by measuring expression of one or more miRNA or small non-coding RNA encapsulated by the purified biological particle, polypeptide encapsulated by the purified biological particle, polypeptide at the surface of the purified biological particle, DNA encapsulated by the purified biological particle, and lipid of the purified biological particle. According to some embodiments, evaluating the cargo comprises determining a cargo profile of the purified biological particle and comparing the cargo profile of the purified biological particle from the subject with cargo profiles from control subjects: (1) individuals who are not infected with coronavirus, (2) individuals who are infected with coronavirus and have mild symptoms, and (3) individuals who are infected with coronavirus and have severe symptoms. According to some embodiments, evaluating the cargo comprises:

[0161] (i) extracting RNA from a population of purified biological particles;

[0162] (ii) identifying and quantifying the expression of one or more small non-coding RNAs, including microRNAs (miRNAs), encapsulated by the population of purified biological particles; and

[0163] (iii) comparing the expression of the identified and quantified small non-coding RNAs, including miRNAs, to the identified and quantified small non-coding RNAs, including miRNAs, from control subjects: i) individuals not infected with the virus, ii) individuals infected with the virus (confirmed by PCR) and with mild disease (meaning they were hospitalized but did not require mechanical ventilation), and iii) individuals infected with the virus (confirmed by PCR) and with severe disease (meaning they showed acute respiratory distress syndrome (ARDS, according to the Berlin classification criteria) and required mechanical ventilation).

[0164] According to some embodiments, the cargo profile of the purified biological particles includes identified and quantified small non-coding RNAs, including miRNAs, from the purified biological particles. According to some embodiments, the cargo profile from the purified biological particles from the subject is compared to cargo profiles from control subjects: i) those who are not infected with the virus, ii) those who are infected with the virus (confirmed by PCR) and have mild disease (meaning they are hospitalized but do not require mechanical ventilation), and iii) those who are infected with the virus (confirmed by PCR) and have severe disease (meaning they exhibit acute respiratory distress syndrome (ARDS, according to the Berlin classification criteria) and require mechanical ventilation).

[0165] According to some embodiments, assessing the cargo of the population of purified biological particles to determine the cargo profile of the purified biological particles is accomplished by (i) extracting RNA from the population of purified biological particles, (ii) identifying and quantifying the expression of small non-coding RNAs, including one or more microRNAs (miRNAs), encapsulated by the population of purified exosomes, and (iii) comparing the cargo profile of the purified biological particles to cargo profiles from control subjects: (1) individuals who are not infected with coronavirus, (2) individuals who are infected with coronavirus and have mild symptoms, and (3) individuals who are infected with coronavirus and have severe symptoms.

[0166] According to some embodiments, the method includes an initial ultrafiltration step, ultracentrifugation step, or both, to provide a pooled heterogeneous population of biological particles.

[0167] According to some embodiments, the severe viral infection is a coronavirus infection. According to some embodiments, the coronavirus infection is a SARS-CoV-1 infection. According to some embodiments, the coronavirus infection is a MERSCoV infection. According to some embodiments, the coronavirus infection is a SARS-CoV-2 viral infection.

[0168] According to some embodiments, the biological sample comprises a bodily fluid, including, but not limited to, blood, urine, sperm (semen), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal discharge, otorrhea, gastric fluid, breast milk, feces, sweat, tears, nipple aspirate, or other secreted bodily fluids. According to some embodiments, the bodily fluid is a circulating / secreted bodily fluid, such as blood (whole blood, serum, or plasma), CSF, or lymph. According to some embodiments, the biological sample comprises a bodily tissue (e.g., diseased tissue).

[0169] According to some embodiments, the method is a high-throughput assay, which allows purification of multiple samples at once, i.e., compatible with 24, 96, 384 well plate formats or other multi-wall formats. According to some embodiments, the biofluid from which biological particles containing a particular biomarker(s) are selected can be reused, e.g., in subsequent rounds to select other biomarkers, select small non-coding RNAs, including miRNAs, isolate RNA, DNA, proteins, lipids, or combinations thereof, etc., without adversely affecting the biofluid.

[0170] According to some embodiments, the binding agent comprises a nucleic acid (e.g., DNA, RNA, or a combination thereof), a polypeptide (e.g., an antibody, an antibody fragment, an aptamer polypeptide, or a combination thereof), or a biodegradable polymer.

[0171] According to some embodiments, the binding agent is directly immobilized to the solid support. According to some embodiments, the binding agent is indirectly immobilized to the solid support. In some such embodiments, the binding agent is conjugated to the nucleic acid and immobilized to the solid support. According to some embodiments, the nucleic acid is immobilized to the solid support by click chemistry.

[0172] According to some embodiments, the binding agent is an antibody specific for the biological particle antigen. According to some embodiments, the biological particle antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers inherited by the biological particle. In this way, the purification process can be tailored to select specific populations of biological particles and scan their contents as needed, such as those infected with viruses, those from cancer cells, those from organs, etc. According to some embodiments, the method comprises an additional cell-specific antibody to enrich a subpopulation of biological particles for detecting low abundance biomarkers. According to some embodiments, the method comprises a larger input of biofluid material than those tested herein. According to some embodiments, the method comprises a preparatory step to improve the qPCR detection threshold. According to some embodiments, the method comprises an additional cell-specific antibody to enrich a subpopulation of biological particles for detecting low abundance biomarkers, a larger input of biofluid material, and a preparatory step to improve the qPCR detection threshold. According to some embodiments, the method comprises an additional cell-specific antibody to enrich a subpopulation of biological particles for detecting low abundance biomarkers, a larger input of biofluid material, and a preparatory step to improve the qPCR detection threshold.

[0173] According to some embodiments, the antibody specific for the biological particle antigen is activated with dibenzocyclooctyl (DBCO) ester, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC, or derivatives thereof. According to some embodiments, the antibody specific for the biological particle antigen is activated with dibenzocyclooctyl (DBCO) ester. According to some embodiments, the DBCO-modified antibody is coupled to a DNA linker by click chemistry. According to some embodiments, the antibody-DNA linker conjugate is coupled to a well plate coated with streptavidin. According to some embodiments, the well plate is pretreated with RNAse A.

[0174] According to some embodiments, the isolation / separation step includes washing, thus separating unbound material in the biological sample from the biological particles bound to the binder. According to some embodiments, the biological particles bound to the binder are enzymatically released from the solid support. According to some embodiments, the release includes enzymatically cleaving the bond between one or more binders and the solid support. According to some embodiments, the population of purified biological particles is released by enzymatic cleavage of the nucleic acid bound to the binder. According to some embodiments, the nucleic acid is DNA. According to some embodiments, the DNA includes one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil, a restriction site sequence, or an RNA / DNA duplex. According to some embodiments, when the RNA and DNA strands are double stranded, the enzyme specifically cleaves the nucleic acid at the ribonucleic acid nucleotide or a specific restriction site sequence, or a specific RNA or DNA strand. According to some embodiments, the ribonucleic acid nucleotide is a uracil nucleotide. According to some embodiments, the enzyme that cleaves the nucleic acid at the uracil nucleotide is a uracil glycosylase. According to some embodiments, the enzyme that cleaves at a specific nucleic acid sequence is a restriction enzyme. According to some embodiments, the enzyme that specifically cleaves an RNA / DNA duplex is a restriction endonuclease or RNase-H.

[0175] According to some embodiments, the population of purified biological particles is released by strand displacement. According to some embodiments, a first existing strand of nucleic acid is separated from a second substrate strand of nucleic acid by an invading strand complementary to the first or second strand of nucleic acid and an enzyme comprising strand displacement activity. According to some embodiments, the enzyme comprising strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. According to some embodiments, the annealed DNA strands are separated using polymerase chain reaction using oligonucleotides complementary to the DNA region bound to the antibody, such that the antibody can be released from the platform without damaging the DNA strand bound to the antibody.

[0176] According to some embodiments, the biological particles are eluted from the binding agent, for example, the biological particles can be eluted by contacting the bound binding agent-biological particle complex with a free ligand specific for the binding agent, such that a population of purified biological particles can be released.

[0177] According to some embodiments, the population of purified biological particles comprises surface protein biomarkers of the biological particles. According to some embodiments, the surface protein biomarkers of the biological particles comprise one or more tetraspanins (e.g., CD9, CD63, CD81, CD37, CD82), Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers inherited by the biological particles. According to some embodiments, the purity of the purified biological particle population is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. According to some embodiments, the population of purified biological particles is homogenous.

[0178] According to some embodiments, RNA species including microRNA (miRNA) small non-coding RNA or DNA encapsulated in purified biological particles can be isolated, identified and quantified. According to some embodiments, small non-coding RNA including miRNA or DNA can be isolated, identified and quantified by next generation sequencing and displayed in a heat map. Briefly, next generation sequencing is a high-throughput method used to determine the nucleotide sequence of an individual's entire genome at once in an automated process. Small non-coding RNA including miRNA or DNA identified by sequencing are displayed using a representation of the data in the form of a map or diagram where the data values ​​generated after statistical analysis are represented as colors ("heat map").

[0179] According to some embodiments, the small non-coding RNAs, including the miRNA content of the isolated biological particles, are compared to control subjects (i.e., normal healthy subjects not infected with the virus, control subjects infected with the virus with mild symptoms, and control subjects infected with the virus with severe symptoms) by next generation sequencing and heat map generation. According to some embodiments, using total RNA from serum biological particles, ten miRNAs are differentially expressed between mild and severe sample groups of SARS-CoV2 patients, including hsa-miR-146a, hsa-miR-126-3p, hsa-miR-424, hsa-miR-151-3p, hsa-miR-126-5p, hsa-miR-627-5p, hsa-miR-145, hsa-miR-205, and hsa-miR-200c. According to some embodiments, miRNA markers of severe COVID infection using total RNA from whole serum include hsa-miR-550-5p, or hsa-miR-629, or both.

[0180] According to some embodiments, the fold change differences in the cargo profiles (ranging from about 0.75 (decreased) to about 1.5 (increased)) are statistically evaluated and identified based on a p-value of <0.05 so that a patient's risk of severe viral infection can be determined. According to some embodiments, the small non-coding RNA or protein cargo profile of the purified biological particle is about 1.5-fold lower or 1.5-fold higher than the cargo profile from a control subject infected with coronavirus and suffering from severe disease.

[0181] According to some embodiments, the therapy includes supportive care such as intravenous fluids, supplemental oxygen, nebulized vapors, etc. According to some embodiments, the therapy includes a pharmaceutical composition including an antiviral agent, an immunomodulatory agent, or both.

[0182] According to some embodiments, the antiviral agent inhibits viral entry, reduces viral load, or both. According to some embodiments, the antiviral agent is selected from acyclovir, ganciclovir, foscarnet, ribavirin, amantadine, azidodeoxythymidine / zidovudine), nevirapine, tetrahydroimidazobenzodiazepinone (TIBO) compounds, efavirenz, remdesivir, lopinavir / ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine.

[0183] According to some embodiments, the immunomodulatory agent is a glucocorticoid or a recombinant interferon. According to some embodiments, the glucocorticoid is a corticosteroid such as prednisone, dexamethasone, azathioprine, mycophenolic acid, mycophenolate mofetil, or a combination thereof.

[0184] According to another aspect, the present disclosure provides a method for enhancing the therapeutic efficacy of convalescent plasma therapy in a patient at risk for severe SARSCoV-2 infection (COVID), the method comprising:

[0185] a) preparing a purified biological particle population from convalescent plasma of a convalescent subject, the preparing comprising:

[0186] (1) Obtaining convalescent serum containing high anti-SARSCoV-2 IgG titers from convalescent subjects;

[0187] (2) contacting the convalescent serum with a binding agent for one or more surface antigens of the biological particles, whereby the binding agent binds to nucleic acid and the nucleic acid is immobilized on the solid support;

[0188] b) isolating the biological particles bound by the binding agent from the biological sample; and

[0189] c) releasing the biological particles bound to the binding agent in (b); and

[0190] (d) eluting the bound biological particles from the binding agent to form a population of free purified biological particles;

[0191] (e) Measuring the neutralization titer of the purified exosome population against the SARS-CoV-2 virus in vitro;

[0192] (f) administering to the patient convalescent serum containing high titer neutralizing biological particles.

[0193] According to some embodiments, the method includes an initial ultrafiltration step, an initial ultracentrifugation step, or both, to provide a pooled heterogeneous population of biological particles.

[0194] According to some embodiments, the convalescent serum samples are derived from normal healthy control subjects (meaning subjects without symptoms or other evidence of severe viral infection, e.g., control subjects not infected with SARS-CoV-2), subjects infected with SARS-CoV-2 (e.g., confirmed by PCR) with mild symptoms and recovered, and subjects infected with SARS-CoV-2 (e.g., confirmed by PCR) with severe symptoms and recovered. According to some embodiments, the convalescent serum samples have high titers of antibodies against the receptor binding protein (RBD) of the SARS-CoV-2 spike protein. According to some embodiments, the titers of antibodies against the receptor binding protein (RBD) of the SARS-CoV-2 spike protein are detectable at least at a 1:10,000 dilution of the convalescent serum. According to some embodiments, the convalescent serum samples have low titers of antibodies against the receptor binding protein (RBD) of the SARS-CoV-2 spike protein. According to some embodiments, sera containing low titers of antibodies against the receptor binding protein (RBD) of the SARS-CoV-2 spike protein range from below a 1:10,000 dilution of convalescent serum to below the limit of quantification.

[0195] According to some embodiments, the biological sample comprises a bodily fluid, including, but not limited to, blood, urine, sperm (semen), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal discharge, otorrhea, gastric fluid, breast milk, feces, sweat, tears, nipple aspirate, or other secreted bodily fluids. According to some embodiments, the bodily fluid is a circulating / secreted bodily fluid, such as blood (whole blood, serum, or plasma), CSF, or lymph. According to some embodiments, the biological sample comprises a bodily tissue (e.g., diseased tissue).

[0196] According to some embodiments, the biofluid from which biological particles containing a particular biomarker(s) are selected can be reused, e.g., in subsequent rounds to select other biomarkers, select small non-coding RNAs including miRNAs, isolate RNA, DNA, proteins, lipids, or combinations thereof, etc., without adversely affecting the biofluid.

[0197] According to some embodiments, the binding agent comprises a nucleic acid (e.g., DNA, RNA, or a combination thereof), a polypeptide (e.g., an antibody, an antibody fragment, an aptamer polypeptide, or a combination thereof), or a biodegradable polymer.

[0198] According to some embodiments, the binding agent is directly immobilized to the solid support. According to some embodiments, the binding agent is indirectly immobilized to the solid support. In some such embodiments, the binding agent is conjugated to the nucleic acid and immobilized to the solid support. According to some embodiments, the nucleic acid is immobilized to the solid support by click chemistry. According to some embodiments, the binding agent is an antibody specific to the biological particle antigen. According to some embodiments, the biological particle antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers carried by the biological particle. According to some embodiments, the method comprises an additional cell-specific antibody to enrich a subpopulation of biological particles for detection of low abundance biomarkers. According to some embodiments, the method comprises a larger input of biofluid material than those tested herein. According to some embodiments, the method comprises a preparatory step to improve the qPCR detection threshold. According to some embodiments, the method includes additional cell-specific antibodies to enrich subpopulations of biological particles for detection of low abundance biomarkers, larger amounts of biofluid material input, and preparatory steps to improve the qPCR detection threshold.

[0199] According to some embodiments, the antibody specific for the biological particle antigen is activated with dibenzocyclooctyl (DBCO) ester, dibenzocyclooctyne (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC, or derivatives thereof. According to some embodiments, the antibody specific for the biological particle antigen is activated with dibenzocyclooctyl (DBCO) ester. According to some embodiments, the DBCO modified antibody is coupled to a DNA linker by click chemistry. According to some embodiments, the antibody-DNA linker conjugate is coupled to a well plate coated with streptavidin. According to some embodiments, the well plate is pre-treated with RNAse A.

[0200] According to some embodiments, the isolation / separation step includes washing, thus separating unbound material from the biological particles bound to the binding agent.

[0201] According to some embodiments, the population of purified biological particles is enzymatically released from the solid support. According to some embodiments, the release comprises enzymatically cleaving a bond between one or more binding agents and the solid support. According to some embodiments, the population of purified biological particles is released by enzymatic cleavage of a nucleic acid bound to the binding agent. According to some embodiments, the nucleic acid is DNA. According to some embodiments, the DNA comprises one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil, a restriction site sequence, or an RNA / DNA duplex. According to some embodiments, when the RNA and DNA strands are double stranded, the enzyme specifically cleaves the nucleic acid at the ribonucleic acid nucleotide or a specific restriction site sequence, or a specific RNA or DNA strand. According to some embodiments, the ribonucleic acid nucleotide is a uracil nucleotide. According to some embodiments, the enzyme that cleaves the nucleic acid at the uracil nucleotide is a uracil glycosylase. According to some embodiments, the enzyme that cleaves at a specific nucleic acid sequence is a restriction enzyme. According to some embodiments, the enzyme that specifically cleaves an RNA / DNA duplex is a restriction endonuclease or RNase-H.

[0202] According to some embodiments, the population of purified biological particles is released by strand displacement. According to some embodiments, a first existing strand of nucleic acid is separated from a second substrate strand of nucleic acid by an invading strand complementary to the first or second strand of nucleic acid and an enzyme comprising strand displacement activity. According to some embodiments, the enzyme comprising strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. According to some embodiments, the annealed DNA strands are separated using polymerase chain reaction using oligonucleotides complementary to the DNA region bound to the antibody, such that the antibody can be released from the platform without damaging the DNA strand bound to the antibody.

[0203] According to some embodiments, the biological particles are eluted from the binding agent, for example, by contacting the bound binding agent-biological particle complex with a free ligand specific for the binding agent, such that a population of purified biological particles can be formed.

[0204] According to some embodiments, the population of purified biological particles comprises exosome surface protein biomarkers. According to some embodiments, the surface protein biomarkers of the biological particles comprise one or more of tetraspanins (e.g., CD9, CD63, CD81, CD37, CD82), Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers inherited by the biological particles. According to some embodiments, the purity of the population of purified exosomes is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. According to some embodiments, the population of purified biological particles is homogenous.

[0205] According to some embodiments, the neutralization is determined in vitro by incubating isolated purified biological particles from convalescent serum with a cell line infected with SARS-CoV-2 in vitro. The presence and size of substances that interfere with viral infectivity are determined by measuring the production of viral particles after an appropriate period of time, e.g., 12 hours, 24 hours, 36 hours, and / or 72 hours. According to some embodiments, cells infected with a neon green SARSCoV-2 reporter virus can be counted and compared to a negative control (infected cells without convalescent purified biological particles). According to some embodiments, the production of viral particles, which can be quantified by fluorescence, is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% when compared to a negative control. According to some embodiments, the neutralizing activity of convalescent EVs containing the ACE2 receptor plays a role in neutralizing the SARS-CoV-2 virus. According to some embodiments, the biological particles may be effective in removing viral proteins from infected cells while retaining them on the surface, thus allowing presentation of antigens to the immune system.

[0206] According to some embodiments, convalescent serum containing neutralizing purified biological particles can increase the efficacy of convalescent plasma therapy by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, and about 10 fold when compared to treatment with convalescent plasma therapy alone.

[0207] According to some embodiments, patients suffering from severe SARSCoV-2 infection may be treated with additional therapies, including supportive care such as intravenous fluids, supplemental oxygen, and nebulized vapor.

[0208] According to some embodiments, the additional therapy includes a pharmaceutical composition comprising an antiviral agent, an immunomodulatory agent, or both. According to some embodiments, the antiviral agent inhibits viral entry, reduces viral load, or both. According to some embodiments, the antiviral agent is selected from acyclovir, ganciclovir, foscarnet, ribavirin, amantadine, azidodeoxythymidine (zidovudine), nevirapine, tetrahydroimidazobenzodiazepinone (TIBO) compounds, efavirenz, remdesivir, lopinavir / ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine.

[0209] In some embodiments, the immunomodulatory agent is a glucocorticoid or a recombinant interferon. In some embodiments, the glucocorticoid is a corticosteroid, such as prednisone, dexamethasone, azathioprine, mycophenolic acid, mycophenolate mofetil, or a combination thereof.

[0210] According to another aspect, the present disclosure further provides a method of treating a patient with a severe coronavirus infection, the method comprising:

[0211] (a) determining in vitro the neutralization titer of a population of purified biological particles from a subject, said determining comprising:

[0212] (1) Obtaining a biological sample containing biological particles from a subject;

[0213] (2) contacting a biological sample containing extracellular vesicles from the subject with a binding agent for one or more surface antigens of the biological particles, wherein the binding agent binds to nucleic acid and the nucleic acid is immobilized on a solid support;

[0214] (3) isolating the biological particles bound by the binding agent from the biological sample by washing;

[0215] (4) releasing the biological particles bound to the binding agent in (3) above;

[0216] (5) eluting the bound biological particles from the binder to form a population of free purified biological particles;

[0217] (6) determining the neutralization titer of the population of purified biological particles in vitro; and

[0218] (b) selecting the subject to receive convalescent plasma therapy comprising neutralizing purified biological particles comprising the ACE2 receptor if the neutralizing titer of the population of purified biological particles from the subject is insufficient to neutralize the viral infection in vitro; and

[0219] (c) preparing a population of purified biological particles from convalescent plasma of a convalescent subject, the preparing comprising:

[0220] (i) obtaining convalescent serum containing high anti-SARSCoV-2 IgG titers from convalescent subjects;

[0221] (ii) contacting the convalescent serum with a binding agent against one or more surface antigens of the biological particles;

[0222] (iii) isolating the biological particles bound by the binding agent from the biological sample by washing;

[0223] (iv) releasing the biological particles bound to the binding agent in (b) above;

[0224] (v) eluting the bound biological particles from the binding agent to form a population of free purified biological particles;

[0225] (d) measuring in vitro the neutralization titer of the population of purified biological particles from step (v) against the SARS-CoV-2 virus; and

[0226] (e) administering to the subject the neutralized purified biological particles or convalescent serum containing the neutralized biological particles of step (d).

[0227] According to some embodiments, the method includes an initial ultrafiltration step, an initial ultracentrifugation step, or both, to provide a pooled heterogeneous population of biological particles.

[0228] According to some embodiments, the biological sample comprises a bodily fluid, including, but not limited to, blood, urine, sperm (semen), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal discharge, otorrhea, gastric fluid, breast milk, feces, sweat, tears, nipple aspirate, or other secreted bodily fluids. According to some embodiments, the bodily fluid is a circulating / secreted bodily fluid, such as blood (whole blood, serum, or plasma), CSF, or lymph. According to some embodiments, the biological sample comprises a bodily tissue (e.g., diseased tissue).

[0229] According to some embodiments, the biofluid from which biological particles containing a particular biomarker(s) are selected can be reused, e.g., in subsequent rounds to select other biomarkers, select small non-coding RNAs including miRNAs, isolate RNA, DNA, proteins, lipids, or combinations thereof, etc., without adversely affecting the biofluid.

[0230] According to some embodiments, the binding agent comprises a nucleic acid (e.g., DNA, RNA, or a combination thereof), a polypeptide (e.g., an antibody, an antibody fragment, an aptamer polypeptide, or a combination thereof), or a biodegradable polymer.

[0231] According to some embodiments, the binding agent is directly immobilized to the solid support. According to some embodiments, the binding agent is indirectly immobilized to the solid support. In some such embodiments, the binding agent is conjugated to a nucleic acid and immobilized to the solid support. According to some embodiments, the nucleic acid is immobilized to the solid support by click chemistry. According to some embodiments, the binding agent is an antibody specific for a biological particle antigen. According to some embodiments, the biological particle antigen is one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers carried by the biological particle.

[0232] According to some embodiments, the antibody specific for the biological particle antigen is activated with dibenzocyclooctyl (DBCO) ester, dibenzocyclooctyne (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC, or derivatives thereof. According to some embodiments, the antibody specific for the biological particle antigen is activated with dibenzocyclooctyl (DBCO) ester. According to some embodiments, the DBCO modified antibody is coupled to a DNA linker by click chemistry. According to some embodiments, the antibody-DNA linker conjugate is coupled to a well plate coated with streptavidin. According to some embodiments, the well plate is pre-treated with RNAse A.

[0233] According to some embodiments, the isolation / separation step includes washing, thus separating unbound material in the biological sample from the biological particles bound to the binding agent.

[0234] According to some embodiments, the population of purified biological particles is enzymatically released from the solid support. According to some embodiments, the release comprises enzymatically cleaving a bond between one or more binding agents and the solid support. According to some embodiments, the population of purified biological particles is released by enzymatic cleavage of a nucleic acid bound to the binding agent. According to some embodiments, the nucleic acid is DNA. According to some embodiments, the DNA comprises one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil, a restriction site sequence, or an RNA / DNA duplex. According to some embodiments, the enzyme specifically cleaves the nucleic acid at the ribonucleic acid nucleotide. According to some embodiments, the ribonucleic acid nucleotide is a uracil nucleotide. According to some embodiments, the enzyme that cleaves the nucleic acid at the uracil nucleotide is a uracil glycosylase. According to some embodiments, the enzyme that cleaves at a specific nucleic acid sequence is a restriction enzyme. According to some embodiments, the enzyme that specifically cleaves an RNA / DNA duplex is a restriction endonuclease or RNase-H.

[0235] According to some embodiments, the population of purified biological particles is released by strand displacement. According to some embodiments, a first existing strand of nucleic acid is separated from a second substrate strand of nucleic acid by an invading strand complementary to the first or second strand of nucleic acid and an enzyme comprising strand displacement activity. According to some embodiments, the enzyme comprising strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. According to some embodiments, the annealed DNA strands are separated using polymerase chain reaction using oligonucleotides complementary to the DNA region bound to the antibody, such that the antibody can be released from the platform without damaging the DNA strand bound to the antibody.

[0236] According to some embodiments, the biological particles are eluted from the binding agent, for example, the biological particles can be eluted by contacting the bound binding agent-biological particle complex with a free ligand specific for the binding agent, such that a population of purified biological particles can be formed.

[0237] According to some embodiments, the population of purified biological particles comprises surface protein biomarkers of the biological particles. According to some embodiments, the surface protein biomarkers of the biological particles comprise one or more of tetraspanins (e.g., CD9, CD63, CD81, CD37, CD82), Alix, ACE-2, Tim4, PLAP, and / or other cell surface markers inherited by the biological particles. According to some embodiments, the purity of the purified biological particle population is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. According to some embodiments, the population of purified biological particles is homogenous.

[0238] According to some embodiments, the neutralization is determined in vitro by incubating isolated purified biological particles from convalescent serum with cells infected with SARS-CoV-2 in vitro. The presence and size of substances that interfere with viral infectivity are determined by measuring the production of viral particles after appropriate periods of time, e.g., 12 hours, 24 hours, 36 hours, and 72 hours. According to some embodiments, cells infected with a neon green SARSCoV-2 reporter virus can be counted and compared to a negative control (infected cells without convalescent purified biological particles). According to some embodiments, to be considered neutralized, the production of viral particles, quantifiable by fluorescence, is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% when compared to a negative control. According to some embodiments, the neutralizing activity of convalescent EVs containing the ACE2 receptor plays a role in neutralizing the SARS-CoV-2 virus. According to some embodiments, the biological particles may be effective in removing viral proteins from infected cells while retaining them on the surface, thus allowing presentation of antigens to the immune system.

[0239] According to some embodiments, convalescent serum samples are derived from normal healthy control subjects (meaning subjects who have no symptoms or other evidence of severe viral infection, e.g., control subjects who are not infected with SARS-CoV-2), subjects who have been infected with SARS-CoV-2 and have developed mild symptoms and recovered, or subjects who have been infected with SARS-CoV-2 and have developed severe symptoms and recovered.

[0240] According to some embodiments, the convalescent serum sample has a high titer of antibodies to the receptor binding protein (RBD) of the SARS-CoV-2 spike protein. According to some embodiments, the titer of antibodies to the receptor binding protein (RBD) of the SARS-CoV-2 spike protein is detectable at least at a 1:10,000 dilution of the convalescent serum. According to some embodiments, the convalescent serum sample has a low titer of antibodies to the receptor binding protein (RBD) of the SARS-CoV-2 spike protein. According to some embodiments, the titer of the serum containing a low titer of antibodies to the receptor binding protein (RBD) of the SARS-CoV-2 spike protein ranges from below a 1:10,000 dilution to below the limit of quantification.

[0241] According to some embodiments, the present disclosure provides a method of preparing a population of purified cells from a biological sample from a subject, the method comprising:

[0242] a) preparing a population of purified cells, said preparing comprising:

[0243] (1) Obtaining a biological sample containing cells from a subject;

[0244] (2) contacting a biological sample from a subject containing cells with a binding agent for one or more cell surface antigens, where the binding agent is attached to the nucleic acid by a linker, and the nucleic acid is immobilized on a solid support;

[0245] b) isolating cells bound by the binding agent from the biological sample; and

[0246] c) releasing the cells bound to the binding agent; and

[0247] and d) eluting the bound cells from the binding agent to form a population of purified cells.

[0248] According to some embodiments, the population of cells is derived from non-eukaryotic and eukaryotic species (e.g., human, mouse, rat, monkey, etc.), healthy normal tissues (e.g., blood, tissue, etc.), including but not limited to endothelial cells, epithelial cells, T cells / fibroblasts, adipocytes, neuronal cells, and certain tumor cell subpopulations. According to some embodiments, the cells can be derived from normal and diseased tissues of human patients, mouse orthotopic / xenograft / PDX organs, blood, or tissue culture, and can be purified using EV-CATCHER targeting unique surface markers, for example, to identify new clones, to purify unique cells that are too sensitive for flow cytometry, or to purify cells that need to be enriched.

[0249] According to some embodiments, the method includes an initial ultrafiltration or ultracentrifugation step to provide an initial pooled heterogeneous population of cells. According to some embodiments, the biological sample is prepared in vivo or in vitro.

[0250] According to some embodiments, the biological sample comprises a bodily fluid. According to some embodiments, the bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymph, urine, feces, sweat, tears, nipple aspirate, or semen, or a secreted biological fluid. According to some embodiments, the bodily fluid is a circulating / secreted bodily fluid. According to some embodiments, the circulating bodily fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF), or lymph.

[0251] According to some embodiments, the method is a high throughput assay, which allows purification of multiple samples at once, i.e., compatible with 24, 96, 384 well plate formats or other multi-wall formats. According to some embodiments, the biofluid from which cells containing a particular biomarker(s) are selected can be reused, e.g., in subsequent rounds to select other biomarkers, select small non-coding RNAs, including miRNAs, isolate RNA, DNA, proteins, lipids, or combinations thereof, etc., without adversely affecting the biofluid.

[0252] According to some embodiments, the binding agent is an antibody specific for a cell surface antigen, which according to some embodiments comprises one or more of CD4, CD8, CD9, CD46, CD63, CD81, CD37, CD82, CD138, CD151, ALix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, heterologous cell surface polypeptides, and / or other cell surface markers. Examples of commercial sources of such antibodies include, but are not limited to, CD4 (Cell Signaling Technologies #27520), CD8 (Cell Signaling Technologies #81575), CD46 (Abcam #ab271871), CD138 (Abcam #ab242394), CD151 (Abcam #ab27406), adiponectin (Abcam #ab227051), and EPCAM (Cell Signaling Technology #46403). Thus, the purification process can be tailored to select specific populations of cells and scan their contents as needed, such as those infected with viruses, those from cancer cells, those from organs, etc. According to some embodiments, the method includes additional cell-specific antibodies to enrich subpopulations of cells for detection of low abundance biomarkers. According to some embodiments, the method includes a larger input of biofluid material than those tested herein. According to some embodiments, the method includes a preparatory step to improve the qPCR detection threshold. According to some embodiments, the method includes additional cell-specific antibodies to enrich for subpopulations of cells for detection of low abundance biomarkers, larger amounts of biofluid material input, and preparatory steps to improve the qPCR detection threshold.

[0253] According to some embodiments, the purity of the population of purified cells is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. According to some embodiments, the population of purified cells is homogenous.

[0254] According to some embodiments, the heterologous cell surface polypeptide is expressed by a chimeric antigen receptor T cell (CAR-T cell).

[0255] According to some embodiments, the binding agent that binds to one or more cell surface antigens is an antibody, an antibody-binding fragment, or an aptamer. According to some embodiments, the aptamer is a nucleic acid or a polypeptide. According to some embodiments, the nucleic acid comprises DNA, RNA, or a combination thereof. According to some embodiments, the nucleic acid comprises DNA. According to some embodiments, the DNA comprises one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil. According to some embodiments, the DNA comprises a restriction enzyme recognition site. According to some embodiments, the nucleic acid is a DNA / RNA duplex that can be degraded by an endonuclease or RNAse (RNase-H). According to some embodiments, the nucleic acid comprises a non-natural nucleotide.

[0256] According to some embodiments, the nucleic acid comprises a binding moiety at a first end of the nucleic acid and a binding moiety at a second end of the nucleic acid, where the binding moiety at the first end of the nucleic acid and the binding moiety at the second end of the nucleic acid are different. According to some embodiments, the binding moiety at the first end of the nucleic acid is an avidin, streptavidin, or a carboxyl binding moiety. According to some embodiments, the binding moiety is biotin. According to some embodiments, the binding moiety at the second end of the nucleic acid is an amine moiety. According to some embodiments, the amine moiety is an azide.

[0257] According to some embodiments, the binding agent for one or more cell surface antigens comprises a dibenzocyclooctyne (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), sulfo-SMCC, or derivatives thereof. According to some embodiments, the DBCO-modified antibody is coupled to a DNA linker by click chemistry. According to some embodiments, the antibody-DNA linker conjugate is coupled to a well plate coated with streptavidin. According to some embodiments, the well plate is pretreated with RNAse A.

[0258] According to some embodiments, the isolation / separation step includes washing, thus separating unbound material in the biological sample from the cells bound to the binding agent. According to some embodiments, the cells bound to the binding agent are enzymatically released from the solid support. According to some embodiments, the release includes enzymatically cleaving the bond between one or more binding agents and the solid support. According to some embodiments, the population of purified cells is released by enzymatic cleavage of the nucleic acid bound to the binding agent. According to some embodiments, the nucleic acid is DNA. According to some embodiments, the DNA includes one or more ribonucleic acid nucleotides. According to some embodiments, the one or more ribonucleic acid nucleotides are uracil, a restriction site sequence, or an RNA / DNA duplex. According to some embodiments, when the RNA and DNA strands are double stranded, the enzyme specifically cleaves the nucleic acid at the ribonucleic acid nucleotide or a specific restriction site sequence, or a specific RNA or DNA strand. According to some embodiments, the ribonucleic acid nucleotide is a uracil nucleotide. According to some embodiments, the enzyme that cleaves the nucleic acid at the uracil nucleotide is a uracil glycosylase. According to some embodiments, the enzyme that cleaves a specific nucleic acid sequence is a restriction enzyme. According to some embodiments, the enzyme that specifically cleaves an RNA / DNA duplex is a restriction endonuclease or RNase-H.

[0259] According to some embodiments, the population of purified cells is released by strand displacement. According to some embodiments, a first existing strand of nucleic acid is separated from a second substrate strand of nucleic acid by an invading strand complementary to the first or second strand of nucleic acid and an enzyme comprising strand displacement activity. According to some embodiments, the enzyme comprising strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase. According to some embodiments, the annealed DNA strands are separated using polymerase chain reaction using oligonucleotides complementary to the DNA region bound to the antibody, such that the antibody can be released from the platform without damaging the DNA strand bound to the antibody.

[0260] According to some embodiments, the binding agent is directly immobilized to the solid support. According to some embodiments, the binding agent is indirectly immobilized to the solid support. In some such embodiments, the binding agent is conjugated to the nucleic acid and immobilized to the solid support. According to some embodiments, the nucleic acid is immobilized to the solid support by click chemistry. According to some embodiments, the solid support is a well plate, a polymer, or a surface.

[0261] According to some embodiments, the release of isolated cells comprises:

[0262] (i) enzymatic cleavage of nucleic acids; or

[0263] (ii) displacing the first strand of the nucleic acid bound to the antibody from the second strand of the nucleic acid bound to the support by strand displacement with a nucleic acid complementary to the first or second strand of the nucleic acid and an enzyme having strand displacement activity to release the antibody from the support; or

[0264] (iii) releasing the antibody from the platform by polymerase chain reaction using oligonucleotides complementary to the region of DNA bound to the antibody to separate the annealed DNA strands without damaging the DNA strand bound to the antibody.

[0265] According to some embodiments, the enzymatic cleavage is performed by uracil glycosylase. According to some embodiments, the enzymatic cleavage is performed using a restriction enzyme. According to some embodiments, the enzymatic cleavage is performed using an endonuclease or an RNAse.

[0266] According to some embodiments, the enzyme with strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase.

[0267] According to some embodiments, the population of purified cells comprises non-eukaryotic cells, eukaryotic cells, or a combination thereof. According to some embodiments, the eukaryotic cells are human, mouse, rat, dog, non-human primate, or feline cells. According to some embodiments, the population of purified cells is derived from a healthy subject or a subject suffering from a disease. According to some embodiments, the disease includes cancer, viral infection, placental abnormalities, inflammation, and other pathologies. According to some embodiments, the cells are endothelial cells, epithelial cells, T cells, fibroblasts, adipocytes, neuronal cells, tumor cells, blood cells, or cardiac cells.

[0268] According to some embodiments, the viral infection is a severe coronavirus infection. According to some embodiments, the severe coronavirus infection is due to SARS-CoV-1, MERS, or SARS-CoV-2. According to some embodiments, the severe coronavirus infection is due to SARS-CoV-2.

[0269] According to some embodiments, the population of cells is derived from (a) non-eukaryotic and eukaryotic species, (b) healthy normal or diseased tissue, (c) mouse orthotopic / xenograft / PDX organs, (d) blood, or (e) tissue culture.

[0270] Pharmaceutical preparations As used herein, the term "pharmaceutical composition" refers to a composition used to prevent, reduce the intensity of, cure, or otherwise treat a targeted disease or disorder. Generally, a pharmaceutical composition comprises an active agent and a pharma- ceutical acceptable carrier.

[0271] The term "carrier" as used herein refers to a material that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the active compounds of the compositions of the described invention. The carrier must be of sufficiently high purity and of sufficiently low toxicity to make it suitable for administration to the mammal receiving the treatment. The carrier may be inert, may have pharmaceutical benefits, cosmetic benefits, or both. The terms "excipient", "carrier", or "vehicle" refer to carrier substances suitable for the formulation and administration of the pharma- ceutically acceptable compositions described herein, and they may be used interchangeably. Carriers and vehicles useful herein include any such substances known in the art that are non-toxic and do not interact with other components. Carriers may be liquid or solid, and are selected with the proposed method of administration in mind, to provide the desired bulk, consistency, etc. when combined with the active agent and other components of a given composition.

[0272] The phrase "pharmaceutical acceptable carrier" is recognized in the art. It is used to mean any substantially non-toxic carrier conventionally usable for the administration of pharmaceutical agents in which the isolated EVs of the present invention remain stable and bioavailable. A pharmaceutical acceptable carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the mammal being treated. In addition, it must maintain the stability and bioavailability of the active agent. A pharmaceutical acceptable carrier may be liquid or solid and is selected with the proposed method of administration in mind to provide the desired bulk, consistency, etc. when combined with the active agent and other components of a given composition. Examples of carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating substances involved in carrying or transporting the subject agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other components of the formulation and not harmful to the patient. Examples of substances which can function as pharma- ceutically acceptable carriers include sugars (e.g., lactose, glucose, and sucrose), starches (e.g., corn starch and potato starch), cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (e.g., cocoa butter and suppository waxes), oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (e.g., propylene glycol), polyols (e.g., glycerin, sorbitol, mannitol, and polyethylene glycol), esters (e.g., ethyl oleate and ethyl laurate), agar, buffers (e.g., magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solutions, and other non-toxic compatible substances used in pharmaceutical formulations. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, incorporated herein by reference in its entirety. In some embodiments, the pharma- ceutically acceptable carrier is sterile pyrogen-free water.In some embodiments, the pharma- ceutically acceptable carrier is lactated Ringer's solution, also known as lactated Ringer's solution.

[0273] Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0274] Examples of pharma- ceutically acceptable antioxidants include water-soluble antioxidants (e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite), oil-soluble antioxidants (e.g., ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol), metal chelating agents (e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like).

[0275] The specific method of administration varies depending on the indication. The selection of the specific route of administration and the dosage should be adjusted or titrated by the clinician according to methods known to the clinician to obtain the optimal clinical response. The amount of the active agent administered is an amount sufficient to provide the intended therapeutic benefit. The dosage administered depends on the characteristics of the treated subject, such as the specific mammal or human being treated, age, weight, health condition, type of concurrent treatment (if any), and frequency of treatment, and can be easily determined by those skilled in the art (e.g., clinician).

[0276] The term "parenteral" and other grammatical forms as used herein refer to administration of a substance that occurs in the body other than through the mouth or digestive tract. For example, the term "parenteral" as used herein refers to introduction into the body by injection (i.e., administration by injection), including, for example, subcutaneous (i.e., injection under the skin), intramuscular (i.e., injection into a muscle), intravenous (i.e., injection into a vein), intrathecal (i.e., injection into the subarachnoid space around the spinal cord or in the brain), or by infusion techniques.

[0277] In some embodiments, the compositions according to the present disclosure are administered several times or as needed at the discretion of the attending physician. In one such embodiment, the composition is administered on the first infusion day, and as needed on the second infusion day, the third infusion day, the fourth infusion day, the fifth infusion day, the sixth infusion day, the seventh infusion day, the eighth infusion day, the ninth infusion day, the tenth infusion day, etc.

[0278] When injected through a catheter, the therapeutic efficacy of the biological particles of the present disclosure depends on the pharmaceutical composition maintaining its efficacy as it passes through the catheter.

[0279] In some embodiments, the method of the present disclosure includes aerosolizing the pharmaceutical composition in a form selected from a dry powder, a suspension, or a solution. According to some embodiments, the composition is a solution. According to some embodiments, the composition is a suspension. According to some embodiments, the composition is a dry powder. For administration by inhalation, the compositions for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. For pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or inhaler can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch. According to some embodiments, the inhalation delivery device is a nebulizer, a metered dose inhaler, or a dry powder inhaler (DPI). According to some embodiments, the size of the respirable particles ranges from about 1 to 10 microns, inclusive. According to some embodiments, particle sizes for nasal administration (inhalation) range from 10 to 500 μM, inclusive.

[0280] According to some embodiments, the pharmaceutical composition of the present disclosure includes one or more therapeutic agents other than the described neutralizing purified biological particles. For example, according to some embodiments, the additional therapy may include a pharmaceutical composition including an antiviral agent, an immunomodulatory agent, or both. According to some embodiments, the antiviral agent inhibits viral entry, reduces viral load, or both. According to some embodiments, the antiviral agent is selected from acyclovir, ganciclovir, foscarnet, ribavirin, amantadine, azidodeoxythymidine / zidovudine), nevirapine, tetrahydroimidazobenzodiazepinone (TIBO) compounds, efavirenz, remdesivir, lopinavir / ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine. According to some embodiments, the immunomodulatory agent is a glucocorticoid or a recombinant interferon. According to some embodiments, the glucocorticoid is a corticosteroid selected from prednisone, dexamethasone, azathioprine, mycophenolic acid, mycophenolate mofetil, and combinations thereof.

[0281] According to the above embodiments, the pharmaceutical composition may be administered once for a limited period of time or as a long-term maintenance therapy, for example, until the condition improves or is cured. The limited period of time may be up to one year, including 1 week, 2 weeks, 3 weeks, 4 weeks, and any period between such values, including the endpoint. According to some embodiments, the pharmaceutical composition may be administered for about 1 day, about 3 days, about 1 week, about 10 days, about 2 weeks, about 18 days, about 3 weeks, or any range of any of those values, including the endpoint.

[0282] According to the foregoing embodiments, the composition or pharmaceutical composition may be administered once a day, twice a day, three times a day, four times a day, or more.

[0283] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within that stated range, is included within the scope of the invention, unless the context clearly dictates otherwise. The upper and lower limits of those smaller ranges can be independently included in the smaller ranges and are also included within the scope of the invention, unless a specifically excluded limit is in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0284] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited.

[0285] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The publications mentioned herein are provided solely for their disclosure prior to the filing date of this application and each is incorporated by reference in its entirety. Any citation herein should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which must be independently confirmed. EXAMPLES

[0286] The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy of numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0287] Materials and Methods Laboratory and clinical specimens Human MCF-7 and mouse RAWS264.7 exosomes were purchased from System Biosciences (catalog numbers: EXOP-100A-1 and EXOP-165A-1, respectively). MCF-7 exosomes for protein titration experiments were obtained by ultracentrifugation. Briefly, 60 ml of tissue culture medium (containing DMEM (Gibco, #10566016) supplemented with 10% exosome-depleted FBS (Gibco, #A2720801) and 1% PenStrep (Gibco, #15140122)) obtained from approximately 80% confluent MCF-7 cells was refreshed for 48 h before collection. Cells, dead cells and cell debris were removed by centrifugation at 500xg for 5 min, 2,000xg for 10 min, followed by 10,000xg for 30 min, respectively. Ultracentrifugation was performed in 10 ml Ultraclear tubes (Beckman Coulter, #344059) in an Optima XE-90 ultracentrifuge (Beckman Coulter) at 100,000xg for 2 h at 4°C. The pellet was washed with 1xPBS (Gibco, #10010023) and then a second ultracentrifugation was performed at 100,000xg for 2 h at 4°C, and the final exosome pellet was resuspended in 50 μl of 1xPBS. Serum specimens from SARS-CoV-2 infected patients (first sample set) were collected at Hackensack University Medical Center (HUMC) under IRB#Pro2018-1022 by the HMH Network. Serum samples were processed at Biorepository (BioR) and stored at -80°C in 500 μl aliquots. After submitting and receiving approval of an internal proposal (proposal number BioRCOV4), we collected 13 serum samples from patients not requiring mechanical ventilation and 17 samples from patients with moderate to severe ARDS on mechanical ventilation, and performed RNA extraction for both whole serum and EV-CATCHER exosome purification. Original convalescent serum specimens (second sample set) were collected from recently recovered Covid-19 patients enrolled in the convalescent plasma study led by Dr. Michele Donato (IRB#Pro2020-0375 / 0378).The remaining samples were de-identified and then transferred to HMH BioR, where they were donated to the Center for Discovery and Innovation (CDI) after review and approval by the Covid-19 Institutional Review Board (Proposal COVID-19#102). The donated samples were stored at -80°C in 1,000 μl aliquots. An enzyme-linked immunosorbent assay (ELISA) developed and optimized to identify high and low IgG serum titers was first performed using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein as the antigen. Serum samples were then stratified into either high IgG (titer >10,000) or low IgG (titer below the limit of quantification (BLQ)). Three serum samples from high IgG convalescent individuals and three serum samples from BLQ IgG convalescent individuals were CD63 purified and assessed for the neutralizing capacity of their exosomal content using the EV-CATCHER assay. Two samples (one high and one BLQ) were ultracentrifuged to assess whole exosomes.

[0288] Extracellular vesicle capture and enzymatic release (EV-CATCHER) assay with selected antibodies The release protocol described by Lof et al. (2017) was further optimized and modified as follows: HPLC purified uracillated oligonucleotides (Integrative DNA Technology) of 5'-azido (5'Az-AAAAAACGAUUCGAGAACGUGACUGCCAUGCCAGCUCGUACUAU CGAA (SEQ ID NO: 1)) and 3'-biotin (5'Bio-CGAUAGUACGAGCUGGCAUGGCAGUCACGUUCUCGAA UCGUUUU (SEQ ID NO: 2)) modified from Lof et al. (2017) were resuspended in RNase-free water at a concentration of 250 ng / μl. Equimolar amounts (1:1 ratio) of each oligo were dissolved in 1x RNA annealing buffer (60 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mM MgCl2After annealing at 90°C for 2 min, 90-42°C for 40 min, and 42°C for 120 min, the fragments were separated on a 15% non-denaturing polyacrylamide (PAGE) gel (0.5xTBE (ThermoFisher, #15581044), 450 volts for 90 min). Double stranded (ds) DNA linkers were visualized on a blue light box using SYBR® Gold™ dye (ThermoFisher, #S11494), excised, crushed centrifugally in a gel breaker tube (IST Engineering, #3388-100), resuspended in 400 mM NaCl, and shaken overnight (O / N) on a thermomixer set at 4°C and 1,100 RPM. The solution was filtered and the dsDNA linkers were purified using a QIAEX® II Gel Extraction Kit (Qiagen, #20021) according to the manufacturer's instructions. The purified dsDNA linkers were assayed on a NanoDrop 2000 and diluted to 250ng / μl. Antibodies (1mg / ml) used for exosome pulls (anti-CD63 (Abcam, #ab59479; RRID:AB_940915), anti-CD81 (Abcam, #ab233692), and anti-CD9 (Abcam, #ab263023)) were activated using 5μl of freshly prepared 4mM DBCO-SS-NHS ester (Sigma Aldrich, #761532), incubated for 30 minutes at room temperature (RT) in the dark, and the reaction was stopped by adding 2.5μl of 1M Tris-Cl (pH 8.0) for 5 minutes at room temperature in the dark. DBCO-activated antibodies were desalted by incubation on a pre-equilibrated Zeba desalting column (ThermoFisher, #89882) for 1 min and centrifugation at 1,500xg for 2 min. Antibodies were quantified on a Nanodrop 2000 instrument using protein A280, and antibody-dsDNA (Ab-dsDNA) stock solutions were prepared by combining 50 μg of activated antibody with 25 μg of purified DNA linker O / N at 4 °C on a rotator.Verification of Ab-dsDNA binding was performed by PAGE, where Ab-dsDNA (1 μg) products were run under non-denaturing and non-reducing conditions, followed by Coomassie (Bio-Rad #1610786) staining to visualize changes in Ab-dsDNA migration. The next day, Ab-dsDNA conjugates were bound to streptavidin-coated 96-well plates (Pierce, #15120). Either a single anti-CD63 antibody (1 μg) or a combination of anti-CD63, anti-CD81, and anti-CD9 (1 μg of each antibody) (linker-conjugated) was added to a single well in 100 μl of 1×PBS. The streptavidin-coated 96-well plates with Ab-dsDNA conjugates were placed on a plate shaker at 4° C., 300 RPM for 8 hours to allow binding to the plate. The solution was carefully removed and the wells were washed three times with cold 1x PBS solution before adding samples (100 μl) treated with RNase-A (12.5 μg / ml). The plate was sealed using microAMP optical adhesive film (Applied Biosystems, #4311971) and placed on a shaker at 300 RPM, 4°C overnight. Samples were carefully removed, wells were washed 3 times with cold 1x PBS, and 100 μl of freshly prepared uracil glycosylase (UNG) enzyme (ThermoFisher, #EN0362) in 1x PBS (1x UNG buffer (200 mM Tris-Cl (pH 8.0), 10 mM EDTA and 100 mM NaCl, containing 1 unit of enzyme) was added to each well. For UNG digestion of dsDNA linkers, plates were incubated at 300 RPM on a shaker at 37°C for 2 hours, and exosomes were collected in this solution for further characterization and downstream analysis.

[0289] RNA extraction Total RNA extraction was performed on whole serum, whole plasma, and isolated exosomes using the miRNeasy serum / plasma kit (Qiagen, # 217184) according to the manufacturer's instructions, with some modifications to improve total RNA yield. Briefly, QIAzol was added to 100 μl of whole plasma, serum, or exosome solution, vortexed and incubated at room temperature for 5 min, after which chloroform was added to each sample. Samples were vortexed again and incubated at room temperature for 5 min. Samples were centrifuged at 12,000xg for 15 min at 4°C, and the upper aqueous phase of each sample was carefully removed and transferred to a new siliconized tube, to which 100% ethanol was added. Prior to column purification, samples were incubated on ice for 40 min. The clear upper phase was then passed twice through the supplied RNeasy minElute column and washed with RPE (wash buffer to wash membrane-bound RNA and remove trace salts with ethanol; Qiagen) and ice-cold 80% ethanol. The column was spun to remove residual ethanol, and total RNA was eluted in 50 μl of RNase-free water. Samples were then speed vacuumed down to 10 μl for small RNA sequencing and 20 μl for quantitative real-time PCR (RT-qPCR). For cDNA small RNA library preparation and next-generation sequencing, 3 ng of size primer (1.5 ng–19 nt and 1.5 ng–24 nt;

[42] ) was spiked into each RNA extract. For RT-qPCR experiments, 1 pg of ath-miR-159a (IDT, rUrUrUGGArUrUGAAGGGAGCrUCrUA (SEQ ID NO: 3)) was added to QIAzol to extract RNA from MCF-7 and clinical specimens (mild vs. severe Covid-19 samples), followed by RNA isolation and column purification, and used as an exogenous technical normalization control.

[0290] Western blot analysis Western blot analysis was performed to assess the presence of exosome surface protein biomarkers from purified exosome fractions. Purified exosomes were lysed and denatured in 1x Laemmli buffer (Bio-Rad, #161-0747) containing 355 mM β-mercaptoethanol and heated at 95°C for 5 min. Denatured lysates were pulse centrifuged and separated on 4-12% polyacrylamide precast mini-PROTEAN TGX gels (Bio-Rad, #4561086) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 5 μl of Precision Plus Protein™ Dual Xtra Prestained Protein Standard (Bio-Rad, #1610377) was loaded and used for gel orientation and molecular weight measurement of separated proteins. Samples were loaded and gels were run (Power Pac 300, Bio-Rad) at 100V and 400mA for 90 minutes in 1xTris / Glycine / SDS buffer (Bio-Rad, #1610732). After SDS-PAGE, proteins were transferred to 0.2μm polyvinylidene fluoride (PVDF) membranes (Bio-Rad, #1704156) using a semi-dry electrotransfer system (TransBlot Turbo™ v1.02, Bio-Rad) at 25V for 30 minutes. Membranes were visualized and protein transfer was assessed using the stain-free blot protocol provided with the Chemi-Doc™ MP (Bio-Rad) system, and non-specific binding was prevented by blocking the membranes for 30 minutes in EveryBlot™ blocking buffer (Bio-Rad, #12010020). Membranes were incubated overnight at 4°C with anti-mouse primary antibodies (1:1000) diluted in TBS-T (1xTBS, pH 6.8, 0.1% Tween® 20) targeting Alix (Life Technologies, #MA183977) and CD63 (Abcam, #ab59479), or anti-rabbit primary antibodies (1:1000) diluted in TBS-T targeting CD81 (Abcam, #ab233692) and CD9 (Abcam, #ab263023).Membranes were washed with TBS-T (3 x 5 min) and then incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:10000) for 1 h at room temperature with gentle agitation. Membranes were washed with TBS-T (3 x 5 min) and then proteins were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate (Pierce, #34095) and protein bands were visualized using ImageLab 4.0 software on a Chemi-Doc MP (Bio-Rad) imaging system.

[0291] Transmission electron microscopy Transmission electron microscopy (TEM) of purified exosomes was performed by Charles River Laboratories (Pathology Associates (PAI), Durham NC). Briefly, purified exosomes were fixed using 2% glutaraldehyde in phosphate buffer (Electron Microscopy Services, #6536-05) and stored at 4 °C. 300 mesh formvar-coated grids were inverted onto 20 μl of fixed exosome suspension for approximately 2 min and allowed to dry. The grids were then inverted onto 40 μl of 2% aqueous uranyl acetate solution for approximately 1 min and allowed to dry. Samples were imaged with a JEOL JEM-1400+ transmission electron microscope (JEOL Ltd., Tokyo, Japan) operating at an accelerating voltage of 80 kV. High-resolution TIFF images were acquired and saved using an AMT 16 MP digital camera system (Advanced Microscopy Techniques Corp., Woburn, MA).

[0292] Nanoparticle Tracking The purified exosomes were assessed for size and particle concentration using a Spectradyne nCS1 instrument. Briefly, 2 μl of purified exosomes were loaded onto a TS400 microfluidic cartridge (Spectradyne, #TS400), which allows for the analysis of particles between 65 and 400 nm. The loaded cartridge was primed in the instrument using 0.2 μm filtered buffer (1xPBS with 1% Tween® 20) and particle acquisition was performed. The acquired statistical files were analyzed for particle concentration and size distribution using Spectradyne Viewer software, and peak filtered CSD graphs were generated.

[0293] Preparation of exosomal small RNA cDNA library Small RNA sequencing from exosomes was performed using the cDNA library preparation protocol described by Loudig et al. (2017) with modifications for exosome purification when RNA amounts could not be quantified. Titration experiments performed in our laboratory showed that robust small RNA profiles could be obtained with as little as 5 ng of exosomes (Figure 5A). For exosomes individually purified from Covid-19 serum, small RNA cDNA library preparation was performed using total RNA recovered from exosomes purified from 200 μl of serum (two wells were prepared for each serum sample (100 μl per well) and pulls were pooled for RNA extraction). Briefly, 15 (Covid-19 library) to 18 (optimization experiments) ligations were individually set up by combining 9.5 μl of total RNA, 8.5 μl of master mix, and 1 μl of 50 μM adenylated barcoded 3' adapter (Integrated DNA Technologies, custom-made). A master mix was prepared using 0.0052 nM calibrator cocktail (40 μl 10x RNA ligase-2 buffer, 120 μl 50% DMSO, and 10 μl calibrator cocktail from

[42] ). Reactions were heated to 90°C for 1 min, incubated on ice for 2 min, and 1 μl (1 / 10 dilution) of truncated K227Q T4 RNA ligase 2 (New England Biolabs, #M0351L) was added to each reaction, which was then incubated on ice overnight in the cold room. The next day, ligations were heat inactivated at 90°C for 1 minute and precipitated individually by adding 1.2 μl Glycoblue™ mix (1 μl Glycoblue™ co-precipitant (15 mg / ml, ThermoFisher, #AM9516) in 26 μl 5M NaCl (ThermoFisher, #AM9579)) and 63 μl 100% ethanol was added to each tube. Reactions were combined, precipitated on ice for 1 hour, and centrifuged at 14,000 RPM, 4°C for 1 hour. Pellets were dried, resuspended in 20 μl nuclease-free water and 20 μl denaturing PAA gel loading solution, and resolved on a 15% urea-PAGE gel.Size marker RNA oligonucleotides (IDT) were used as a guide for gel excision. Gel pieces were crushed using gel breaker tubes (IST Engineering, #3388-100) and incubated overnight at 4°C, 1,100 RPM in 400 mM NaCl on a thermomixer. The next day, the solution was filtered and precipitated in 100% ethanol on ice for 1 hour. RNA pellets were obtained by centrifugation at 4°C, 14,000 RPM for 1 hour. 5' adapters were added to the resuspended pellets and T4 RNA Ligase 1 (New England Biolabs, #M0204L) was added for 1 hour at 37°C. Ligated products were resolved on a 12% urea-PAGE gel in the presence of 5' ligated size markers as a guide for size selection. The gel was spun in a gel breaker tube, after which the broken gel was resuspended in 1 μl of 300 mM NaCl solution containing 100 μM 3′ PCR primer and incubated overnight at 1,100 RPM on a thermomixer at 4° C. The solution was then filtered, precipitated with 100% ethanol, incubated on ice for 1 hour, and pelleted by centrifugation at 14,000 RPM for 1 hour at 4° C. The RNA pellet was resuspended in nuclease-free water (3 μl of 5× first strand buffer, 1.5 μl of 0.1 M DTT, and 4.2 μl of dNTP Mix (2 mM each, ThermoFisher, #R0241)) for reverse transcription with 0.75 μl of SuperScript® III Reverse Transcriptase (ThermoFisher, #18080-093) and incubated at 50° C. for 30 minutes. After inactivating the reverse transcription at 95° C. for 1 min, 95 μl of nuclease-free water was added. A pilot PCR reaction was set up (10 μl 10×PCR buffer, 10 μl dNTP Mix (2 mM each), 10 μl cDNA, 67 μl nuclease-free water, 0.5 μl 3′ PCR primer, 0.5 μl 5′ PCR primer, and 2 μl Titanium® Taq DNA polymerase (Clontech Laboratories, #639208)).Aliquots of 12 μl were withdrawn at cycles 10, 12, 14, 16, 18, 20, and 22 for analysis on a 2.5% agarose gel and identification of optimal PCR amplification cycles. Six PCR reactions were then set up and run at the optimal number of amplification cycles, and a portion (10 μl) was evaluated on a 2.5% agarose gel. The remaining solutions were combined, precipitated, digested with PmeI to remove size markers, and separated on a 2.5% gel. The 100 nt PCR library products were excised, purified with a QIAquick Gel Extraction Kit (Qiagen, #28704), and quantified using the Qubit® dsDNA HS Assay Kit (ThermoFisher, #Q32854). The cDNA libraries were then sequenced (single read 50 cycles) on a HiSeq2500 sequencing system (Illumina, #SY-401-2501), and the FASTQ files containing the raw sequencing data were then processed for adaptor trimming and small RNA alignment to the hg-19 genome. Read counts were normalized to total counts and statistical analyses were performed (see below).

[0294] Quantitative PCR experiments Quantitative PCR (qPCR) experiments were performed using TaqMan® microRNA Reverse Transcription Kit, TaqMan® microRNA Master Mix PCR Kit, and individual TaqMan® microRNA assays according to the manufacturer's instructions (ThermoFisher). For evaluation of non-specific exosome binding to Dynabeads® and streptavidin-coated 96-well plates, ath-miR-159a RNA oligos (Integrated DNA Technologies) were used. One picogram of ath-miR-159a was also added during RNA extraction of exosomes purified from Covid-19-infected serum samples to serve as a technical normalization control. In optimization experiments, RNA extracted from MCF-7 exosomes was used to quantify hsa-miR-21 (ThermoFisher, #000397) and hsa-miR-200c (ThermoFisher, #000505). For validation of differentially expressed miRNAs identified by sequencing of exosomal RNA from Covid-19 serum samples, TaqMan® miRNA primer assays for hsa-miR-126-3p (#002228), hsa-miR-146a (#002163), hsa-miR-126-5p (#000451), and hsa-miR-205 (#000509) were used. In these qPCR reactions, reverse transcription was set up using 10% (2 μl in 20 μl) of the RNA extracted from combined CD63 / CD81 / CD9 exosome purification from 100 μl serum. For quantification of individual transcripts, three individual qPCR experiments were set up using 1 / 3 (5 μl) of the RT reaction. Quantitative PCR measurements were performed on a Step-One-Plus instrument using streptavidin-coated 96-well plates and covers as recommended by the manufacturer. Quantitative data was transferred to an Excel sheet for statistical analysis as described below. Exemplary miRNA sequences are listed in Table 2 below. [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

[0295] BSL-3 Tissue Culture and Neutralization of SARS-CoV-2 Data Analysis For sequencing data analysis, raw FASTQ data files obtained from an Illumina HiSeq2500 sequencer were processed using the RNAworld server, including adapter trimming, read alignment, and annotation. miRNA counts were exported to a spreadsheet for data analysis. Statistical analysis of miRNA counts was performed using dedicated Bioconductor packages in the R platform, as detailed below. Heatmaps were generated from the transformed counts using the “NMF” package (heatmap function). Differential expression was assessed using “DESeq2” and “edgeR”. The differential expression model included a batch variable (library) to reduce batch bias. Interactions with sex and age were tested

[43] . To maximize the discrimination ability of miRNAs, we calculated a score for each sample (the “miRNA score”,

[44] ), which was compiled by summing the normalized levels (z-values) of all significantly upregulated miRNAs and the negative z-values ​​of all significantly downregulated miRNAs. For quantitative PCR (qPCR), the threshold cycle (Ct) value of ath-miR-159a (exogenous normalization control) was subtracted from the Ct values ​​of individual miRNAs to calculate the ΔΔCt comparison value. qPCR differential expression was evaluated by t-test or Mann-Whitney U-test of ΔΔCt values. For convalescent exosome neutralization, statistical analysis was performed using Graphpad Prism 8.0 software and data were evaluated using the mean ± standard error of the mean (SEM). All in vitro experiments consisted of both technical replicates (three separate wells) and biological replicates (three separate experiments). One-way analysis of variance (ANOVA) with Bonferroni post-hoc test was performed. A p-value <0.05 was considered statistically significant.

[0296] result Development of the EV-CATCHER assay for purifying exosomes from human biofluids We sought to optimize the customizable conjugation of selective antibodies to the conjugation platform to specifically purify exosomes from biofluids. Thus, using the design described by Lof et al., 2017 (Figure 2A), we first established the optimal hybridization conditions to generate the highest amount of double-stranded DNA linker for the subsequent assay (Figure 2B, lane 4). To increase the purity of dsDNA and prevent the expression of single-stranded 5' azido oligonucleotides that can bind to DBCO-activated antibodies and prevent their binding to the platform, the annealed dsDNA products were purified on a non-denaturing acrylamide gel. Uracil-glycosylase (UNG) was used to verify the degradable nature of the dsDNA linker through enzymatic digestion (Figure 2B, lane 5). We then determined the optimal ratio between dsDNA linker and DBCO-activated antibody, which was evaluated by 12% PAGE (Figure 2C, compare lanes c, d, and e). The attachment of the ds-DNA linker altered the migration pattern of the antibody, which could be restored by UNG glycosylase treatment and digestion of the dsDNA linker (Figure 2C, lane f). These experiments were repeated with different antibodies to verify the optimized conditions for the preparation of the ds-DNA linker and antibody duplex.

[0297] Identification of a low non-specific nucleic acid binding platform Although magnetic beads are widely used to isolate exosomes prior to cargo evaluation, repeated experiments performed in our laboratory suggested high levels of nonspecific small RNA binding in the absence of capture antibodies (Figure 8). Therefore, we sought to investigate the nonspecific binding capacity of magnetic beads by comparing nonmagnetic streptavidin-coated 96-well plates (wells, mainly used for ELISA assays) with Dynabeads™ (magnetic beads) MyOne™ Streptavidin T1 Beads (beads). We chose to use ath-miR-159a RNA oligonucleotides, an Arabidopsis specific miRNA that was not detected as a contaminant in our laboratory (control PCR experiments), as a source of small RNA for the evaluation of the two platforms (beads vs. wells). Using qPCR as a sensitive measurement tool, we determined that 1 ng of ath-miR-159a (Ct of 14 cycles, see Figure 3A and Figure 3B - black bars in all graphs) was optimal for quantifying nonspecific binding to wells and beads. For the assessment of nonspecific ath-159a RNA binding, four fractions were collected from the wells (Figure 3A, top left) or beads (Figure 3A, bottom left), including three subsequent PBS washes and one final elution after UNG digestion. Our data indicate that the wells retained the least amount of ath-miR-159a RNA, especially in the final elution (Figure 3A top left and bottom left, light gray bars), and that a large amount of ath-miR-159a RNA was nonspecifically bound and released at all steps, but especially during the final elution by the beads. We found that treatment of the wells and beads with RNase-A prior to incubation with ath-miR-159a (Figure 3A, top right and bottom right) largely eliminated nonspecific binding of ath-159a RNA and its high expression in the final elution with beads. This pretreatment suggests carryover of RNase-A activity in the elution and may not be suitable for all subsequent experiments, but may be necessary when using magnetic beads.Next, we sought to determine whether either the wells or the beads could also bind exosomes nonspecifically (Figure 3B). We performed qPCR assessment of hsa-miR-21 (Ct=24) and hsa-miR-200c (Ct=27), which we had previously detected in exosomal RNA extracts from MCF-7 cells. Given that commercially purified MCF-7 exosomes are obtained by ultracentrifugation, we expected exosome-free hsa-miR-21 and hsa-miR-200c, and therefore pretreated all reactions with RNase-A. Our data showed that approximately half of the miRNA signal remained, possibly suggesting protection from RNase-A activity by the exosomal lipid bilayer (Figure 3B, dark grey bars). The RNase-A-treated exosome solution was removed after incubation in the wells or with the beads, and then the hsa-miR-21 and hsa-miR-200c RNA levels remaining in the solution were assessed. Our data systematically confirmed that after incubation of the exosome solution on the wells, both miRNAs maintained the same detectable levels, while the majority of the miRNA signal was lost after incubation and removal of the beads (Figure 3B, light gray bars). These data suggest that the magnetic beads have the ability to nonspecifically bind both small RNAs and exosomes, which may contribute to the nonspecific RNA background observed when performing small RNA sequencing of beads incubated in serum (Figure 8). Therefore, in all subsequent experiments, we chose streptavidin-coated 96-well plates for immunopurification of exosomes from all biofluids evaluated.

[0298] Reproducibility and sensitivity of the EV-CATCHER assay for exosome purification from biological fluids In these proof-of-principle experiments, we assessed the purification of exosomes from three different biological sources: tissue culture medium of a human cell line (MCF-7), human plasma, and human serum, using anti-CD63 antibodies in the EV-CATCHER assay (Figure 4A-E). These exosome purifications were performed under decreasing total protein input (quantified with a NanoDrop spectrophotometer), and the reproducibility of CD63 pulldown was assessed by Western blotting using anti-Alix, -CD63, -CD9, and -CD81 antibodies for protein expression in exosome eluates (Figure 4A-C). Exosome purifications were performed using a single volume of serum (100 μl) due to limited availability. Transmission electron microscopy (TEM, Figure 4D) was used to reveal the presence of intact EVs with a size and morphology consistent with previous exosome evaluations (36). Finally, a Spectradyne nCS1 nanoparticle tracker was used to identify CD63 + The size distribution of purified and released exosomes was assessed, with MCF-7 cell culture medium (3.30 × 10 10 particles / ml (n=20,058)), plasma (6.93×10 9 particles / ml (n = 340,956)), and serum (2.27 × 10 10 The results revealed the presence of nanoparticles with sizes ranging from 65 to 150 nm for exosomes from 1000 μg / ml (n = 12,516) (Figure 4E). Collectively, these analyses revealed the uniform reproducibility of the EV-CATCHER assay for purifying exosomes from various biofluids.

[0299] CD63 purified by EV-CATCHER + Small RNA sequencing using exosomes Given that previous small RNA sequencing experiments using total RNA extracted from magnetic bead-purified exosomes (Figure 8) showed the presence of a high small RNA background, we sought to assess the specificity of the EV-CATCHER assay using the same small RNA cDNA library preparation protocol

[42] . As a first test, we verified the applicability of the cDNA library preparation protocol for the detection of circulating small RNAs by decreasing the amount of total RNA extracted from whole serum (Figure 5A). Repeated experiments demonstrated the reproducibility of the sequencing pipeline using low amounts of circulating small RNAs. We then compared small RNA sequencing data from RNA extracted from ultracentrifuged human plasma (classical purification of whole exosomes) and whole plasma and identified differences in specific miRNA expression (Figure 5B), further supporting that the RNA extraction method influences expression levels

[45] . As a measure of control, we confirmed the presence of specific small RNA expression differences between mouse and human exosomes (Figure 5B). Finally, we sought to determine whether EV-CATCHER could be used to specifically select a subpopulation of exosomes from biofluids. In these experiments, we chose to spike human plasma with mouse Raws264.7 exosomes. We customized the EV-CATCHER assay using an anti-mouse CD63 antibody that has no cross-reactivity with human CD63 to perform the purification of mouse exosomes from human plasma. For a sensitive assessment of the purified miRNA cargo, we used a cDNA library preparation protocol and sequenced the small RNA content of selected exosomes (Figure 5C). In these experiments, all libraries were cloned using new (repeat #1, 3 years) and old (repeat #2, 6 years) 3' barcoded adapters to highlight the inherent variability of cDNA library preparation protocols. As can be seen in Figure 5C, mouse CD63 captured from human plasma using the EV-CATCHER assay was sequenced using a cDNA library preparation protocol. +Small RNA expression data from exosomes contained mouse-specific small RNA sequences (tags) that were identified only in mouse RAWS264.7 whole exosomes (Figure 5C, columns 2 and 3). Additionally, miRNAs differentially detected in human whole plasma (but not in mouse RAWS264.7 exosomes) were not identified in mouse CD63 + We also noted that it was not detectable in purified exosomes. Interestingly, both whole RawS264.7 exosomes (representing a pool of exosomes) and CD63 + Differences in miRNA expression were observed among purified exosomes, and certain miRNAs were only detectable in pooled exosomes from murine RAWS264.7 cells. These next-generation sequencing experiments demonstrate the specificity of the EV-CATCHER assay for reproducible detection of circulating small RNAs and selective capture of exosome subpopulations from biofluids.

[0300] miRNA analysis of serum exosomes from mild and severe Covid-19 patients The location of our institute and its parent hospital network during the early days of the SARS-CoV-2 pandemic in the United States provided us with a unique opportunity to investigate pressing biological questions and further explore the clinical utility of the EV-CATCHER assay to identify miRNA biomarkers in serum samples from Covid-19 patients treated within our network. A key question regarding Covid-19 infection (which is associated with a wide range of patient outcomes) was to evaluate whether changes in circulating miRNA expression could help to identify patients at risk for severe disease early. We therefore established the first pilot study to purify circulating exosomes from serum of mild and severe Covid-19 patients (our set of SARS-CoV-2-infected serum samples) and evaluate their miRNA expression levels by next-generation sequencing. As a proof-of-principle, we chose to use a combination of anti-CD63, -CD81, and -CD9 antibodies to capture circulating exosomes from serum in an unbiased manner. To measure the sensitivity of the EV-CATCHER assay, we measured the differences in miRNA expression between mild and severe patients using total RNA extracted from low-level EVs (EV-CATCHER) and whole serum. Mild patients were hospitalized but did not require mechanical ventilation (n = 13), whereas severe patients showed acute respiratory distress syndrome (ARDS; according to the Berlin classification criteria

[46] ) and required mechanical ventilation (n = 17), and both groups had PCR-confirmed SARS-CoV-2 virus infection. Clinical data were obtained from these patients (Table 1). To minimize batch effects, small RNA cDNA libraries were prepared from RNA extracted from whole serum (30 samples; 2 libraries) or from serum exosomes (30 samples; 2 libraries) using 15 independent samples with an equal distribution of mild and severe RNA samples in each library.In our expression analysis, using total RNA from serum exosomes, 10 differentially expressed miRNAs were identified between the mild and severe sample groups (Figure 6A; hsa-miR-146a (p-val=0.00041), hsa-miR-126-3p (p-val=0.024), hsa-miR-424, (p-val=0.00454)), hsa-miR-151-3p (p-val=0.012), hsa-miR-126-5p (p-val=0.00017), hs a-miR-627-5p (p-val=0.011), hsa-miR-145 (p-val=0.015), hsa-miR-205 (0.00049), and hsa-miR-200c (p-val=0.01). Also, using total RNA from whole serum, only two differentially expressed miRNAs were identified between the mild and severe sample groups (Figure 6A; hsa-miR-550-5p (p-val=0.026), hsa-miR-629* (p-val=0.0088)). As shown in Figure 6B, the 10 exosomal miRNAs differentially expressed between the two groups could be reproducibly detected, although their abundance was low. We established an integrative miRNA signature using the 10 miRNAs differentially expressed in serum exosomes of mild and severe patients (Figure 6C). Evaluation of this signature between both exosomal small RNA libraries confirmed the expression differences observed between the mild and severe sample groups (Figure 6C). Interestingly, when this miRNA integration signature was evaluated for both serum exosomes and total serum (Figure 6D), significant differences were confirmed in serum purified exosomes between the mild and severe sample groups (Figure 6D, p-val=2e-07). Surprisingly, these 10 miRNAs showed no expression differences individually between the two patient groups across all serotypes (p-val=0.011). Finally, a gold standard quantitative PCR approach was utilized to evaluate the top four differentially expressed miRNAs identified in serum exosomes (Figure 6E).To allow a full technical validation of the obtained findings, we activated the novel anti-CD63, -CD81 and -CD9 antibodies initially used for purification of serum exosomes, purified novel exosomes using EV-CATCHER from the same 30 total serum samples (mild n=13, severe n=17) and extracted total RNA. Differential expression of hsa-miR-146a, hsa-miR-126-5p, hsa-miR-126-3p, and hsa-miR-205 was evaluated using TaqMan® assays, and the top two differentially expressed miRNAs, hsa-miR-146a (pval=0.0023) and hsa-miR-1226-3p (pval=0.036), were successfully validated (Figure 6E, top two box plots). To evaluate possible reasons for the lack of validation of the other two miRNAs, the average qPCR Ct values ​​of 30 samples for all four miRNAs are shown. Ct values ​​were We observed that only the lowest (highest expressed) miRNAs could be validated (see upper box plot, Ct values ​​in Figure 6E), which may suggest that these miRNA transcripts were below the qPCR detection threshold. Overall, our sequencing analysis demonstrates that serum exosomes contain low abundance specific miRNAs that can be captured using the EV-CATCHER assay and validated by qPCR. Our analysis also suggests that there are differences in miRNA expression between serum exosomes from mild and severe Covid-19 patients. [Table 3]

[0301] EV-CATCHER purified exosomes maintain functional properties for in vitro analysis Given that previous TEM analysis suggested that exosomes released after EV-CATCHER purification were largely intact, we addressed whether exosomes purified from biofluids using EV-CATCHER maintained their biological properties in in vitro analyses. For these proof-of-concept experiments, we established a second pilot study (convalescent serum sample set) in which we purified CD63+ circulating exosomes from the serum of six individuals who had recently recovered from SARS-CoV-2 infection (enrolled in HUMC's convalescent plasma study and assessed anti-SARS-CoV-2 by ELISA using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein as antigen (IgG)). As shown in Figure 7A, three of the serum samples were obtained from individuals with high IgG titers (High; defined as >10,000-fold dilution of serum) and three were obtained from serum of individuals with IgG levels below the limit of quantification (BLQ; undetectable). Using the EV-CATCHER assay, we purified exosomes from different serum samples and confirmed their identity by Western blot analysis using anti-Alix, -CD9, -CD63, and -CD81 antibodies (Figure 7B). These experiments revealed homogeneous purification of exosomes between high serum (Figure 7B, lanes 1-3) and BLQ serum (Figure 7B, lanes 4-6). Using TEM, we assessed the size, morphology, and integrity of exosomes purified from both high and BLQ serum samples (Figure 7C). The size distribution of exosomes was also assessed by nanoparticle tracking (Figure 7D), showing that exosomes at high concentrations (mean concentration: 1.99 × 10 10 / ml (n = 20.481) and BLQ (1.55 × 10 10We demonstrated a similar nanoparticle distribution (65–150 nm) among serum exosomes at 1000 / ml (n=20.449). We performed infection and propagation of the mNeonGreen SARS-CoV-2 reporter virus (viral replication produces a measurable green fluorescent protein signal) in gold standard Vero E6 (African green monkey kidney) cells, which harbor high levels of the ACE-2 receptor

[47] , thereby evaluating the neutralizing properties of different sera and purified circulating serum exosomes (Figure 7E) [48–50]. Treatment of VeroE6 cells with whole convalescent serum from an individual with high IgG titers resulted in neutralization of the SARS-CoV-2 virus, as expected (Figure 7E, high IgG), but not observed when whole serum from an individual with BLQ IgG titers was used (Figure 7E, BLQ IgG). To evaluate the potential neutralizing properties of circulating exosomes, we first obtained highly purified exosomes by the gold-standard ultracentrifugation (UC) method

[35] . To prevent carryover of serum IgG, successive ultracentrifugation steps and washes were performed, which allowed us to obtain approximately 1–2 × 10 of the initial serum. 7We estimated that this would lead to a 2-fold dilution (Figure 7E; 1 high, 1 BLQ). Our experiments revealed that Vero E6 cells pretreated with UC exosomes derived from high IgG titer serum samples resulted in a neutralizing effect on the SARS-CoV-2 virus similar to that seen with treatment of whole serum (Figure 7E, third column), whereas cells treated with UC exosomes derived from BLQ IgG titer serum samples showed no neutralizing effect against the virus (Figure 7E, fourth column). Based on the very high dilution obtained from UC of serum, our data suggest that the neutralizing effect observed with UC exosomes from serum with high IgG titer may be exosome-mediated. Therefore, we sought to confirm these unexpected results using serum exosomes purified and released by the EV-CATCHER assay. Our data showed that exosomes purified from high IgG titer serum (Figure 7E. Samples 1-3) showed neutralizing activity against SARS-CoV-2 similar to that of UC exosomes from high IgG titer serum, but this was not observed in cells treated with BLQ IgG titer serum samples. This was not observed in the IgG serum (Figure 7E, samples 4-6). These observations were verified by the detection of fluorescent viral particles by imaging (Figure 7F). These unexpected data suggest neutralizing properties of exosomes purified from convalescent serum with high IgG titers, although identification and confirmation of the mechanism of action remains to be determined. However, our analysis demonstrates that exosomes purified using the EV-CATCHER assay are comparable to those observed with highly purified UC exosomes from one of the same serum samples.

[0302] Figure 9A shows SDS PAGE analysis of exosome extracts from COVID19 convalescent serum (left, high IgG titer convalescent serum, RBD IgG>10,000; right, BLQ convalescent serum) stained for total protein and ACE-2. Figure 9B shows a graph of ACE-2 protein expression normalized to total protein for convalescent serum (left, high IgG titer convalescent serum, RBD IgG>10,000; right, BLQ convalescent serum), demonstrating a significant increase in ACE-2 in convalescent plasma from individuals with high IgG titers.

[0303] Consideration In this study, we developed a sensitive and customizable antibody-based exosome purification procedure, EV-CATCHER, to identify exosomal small RNA cargo in a high-throughput manner and release purified exosomes intact to evaluate their biological properties.

[0304] A key issue in identifying circulating miRNA biomarkers is the inherently high level of ambient miRNA noise, resulting in their signal being “attenuated” in biofluids, especially in detecting miRNA expression changes from cell populations within larger organisms

[32] . Evaluation of circulating exosomes offers a unique opportunity to evaluate compartmentalized miRNA cargo from specific cells, but current laboratory-based techniques have significant caveats [25–31]. We therefore optimized the EV-CATCHER assay with the intention of minimizing RNA background / noise during exosome purification, choosing to use streptavidin-coated 96-well plates as the binding platform, as we determined that magnetic beads would essentially nonspecifically capture RNA and exosomes, impeding our analysis. We proved the specificity of EV-CATCHER by sequencing RNA extracted from selectively purified CD63+ mouse RAWS264.7 exosomes spiked into human plasma using an optimized small RNA cDNA library generation protocol. Moreover, next-generation sequencing analysis of different circulating RNA fractions from the same biofluid (whole plasma, plasma-derived ultracentrifuged exosomes, and antibody-elicited exosomes) further validated the principle that differences in miRNA expression can be systematically identified based on RNA source and purification method. These data demonstrate the great potential of using the EV-CATCHER assay for the assessment of human disease pathogenesis by adapting antibody selection to the capture of cell-specific surface proteins present on circulating exosomes.

[0305] We proposed that by selectively selecting the biomarkers included, finely tuned, low abundance small RNAs could be detected. Our analysis supports this principle because: CD63 + / CD81 + / CD9 + Small RNA sequencing of purified exosomes allowed the identification of 10 miRNAs that were differentially expressed between mild and severe Covid-19 hospitalized patients, whereas none of these miRNAs could be detected as differentially expressed individually when whole serum (first serum sample set) was evaluated. Interestingly, when these 10 miRNAs were evaluated as an integrated miRNA signature using whole serum miRNA expression sequencing data, they were found to be able to distinguish the two patient groups (individual pval>0.005). These observations suggest that differences in unique miRNA expression from encapsulated exosomes are attenuated when evaluated within whole biofluids, further demonstrating the need for focused evaluation of specifically purified exosomes to discover low-expressing, potentially robust and clinically relevant miRNA biomarkers. Considering that qPCR is one of the gold standards for detection of RNA transcripts in clinical assays, it is important to note that qPCR has major limitations when dealing with total RNA extracted from circulating exosomes purified from small volumes of serum samples (100 μl). For example, during qPCR validation, the top two differentially expressed miRNAs (hsa-miR-146a and hsa-miR126-3p) retained statistical significance between the two patient groups (mild vs. severe), while hsa-miR-205 lost significance at a comparative detection threshold of ~38–39 amplification cycles. These evaluations suggest that detection of low-abundance biomarkers requires: 1) the use of more cell-specific antibodies for exosome subpopulation enrichment; 2) the input of larger amounts of biofluid material; and 3) a preamplification step to improve the qPCR detection threshold.

[0306] As we developed the EV-CATCHER assay to discover disease-associated small RNA biomarkers, we wanted to ensure the release of intact exosomes for accurate molecular analysis. Therefore, we decided to include an enzymatically degradable DNA linker between the capture antibody and the low nonspecific small RNA binding platform. To evaluate the intact release of exosomes, we used TEM and nanoparticle tracking experiments to evaluate the enrichment of size-specific intact exosome subpopulations. As our experiments suggest, this enzymatic release of intact exosomes may also be an opportunity to explore the functional properties of EV-CATCHER-purified exosomes using in vitro assays without interfering with in vivo analysis. As a proof of principle for the validation of the assay, we used a pool of well-recognized selection antibodies (anti-CD63 / CD81 / CD9) for the purification of exosomes, but our data suggest that customization with specific monoclonal antibodies has great potential for the selection and analysis of cell-specific exosomes circulating in biofluids. Indeed, recent studies have already shown that organ / tissue-specific exosomes can be purified from the circulation using monoclonal antibodies and then used for in vitro analysis [51-54].To increase the specificity of exosome capture, we proposed that an initial ultrafiltration or ultracentrifugation step could provide intact pooled exosomes, which could then be used to select single antibodies using the EV-CATCHER assay to assess miRNA biomarkers and functional properties.

[0307] As our institution and its parent institution were located in the midst of the early SARS-CoV-2 pandemic in the United States, we sought to determine whether our molecular assays could be utilized to evaluate sera from Covid-19-infected patients. Given the lack of molecular studies evaluating risk-predictive biomarkers for severe disease stratification in Covid-19 serum samples, we used the EV-CATCHER assay as a proof-of-principle to determine whether it could detect circulating exosomal miRNA expression changes associated with the severity of infection (first set of serum samples). Although small in scale, comparative exosomal miRNA expression analysis between mildly affected Covid-19 (n=13) and severely affected hospitalized patients requiring mechanical ventilation (n=17) allowed us to detect and validate by qPCR at least two downregulated miRNAs (miR-146a and miR-126-3p) that were not detectable in whole serum. These analyses not only validated the sensitivity of the EV-CATCHER assay, but also suggested that putative prognostic biomarkers associated with disease severity could be detected in serum exosomes from Covid-19 patients. Interestingly, previous studies suggested that both of these miRNAs are immune- and vascular-regulatory miRNAs, and are associated with inflammation and vascular-damaging events [55–60]. For example, a study by Taganov et al. suggested that hsa-miR-146a is a molecular brake on inflammation

[56] . A study of hsa-miR-126 revealed that it is a vascular miRNA that regulates angiogenesis

[61] , in part through the activation of VEGF signaling

[62] . More precisely, a study demonstrated that exosomes secreted from human endothelial progenitor cells were beneficial for lipopolysaccharide-induced acute lung injury in mice by delivering hsa-miRNA-126 (both miR-126-3p and miR-126-5p) to damaged alveoli

[63] . Although small, our observation that both hsa-miR-146a and hsa-miR-126-3p may be downregulated in hospitalized COVID-19 patients with severe disease on mechanical ventilation compared with those with milder disease is consistent with their reported anti-inflammatory and vascular health-promoting properties.These observations suggest that a larger-scale evaluation of serum exosomes from hospitalized Covid-19 patients could identify robust prognostic circulating miRNAs, which may help strategically improve clinical outcomes by identifying patients who would benefit most from convalescent plasma therapy.

[0308] Circulating exosomes are involved in immune responses during viral infections [64–68], and studies have demonstrated that exosomes can harbor proteins that inhibit viral replication (e.g., against HIV

[69] ) or that can induce B-lymphocyte proliferation (e.g., exosomes released from Epstein-Barr virus (EBV)-infected cells

[34] ). However, so far the potential role of circulating exosomes in virus neutralization of SARS-CoV2 has not been evaluated. In initial experiments, highly purified exosomes obtained by sequential ultracentrifugation from high IgG titer sera showed neutralizing activity against SARS-CoV-2 infection in vitro, whereas exosomes purified from sera with IgG titers below the quantification level did not show neutralizing activity. We decided to use the EV-CATCHER assay to replicate in vitro the neutralizing properties of Covid-19 recovery exosomes (second serum sample set). We confirmed this neutralizing activity in vitro using exosomes captured and released by the EV-CATCHER assay from sera with high IgG titers, which could not be observed with exosomes from sera with IgG titers below quantifiable levels. Although we may not be able to completely exclude the possibility of minimal IgG carryover (from all convalescent sera) using a highly specific purification assay, we found that convalescent exosomes highly purified by ultracentrifugation (at a dilution factor of approximately 1–2 × 10 7Exosomes (estimated to be 10–12 nm) showed virus-neutralizing properties similar to those observed with whole serum. Interestingly, recent studies have demonstrated that exosomes may transport immunoglobulins, as their estimated size of 10–12 nm is compatible with compartmentalization within circulating exosomes [35, 36]. Such exosomal properties may explain the strong association between exosomal neutralization and the presence of high neutralizing IgG titers, but further evaluation is required. However, additional mechanisms of virus neutralization have also been proposed, which may include an indirect cloaking strategy in which convalescent exosomes bind to the virus-targeted cells and prevent virus entry

[65] , or a direct cloaking strategy in which exosomes themselves harbor viral receptors (i.e., ACE2, the SARS-CoV-2 receptor) and cloak the viral particles

[70] . Although the exact neutralizing mechanism of Covid-19 convalescent exosomes requires further evaluation, the observation that exosomes isolated from high IgG titer sera have anti-SARS-CoV-2 neutralizing activity has important implications for the potential of purified exosomes as an augmentation to convalescent plasma therapy.

[0309] In summary, our study shows that the combination of highly sensitive small RNA cDNA library preparation, low small RNA background, and a reproducible and customizable exosome selection assay, EV-CATCHER, may help identify low-expression exosome-specific circulating biomarkers associated with disease. We also propose that this assay may provide a useful tool to evaluate the biological properties of different exosome subpopulations, both in vitro and potentially in vivo. Considering the 96-plex format of this assay, we propose that the purification and evaluation of circulating exosomes can be easily automated, offering great prospects for the sequential analysis of exome subpopulations from a single biofluid sample.

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Claims

1. 1. A method for preparing a purified population of biological particles from a biological sample from a subject, and a method for evaluating the cargo of said purified population of biological particles, comprising: a) (1) obtaining a biological sample containing biological particles; (2) contacting the biological sample containing biological particles from the subject with a binding agent directed to one or more biological particle surface antigens, where the binding agent is linked to a nucleic acid, and the nucleic acid is immobilized on a solid support; Preparing a purified population of biological particles; b) isolating the biological particles bound by the binding agent from the biological sample; and c) releasing the biological particles bound to the binding agent; and d) eluting the bound biological particles from the binding agent to form a population of free purified biological particles; e) evaluating the cargo and surface molecules, including proteins, DNA, RNA, or lipids, of the purified population of biological particles.

2. (A) Step e) of evaluating cargo and surface molecules comprises: (i) identifying specific proteins and / or lipids on the surface of said biological particles by mass spectrometry; or (ii) identifying the protein and / or lipid cargo by mass spectrometry; or (iii) identifying the DNA molecule by sequencing or quantitative PCR; or (iv) extracting RNA from the purified population of biological particles and identifying and quantifying expression of small non-coding RNA, including microRNA (miRNA), encapsulated by the purified population of biological particles; (B) Step c) of releasing the isolated biological particles comprises: (i) enzymatically cleaving the nucleic acid; or (ii) displacing the first strand of the nucleic acid bound to the antibody from the second strand of the nucleic acid bound to the support by strand displacement using a nucleic acid complementary to the first strand or the second strand of the nucleic acid and an enzyme having strand displacement activity that releases the antibody from the support; or (iii) separating the annealed DNA strands so that the antibody can be released from the platform without damaging the DNA strand bound to the antibody by polymerase chain reaction using an oligonucleotide complementary to the region of the DNA bound to the antibody; or 13. The method of claim 1, comprising (C) an initial ultrafiltration or ultracentrifugation step to provide a pooled heterogeneous population of source biological particles. (a) the biological sample comprises a bodily fluid; or (b) the solid support is a well plate, a polymer, or a surface; or (c) the isolated biological particle is derived from a healthy subject or a diseased subject; or (d) the binding agent that binds to one or more biological particle surface antigens is an antibody, an antibody-binding fragment, or an aptamer; or (e) the biological particle surface antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, or a heterologous cell surface polypeptide; or (f) the nucleic acid comprises DNA, RNA, or a combination thereof; or (g) the nucleic acid comprises non-naturally occurring nucleotides; or (h) the binding agent for one or more biological particle surface antigens is a dibenzocyclooctiine (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), Sulfo-SMCC, or a derivative thereof; The method of claim 1.

4. (a) the bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymphatic fluid, urine, stool, sweat, tears, nipple aspirate, or semen or a secreted biological fluid; or (b) the method of claim 3, wherein the body fluid is a circulating or secreted body fluid.

5. 5. The method of claim 4, wherein the circulating fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF) or lymphatic fluid.

6. The method of claim 1 , wherein the aptamer is a nucleic acid or a polypeptide.

7. The method of claim 1 , wherein the nucleic acid comprises DNA. (a) the DNA comprises one or more ribonucleic acid nucleotides; or (b) the DNA comprises a restriction enzyme recognition site; or (c) The method of claim 7, wherein the nucleic acid is a DNA / RNA duplex that can be degraded by an endonuclease or an RNAse (RNase-H).

9. 9. The method of claim 8, wherein the one or more ribonucleic acid nucleotides are uracil.

10. 4. The method of claim 3, wherein the nucleic acid further comprises a binding site on a first end of the nucleic acid and a binding site on a second end of the nucleic acid, and the binding site on the first end of the nucleic acid and the binding site on the second end of the nucleic acid are different. (a) the binding site on the first end of the nucleic acid is an avidin, streptavidin or carboxyl binding moiety; and / or (b) the binding site on the second end of the nucleic acid is an amine site; The method of claim 10.

12. The method of claim 11, wherein the binding site on the first end is biotin.

13. The method of claim 11 , wherein the amine moiety is an azide.

14. (a) the enzymatic cleavage is performed by uracil glycosylase; or (b) the enzymatic cleavage is performed by a restriction enzyme; or (c) the enzymatic cleavage is carried out by an endonuclease or an RNAse; or (d) The method according to claim 2, wherein the enzyme having strand displacement activity is a DNA polymerase, a topoisomerase or a helicase.

15. identifying said one or more small non-coding RNAs, including miRNAs, encapsulated in said one or more biological particles by next generation sequencing; or The method of claim 2 , wherein the isolated biological particle is derived from a healthy subject or a diseased subject.

16. 16. The method of claim 15, wherein the disease comprises viral infection, cancer, placental abnormalities, exercise-induced muscle damage, heart failure, Alzheimer's disease, liver cirrhosis, viral and bacterial infections, kidney disease, bone remodeling after injury, wound healing or COPD and grey matter.

17. 17. The method of claim 16, wherein the viral infection is a severe coronavirus infection.

18. 18. The method of claim 17, wherein the severe coronavirus infection is due to SARS-CoV-1, MERS or SARS-CoV-2. (a) the severe coronavirus infection is caused by SARS-CoV-2; and (b) the isolated and quantified miRNAs from subjects suffering from severe coronavirus infection by SARS-CoV-2 comprise one or more of hsa-miR-146a, hsa-miR-126-3p, hsa-miR-15a, hsa-miR-424, hsa-miR-151-3p, hsa-miR-126-5p, hsa-miR-627-5p, hsa-miR-145, hsa-miR-205, hsa-miR-200c, hsa-miR-550-5p, and hsa-miR-629.

20. 20. The method of claim 19, wherein the miRNA markers of severe SARSCoV-2 disease include hsa-miR-146a, hsa-miR126-3p, or both.

21. 1. An in vitro method for assessing risk of developing severe coronavirus infection in a subject and for treating the subject, comprising: a) (1) obtaining a biological sample containing biological particles from the subject; (2) contacting the biological sample containing biological particles from the subject with a binding agent for one or more biological particle surface antigens, where the binding agent is linked to a nucleic acid, and the nucleic acid is immobilized on a solid support; Preparing a purified population of biological particles; b) isolating the biological particles bound by the binding agent from the biological sample; and c) releasing the biological particles bound to the binding agent; and d) eluting the bound biological particles from the binding agent to form a population of free purified biological particles; e) (i) extracting RNA from the purified population of biological particles; (ii) identifying and quantifying the expression of small non-coding RNAs, including one or more microRNAs (miRNAs), encapsulated by the purified exosome population; and (iii) comparing the cargo profile of the purified biological particles with the cargo profiles of (1) control subjects not infected with the coronavirus, (2) control subjects infected with the coronavirus and suffering from mild symptoms, and (3) control subjects infected with the coronavirus and suffering from severe symptoms. determining a cargo profile of the purified biological particles by evaluating the cargo of the population of purified biological particles; f) determining the patient's risk of developing a severe viral infection, wherein the small non-coding RNA or protein cargo profile of the purified biological particle is about 1.5-fold lower or about 1.5-fold higher than the cargo profile from a coronavirus-infected control subject who developed a severe disease; g) administering an appropriate treatment to a patient at risk for said severe viral infection.

22. (a) comprising an initial ultrafiltration or ultracentrifugation step to provide a pooled heterogeneous population of biological particles; or (b) the step c) of releasing the isolated biological particles comprises: (i) enzymatically cleaving the nucleic acid; or (ii) displacing the first strand of the nucleic acid bound to the antibody from the second strand of the nucleic acid bound to the support by strand displacement using a nucleic acid complementary to the first strand or the second strand of the nucleic acid and an enzyme having strand displacement activity that releases the antibody from the support; or (iii) separating the annealed DNA strands so that the antibody can be released from the platform by polymerase chain reaction using an oligonucleotide complementary to the region of the DNA bound to the antibody, without damaging the DNA strand bound to the antibody; or (c) identifying and quantifying small non-coding RNAs, including one or more microRNAs (miRNAs), encapsulated by the population of purified biological particles, step d)(ii) is performed by next generation sequencing; 22. The method of claim 21.

23. (a) the biological sample comprises a bodily fluid; or (b) the severe coronavirus infection is due to SARS-CoV-1, MERS or SARS-CoV-2; or (c) the binding agent that binds to one or more biological particle surface antigens is an antibody, an antibody fragment, or an aptamer; or (d) the exosome surface antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, or a heterologous cell surface polypeptide; or (e) the nucleic acid comprises DNA comprising one or more ribonucleic acid nucleotides, wherein the ribonucleic acid nucleotides are uracil; or (f) the treatment appropriate for the patient at risk for severe viral infection comprises one or more of supportive care, antiviral agents, and immunomodulatory agents; 22. The method of claim 21.

24. (a) The bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymphatic fluid, urine, stool, sweat, tears, nipple aspirate, semen, or a secreted biological fluid; or (b) the body fluid is a circulating or secreted body fluid; 24. The method of claim 23.

25. 25. The method of claim 24, wherein the circulating fluid is whole blood, serum, plasma, CSF or lymphatic fluid.

26. 24. The method of claim 23, wherein the severe coronavirus infection is caused by SARS-CoV-2. (a) the population of miRNAs in the subject infected with SARS-Cov-2 compared to a control comprises one or more of hsa-miR-146a, hsa-miR-126-3p, hsa-miR-15a, hsa-miR-424, hsa-miR-151-3p, hsa-miR-126-5p, hsa-miR-627-5p, hsa-miR-145, hsa-miR-205, hsa-miR-200c, hsa-miR-550-5p, or hsa-miR-629; or (b) miRNA markers of severe disease caused by SARS-CoV-2 include hsa-miR-146a, hsa-miR126-3p, or both hsa-miR-146a and hsa-miR126-3p, when compared to controls; 27. The method of claim 26.

28. (a) the enzymatic cleavage is performed by uracil glycosylase; or (b) the enzymatic cleavage is performed by a restriction enzyme; or (c) the enzymatic cleavage is carried out by an endonuclease or an RNAse; or (d) the enzyme having strand displacement activity is a DNA polymerase, a topoisomerase or a helicase; 23. The method of claim 22.

29. (a) the supportive care includes intravenous fluids, supplemental oxygen, or nebulized steam; or (b) the antiviral agent inhibits viral entry, reduces viral load, or both; or (c) the antiviral agent is selected from acyclovir, gancidvir, foscarnet; ribavirin; amantadine, azidodeoxythymidine (zidovudine), nevirapine, tetrahydroimidazobenzodiazepinone (TIBO) compounds; efavirenz; remdesivir, lopinavir / ritonavir, umifenovir, favipiravir, ivermectin, and delavirdine; or (d) the immunomodulatory agent is a glucocorticoid or a recombinant interferon; or 24. The method of claim 23, wherein (e) the immunomodulatory agent is a glucocorticoid, and the glucocorticoid is a corticosteroid.

30. 30. The method of claim 29, wherein the glucocorticoid comprises prednisone, dexamethasone, azathioprine, mycophenolic acid, mycophenolate mofetil, or a combination thereof.

31. 1. A method for enhancing the therapeutic effect of convalescent plasma therapy for treating a patient at risk for severe coronavirus infection, comprising: a) (1) obtaining convalescent serum from the convalescent subject having a high IgG titer against the coronavirus; (2) contacting the convalescent serum with a binding agent directed to one or more biological particle surface antigens, where the binding agent is linked to a nucleic acid, and the nucleic acid is immobilized on a solid support; preparing said purified population of biological particles from convalescent serum of a convalescent subject; b) isolating the biological particles bound by the binding agent from the biological sample; and c) releasing the biological particles bound to the binding agent; and d) eluting the bound biological particles from the binding agent to form a population of free purified biological particles; e) measuring in vitro the neutralization titer of said purified population of biological particles against said coronavirus; f) administering to the subject the convalescent serum containing high neutralizing titer biological particles and high neutralizing titer IgG.

32. (a) comprising an initial ultrafiltration or ultracentrifugation step to provide a pooled heterogeneous population of biological particles; or (b) the step c) of releasing the isolated biological particles comprises: (i) enzymatically cleaving the nucleic acid; or (ii) displacing the first strand of the nucleic acid bound to the antibody from the second strand of the nucleic acid bound to the support by strand displacement using a nucleic acid complementary to the first strand or the second strand of the nucleic acid and an enzyme having strand displacement activity that releases the antibody from the support; or (iii) separating the annealed DNA strands so that the antibody can be released from the platform by polymerase chain reaction using an oligonucleotide complementary to the region of the DNA bound to the antibody, without damaging the DNA strand bound to the antibody; or (c) step e) of measuring the neutralization titer of the purified population of biological particles against SARS-CoV-2 virus further comprises incubating mammalian cells infected with coronavirus in vitro with a dilution series of the isolated and purified biological particles from the convalescent serum having a high IgG titer, and measuring viral particle production in comparison to a negative control (infected cells without the purified biological particles from the convalescent serum); 32. The method of claim 31 .

33. (a) the biological sample comprises a bodily fluid; or (b) the binding agent that binds to one or more biological particle surface antigens is an antibody, an antibody fragment, or an aptamer; or (c) the biological particle surface antigen comprises one or more of CD9, CD63, CD81, CD37, CD82, Alix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, or a heterologous cell surface polypeptide; or (d) the nucleic acid comprises DNA comprising a ribonucleic acid nucleotide, the ribonucleic acid nucleotide being uracil, or the DNA comprises a restriction enzyme recognition site; or (e) the severe coronavirus infection is due to SARS-CoV-1, MERS or SARS-CoV-2; or (f) the neutralized purified population of biological particles derived from the convalescent serum with high IgG titers contains the ACE2-receptor; or (g) the convalescent serum containing the neutralized purified biological particles can improve the efficacy of convalescent plasma therapy by at least about 2-fold compared to treatment with convalescent plasma therapy alone; and / or (h) the neutralized purified biological particles can present viral antigens to the immune system; 32. The method of claim 31 .

34. (a) The bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymphatic fluid, urine, stool, sweat, tears, nipple aspirate, semen, or a secreted biological fluid; or (b) the body fluid is a circulating body fluid; 34. The method of claim 33.

35. 35. The method of claim 34, wherein the circulating fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF) or lymphatic fluid.

36. the enzymatic cleavage is performed by uracil glycosylase; or The enzymatic cleavage is performed by a restriction enzyme; or The enzymatic cleavage is carried out by an endonuclease or an RNAse; or The enzyme having strand displacement activity is a DNA polymerase, a topoisomerase, or a helicase.

33. The method of claim 32.

37. 34. The method of claim 33, wherein the severe coronavirus infection is caused by SARS-CoV-2.

38. 33. The method of claim 32, wherein the viral particle production is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% as compared to a negative control.

39. 1. A method for preparing a purified cell population from a biological sample from a subject, comprising: a) (1) obtaining a biological sample from the subject comprising a cell; and (2) contacting the biological sample from the subject containing cells with a binding agent directed to one or more cell surface antigens, where the binding agent is linked to a nucleic acid, and the nucleic acid is immobilized on a solid support; Preparing a population of purified cells; b) isolating the cells bound by the binding agent from the biological sample; and c) releasing the cells bound to the binding agent; and and d) eluting the bound cells from the binding agent to form a purified population of cells.

40. (a) including an initial ultrafiltration or ultracentrifugation step to provide a pooled heterogeneous population of source cells; and / or (b) the step c) of releasing the isolated cells comprises: (i) enzymatically cleaving the nucleic acid; or (ii) displacing the first strand of the nucleic acid bound to the antibody from the second strand of the nucleic acid bound to the support by strand displacement using a nucleic acid complementary to the first strand or the second strand of the nucleic acid and an enzyme having strand displacement activity that releases the antibody from the support; or (iii) separating the annealed DNA strands so that the antibody can be released from the platform by polymerase chain reaction using an oligonucleotide complementary to the region of the DNA bound to the antibody, without damaging the DNA strand bound to the antibody; 40. The method of claim 39.

41. (a) the biological sample is prepared in vivo or in vitro; and / or (b) the one or more cell surface antigens comprise CD4, CD8, CD9, CD46, CD63, CD81, CD37, CD82, CD138, CD151, ALix, ACE-2, Tim4, PLAP, adiponectin, FABP4, caveolin-1, cytokeratin, EPCAM, E-cadherin, P63, or a heterologous cell surface polypeptide; and / or (c) the binding agent that binds to one or more cell surface antigens is an antibody, an antibody-binding fragment, or an aptamer; and / or (d) the nucleic acid comprises DNA, RNA or a combination thereof; and / or (e) the nucleic acid comprises non-naturally occurring nucleotides; and / or (f) the nucleic acid further comprises a binding site on a first end of the nucleic acid and a binding site on a second end of the nucleic acid, the binding site on the first end of the nucleic acid and the binding site on the second end of the nucleic acid being different; and / or (g) the binding agent for one or more cell surface antigens is a dibenzocyclooctiine (DBCO) molecule, 2-IT (2-iminothiolane), MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), SATA (N-succinimidyl S-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), Sulfo-SMCC, or a derivative thereof; and / or (h) the solid support is a well plate, a polymer, or a surface; and / or (i) the population of purified cells comprises non-eukaryotic cells, eukaryotic cells, or a combination thereof; and / or (j) the purified population of cells is derived from a healthy subject or a diseased subject; 40. The method of claim 39.

42. 40. The method of claim 39, wherein the biological sample comprises a bodily fluid.

43. The bodily fluid comprises whole blood, serum, plasma, cerebrospinal fluid (CSF), lymphatic fluid, urine, stool, sweat, tears, nipple aspirate, semen or secreted biological fluids; or The body fluid is a circulating body fluid or a secreted body fluid.

43. The method of claim 42.

44. 44. The method of claim 43, wherein the circulating fluid is whole blood, serum, plasma, cerebrospinal fluid (CSF) or lymphatic fluid.

45. 42. The method of claim 41 , wherein the heterologous cell surface polypeptide is expressed by a chimeric antigen receptor T cell (CAR-T cell).

46. 42. The method of claim 41 , wherein the aptamer is a nucleic acid or a polypeptide.

47. the nucleic acid comprises DNA; or The nucleic acid is a DNA / RNA duplex that can be degraded by an endonuclease or an RNAse (RNase-H), 42. The method of claim 41.

48. the DNA comprises one or more ribonucleic acid nucleotides; or the DNA comprises a restriction enzyme recognition site; 48. The method of claim 47.

49. 49. The method of claim 48, wherein said one or more ribonucleic acid nucleotides is uracil.

50. the binding site on the first end of the nucleic acid is avidin, streptavidin or a carboxyl binding moiety; and / or the binding site on the second end of the nucleic acid is an amine site; 42. The method of claim 41.

51. The binding site on the first end is biotin; and / or the amine moiety is an azide; 51. The method of claim 50.

52. (a) the enzymatic cleavage is performed by uracil glycosylase; or (b) the enzymatic cleavage is performed by a restriction enzyme; or (c) the enzymatic cleavage is carried out by an endonuclease or an RNAse; or (d) the enzyme having strand displacement activity is a DNA polymerase, a topoisomerase or a helicase; 40. The method of claim 39.

53. 42. The method of claim 41, wherein the eukaryotic cell is human, mouse, rat, dog, non-human primate or feline.

54. 40. The method of claim 39, wherein the disease comprises cancer, viral infection, placental abnormality, inflammation.

55. (a) the cell is an endothelial cell, an epithelial cell, a T cell, a fibroblast, an adipocyte, a neuronal cell, a tumor cell, a blood cell, or a cardiac cell; or (b) the population of cells is (i) non-eukaryotic and eukaryotic species; or (ii) healthy normal tissue, or diseased tissue, or (iii) mouse orthotopic / xenograft / PDX organs, or (iv) blood, or (v) derived from tissue culture; 40. The method of claim 39.

56. 55. The method of claim 54, wherein the viral infection is a severe coronavirus infection.

57. 57. The method of claim 56, wherein the severe coronavirus infection is due to SARS-CoV-1, MERS or SARS-CoV-2.

58. 57. The method of claim 56, wherein the severe coronavirus infection is due to SARS-CoV-2.

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