Methods of analyzing immunoglobulin autoantibodies for diagnosing risk of microbial disease severity and guiding therapy

Abstract

Disclosed herein are methods of diagnosing a subject for a risk of developing severe coronavirus disease as correlated to the presence of IgA anti-IFN-alpha autoantibodies. In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of IgA1 and / or IgA2 anti-IFN-alpha autoantibodies and simultaneous measurement of IFN-α and CXCL10 in the same nasal sample. In certain embodiments, methods further comprise reporting the diagnosis to the subject or medical professional. In certain embodiment, methods further comprise administering a pharmaceutical agent to the subject.

Description

[0001] METHODS OF ANALYZING IMMUNOGLOBULIN AUTOANTIBODIES FOR DIAGNOSING RISK OF MICROBIAL DISEASE SEVERITY AND GUIDING THERAPY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 715,902 filed November 4, 2024. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant number All 67032 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] COVID- 19 infections, otherwise known as Severe Acute Respiratory Syndrome of Coronavirus 2 or SARS-CoV-2, can become life-threatening for certain individuals. Lifethreatening SARS-CoV-2 is reported to be associated with hyper-inflammatory responses in the airways as opposed to uncontrolled viral load. See e.g., Hadjadj et al., Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369, 718-724 (2020). A number of antiviral medications are available; however, their effectiveness varies and can potentially lead to the development of resistant SARS-CoV-2 strains. See Dinata et al. Viruses, 2025, 18; 17(5):722. Thus, there is a need to identify improved methods for diagnosing whether an infected individual will develop life threatening complications from a SARS-CoV-2 infection.

[0008] Channappanavar et al. report dysregulated type I interferon and inflammatory monocytemacrophage responses cause lethal pneumonia in SARS-CoV-infected mice. Cell Host Microbe 19, 181-193 (2016).

[0009] Israelow et al. report a mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling. J Exp Med. 217, e20201241 (2020).

[0010] Vox et al. report modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques. Sci Immunol, 8, eadg0033 (2023). Liechti et al. report immune phenotypes that are associated with subsequent COVID-19 severity inferred from post-recovery samples. Nat Commun, 13, 7255 (2022).

[0011] Wijst et al. report Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19. Sci Transl Med, 13, eabh2624 (2021).

[0012] Bastard et al. report autoantibodies neutralizing type I IFNs are present in 4% of uninfected individuals over 70 years old and account for 20% of COVID- 19 deaths. Sci Immunol, 6, eabl4340 (2021).

[0013] Bastard et al. report preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1. J Exp Med. 218, e20210554 (2021).

[0014] Wang et al. report diverse functional autoantibodies in patients with COVID-19. Nature 595, 283-288 (2021).

[0015] Vazquez et al. report neutralizing autoantibodies to type I interferons in COVID-19 convalescent donor plasma. J Clin Immunol, 41, 1169-1171 (2021).

[0016] Meisel et al. report mild COVID-19 despite autoantibodies against type I IFNs in autoimmune polyendocrine syndrome type 1. J Clin Invest, 2021, 13 l(14):el 50867.

[0017] Muri et al. report autoantibodies against chemokines post-SARS-CoV-2 infection correlate with disease course. Nature Immunol, volume 24, pages 604-611 (2023).

[0018] Iyer et al. report using a QuantiBRITE™ bead method for quantitation of CD38 expression on CD8 positive T-lymphocytes. Cytometry, 33:206-212 (1998).

[0019] Yan et al. report microsphere-based multiplexed flow cytometric immunoassay for influenza virus detection and differentiation. Anal Chem, 2005, 77, 7673-7678.

[0020] Perfetto et al. report procedures using a variety of bead particles to detect fluorescent marker combinations on cells used for polychromatic analysis using flow cytometry. Nat Protoc, 2006, 1(3): 1522-30.

[0021] See also US Patent Nos. 5,286,452, 5,981,180, and 6,159,748. US Patent App. No. 2021 / 0088517, and WO W02007 / 118120

[0022] References cited herein are not an admission of prior art.

[0023] SUMMARY

[0024] Disclosed herein are methods of diagnosing a subject at risk of developing severe coronaviral, influenza, viral, or other microbial disease comprising: quantifying IgA anti-IFN- alpha autoantibodies from sample of a subject exhibiting symptoms of a coronavirus, influenza, or other viral or microbial disease (e.g. bacterial or parasitic infection). In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of IgA (IgAl and / or IgA2) anti-IFN-alpha autoantibodies and simultaneous measurement of IFN-a and CXCL10 in the same nasal sample.

[0025] In certain embodiments, methods include determining the quantity of nasal or airway IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value, e.g.. a typical sample from a subject without microbial disease or ii) greater than a reference or normal value; and diagnosing the subject exhibiting symptoms of a coronavirus, influenza, or other viral or microbial disease as at high risk of developing severe coronavirus, influenza, or other viral or microbial disease if quantifying nasal or airway IgAl anti-IFN-alpha autoantibodies from the nasal or airway sample is similar to a reference or normal value; or diagnosing the subject exhibiting symptoms of microbial disease as a low risk of developing severe microbial disease if quantifying nasal or airway IgAl anti-IFN-alpha autoantibodies from the nasal or airway sample is greater than a reference or normal value.

[0026] In certain embodiments, methods further comprise providing particles coated with antiIgA 1 antibodies or other IgAl specific binding agents, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl antibodies or other specific finding agents coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the sample from the subject diagnosed with an infection with the fluorescent particles coated with anti-IgAl antibodies that specifically bind with the anti-IgAl fluorescent coated particles providing IgAl anti-IFN-alpha autoantibody fluorescent particles in the sample; analyzing the IgAl anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of IgAl anti-IFN-alpha autoantibodies in the sample of the subject. In certain embodiments, the sample is from nasal aspirates or endotracheal aspirates (ETAs).

[0027] In certain embodiments, methods further comprise detecting elevated levels of INF-alpha in the nasal and / or airway sample, wherein if the subject does not produce nasal IgA (IgAl or IgA2) anti-IFN-alpha autoantibodies and does not produce IFN-alpha in the nasal or airway, then those subjects are at greater risk of developing severe coronaviral disease, severe influenza disease, or other severe viral or microbial disease. In certain embodiments, method further comprises detecting elevated levels of CXCL10 in the nasal and / or airway sample, wherein if the subject does not produce nasal IgA (e.g. IgAl or IgA2) anti-IFN-alpha autoantibodies and does not produce CXCL10 in the nasal or airway, then the subject is at greater risk of developing severe coronaviral disease, severe influenza disease, or other severe viral or microbial disease.

[0028] In certain embodiments, the IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2 to 4 micrometers or 1 to 5 micrometers. In certain embodiments, IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.5 pm, is exposed to a 561 nm wavelength, and a fluorescent signal is measured from 586 to 610 nm in wavelength. In certain embodiments, the IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.8 pm, is exposed to a 488 nm wavelength, and a fluorescent signal is measured from 475 to 540 nm in wavelength.

[0029] In certain embodiments, methods further comprise recording a quantification and / or the risk of developing microbial disease severity on a non-transitory computer readable medium. In certain embodiments, the risk of developing a coronaviral disease severity is a relative, high, low, medium, or quantitative number.

[0030] In certain embodiments, methods further comprise communicating the risk of developing a coronaviral disease severity to a medical professional or the subject. In certain embodiments, if the subject is diagnosed with a high risk of severe disease, then method comprise administering an antiviral, antimicrobial or coronaviral drug to the subject. In certain embodiments, if the subject is diagnosed with a high risk of severe disease, methods further comprise administering mechanical assisted oxygen to the airways and lungs of the subject.

[0031] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0032] Figure 1A illustrates FlowBEAT™ sampling from different tissues to reveal antibody isotypes and subclasses and antigen specificities.

[0033] Figure IB shows a dot plot data indicating the linear range of the anti-RBD MFI signal by FlowBEAT™ serial dilution of seropositive NIH standard (n = 3 independent replicates) and monoclonal human IgGl anti-RBD antibodies.

[0034] Figure 2A shows data on the types and specificities of antibodies against SARS-CoV-2 structural and nonstructural proteins change with disease severity. Longitudinal plots showing isotype-specific anti-spike protein MFT signal (y axis) across infection including and up to four doses of mRNA vaccination (x axis) in the nasal mucosa and blood of patients with mild and moderate COVID-19 (n = 36 nasal and 107 longitudinal samples; n = 52 systemic and 116 longitudinal samples). Solid lines connect longitudinal samples from individual patients.

[0035] Figure 2B shows data on tissue-specific antibody signatures against SARS-CoV-2 NSPs, including ORF proteins (NSP signature). Pie charts show the percentage of donors with a detectable anti-SARS-CoV-2 NSP signal, defined by MFI > 102. The shading in the pie chart corresponds to peak antibody MFI signal during infection.

[0036] Figures 3A and 3B show data indicating anti-IFN-alpha autoantibodies are transiently induced in the nasal mucosa after infection and are associated with viral load and local IFN-alpha secretion.

[0037] Figure 3A shows Longitudinal plot data of the post-onset induction of nasal IgAl, IgA2, and IgGl anti-IFN-alpha2a autoantibodies in nasal swabs and blood samples from individuals with mild and moderate COVID-19. Days after onset are on the x axis, and longitudinal samples from individual donors are linked by solid lines. Loess regression smooth curves of the same data are overlaid with the confidence interval shown as a shaded area, and corresponds to the antibody isotype. Pie charts show the percentage of donors with detectable anti-IFN-alpha2a in nasal swabs and blood samples. The gradient shading in the pie chart corresponds to the peak antibody MFI signal during acute infection.

[0038] Figure 3B show data on longitudinal viral load [viral copies per milliliter, quantitative PCR (qPCR)] in nasal swabs. Patients were grouped by the presence (IgA producers) or absence (IgA nonproducers) of anti-IFN-alpha2a autoantibodies.

[0039] Figures 4A and B shows data from patients with severe COVID-19 indicating persistent IFN-alpha and anti-IFN-alpha responses associated with viral load, hyperinflammation, and lower anti-SARS-CoV-2 humoral immunity.

[0040] Figure 4A shows Pearson correlation between ETA IgAl and IgA2 anti-IFN-alpha autoantibodies and IFN-alpha2a cytokine.

[0041] Figure 4B shows a bar chart comparing the relative IgAl and IgA2 subclass usage in the airways of patients with mild or moderate (nasal swabs) and severe (ETAs) COVID- 19.

[0042] Figure 5A shows data indicating nasal anti-IFN-a autoantibodies emerge before the peak of anti-viral (HA) response. Figure 5B shows data in mice for influenza.

[0043] DETAILED DESCRIPTION

[0044] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0046] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0047] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0048] An “embodiment” refers to a specific or contemplate example but is not necessarily limited to such an example. Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0049] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0050] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0051] "Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods.

[0052] “Subject” refers to any animal, preferably a human patient, livestock, rodent, monkey, or domestic pet.

[0053] As used herein, the term "predicting" refers to making a finding that an individual has a significantly probability of developing or enhanced symptoms associated with a coronavirus, other virus, or microbial agent.

[0054] A "normal" individual or sample from a "normal" individual as used herein for quantitative and qualitative data refers to an individual not diagnoses as having or would be assessed by a physician as not having a coronavirus, virus, or other microbial infection.

[0055] A “normalized measured” value refers to a measurement taken and adjusted to take background into consideration. Background subtraction to obtain total fluorescence is considered a normalized measurement. The background subtraction allows for the correction of background fluorescence that is inherent in the optical systems and assay buffers.

[0056] As used herein, a “reference value” can be an absolute value; a relative value; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample or a large number of samples, such as from patients or normal individuals.

[0057] A “diagnosis” of COVID- 19, coronavirus, virus, or other microbial can made be by a medical professional or by laboratory techniques or by using commercially available test kits, e.g., to detect the associated proteins or particles derived, e.g., fluorescent antibodies conjugated to particles evaluated by flow cytometry from the microbe from a sample, or by PCR or other nucleic acid based techniques. Such a diagnosis may be performed at the office of a doctor, a pharmacy, or by using an at-home testing kit. The molecular tests and antigen tests may include using a swab obtained from the inside of the nose or mouth. Symptoms of coronavirus and other microbial agents may include fever, chills or shivering, cough, shortness of breath or difficulty breathing, fatigue (physically and mentally tired), muscle pain or body aches, headache, confusion, changes or loss of taste or smell, sore throat, stuffy or runny nose, nasal congestion, nausea or vomiting, diarrhea, chest pain or chest pressure , trouble breathing, persistent pain or pressure in the chest, inability to wake or stay awake, pale, gray, or blue-colored skin, lips, or nail beds.

[0058] An individual with “mild” or “moderate” CO VID- 19, coronavirus, virus, or other microbial infection" and the like, is an individual with COVID-19, coronavirus, virus, or other microbial infection or has symptoms such as cough, sore throat, and / or fatigue but does not have a substantial shortness of breath necessitating the use of an assisted breathing device / ventilator (air and / or oxygen) or as diagnosed by a medical professional.

[0059] An individual with “severe” COVID-19, coronavirus, virus, or other microbial infection" and the like, is an individual who has been diagnosed with COVID-19, coronavirus, virus, or other microbial infection or has symptoms such as cough, sore throat, and / or fatigue and has a substantial shortness of breath or requires an assisted breathing device / ventilator such as from an oxygen tank or as diagnosed by a medical professional.

[0060] As used herein, unless the context suggests otherwise, “separating” refers to purify the particles or cells from other particles or impurities that do not contain or contain less of a target molecule on the surface of the particle. One method of selecting proteins that are on the outside of a particle is to conjugate the protein directly to the particle surface using coupling reagents or by providing a specific binding agent on the surface, or by use of a primary antibody that binds a target, and further trap the primarily antibody bound to the particle or cell using a secondary antibody that is conjugated to beads or cells. The beads can be magnetic and captured by a magnetic field and separated from the rest of a solution. In another method, beads may have a fluorescent signature wherein secondary antibodies contain a fluorescent marker and the particles can be separated using flow cytometry or fluorescence activated sorting. Flow cytometry (FC) is method for detecting and measuring physical and chemical characteristics of a population of particles such as a micro sized particles or cells. A sample containing cells or particles is typically suspended in a fluid and injected into a flow cytometer instrument. Measurements of cells or particles are taken as they travel in a stream by a light source / a laser beam, e.g., diode laser, which is focused on the stream as cells or particles travel single-file through the stream at the point of light or laser interrogation referred to as hydrodynamic focusing. Within the flow cell, a slow-moving sample stream is injected into a faster moving “sheath” stream. The sample stream is integrated at the injection point into the faster moving sheath stream. Adjusting the velocity of the streams controls the width of the center stream aligning of the cells or particles within the center stream. Photons from the laser beam diverge, e.g., by light diffraction, from their path as they contact the passing cells or particles. A detector to collect “scattered” light referred to as Small Angle Light Scatter (SALS), Forward Angle Lights Scatter (FALS), or Forward Scatter (FSC). Forward scatter is proportional to cell or particle size or the presence other constituents that make up their composition as photons pass through the cell or translucent particle. If the photon strikes an obstacle, e.g. large protein complex, organelle, nucleus, etc., the photon will be reflected at a larger angle than those generated by the forward scatter phenomenon. In a typical cytometer, a second detector is placed perpendicular to the laser path to collect light scattered in this manner. This is known as Wide Angle Light Scatter (WALS), Orthogonal Light Scatter (OLS), 90° Lights Scatter, or, commonly, Side Scatter (SSC). Side scatter is proportional to complexity of the cell or particle; the more bits inside the cells or particles, the more of light scatter, the higher the detected signal.

[0061] One can conjugate a fluorescent molecule to the particles with fluorescent core shell properties to further distinguish or “stain” the particles within a tag. As these particles passed through the stream, the laser light would excite the fluorescent core, tag, or fluorochrome to emit photons of light, e.g., at a higher wavelength (e.g., fluorescein isothiocyanate emits light at -530 nm when excited by a 488 nm laser). This light can be collected and used to further categorize the cells or particles in one or more a detectable fluorescent pattem(s) in addition to forward scatter and side scatter depending on the position of detectors of the fluorescent light or scattered light. Such designs may contain light filters and mirrors to direct signals to desired detectors.

[0062] The term “fluorescence-activated sorting,” “fluorescence-activated cell sorting” or “FACS” refers to a method of sorting a mixture of particles or cells into two or more areas, typically one particle or cell at a time, based upon the fluorescent characteristics of each particle or cell. It is typically accomplished by applying an electrical charge and separating by movement through an electrostatic field. Typically, a vibrating mechanism causes a stream of particles or cells to break into individual droplets. Just prior to droplet formation, a particle or cell in a fluid pass through an area for measuring fluorescence. An electrical charging mechanism is configured at the point where the stream breaks into droplets. Based on the fluorescence intensity measurement, a respective electrical charge is imposed on the droplet as it breaks from the stream. The charged droplets then move through an electrostatic deflection system that diverts droplets into areas based upon their relative charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. In other systems, a charge is provided on a conduit inducing an opposite charge on the droplet.

[0063] Method of Diagnosis

[0064] In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of IgAl and / or IgA2 anti-IFN-alpha autoantibodies IgA and simultaneous measurement of IFN-a and CXCL10 in the same nasal sample.

[0065] In certain embodiments, this disclosure relates to methods of diagnosing a subject for a risk of developing severe viral or microbial disease comprising: quantifying IgAl anti-IFN-alpha autoantibodies from a nasal sample of a subject diagnosed with a viral or microbial infection; determining the quantity of nasal IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value or ii) greater than a reference or normal value; and diagnosing the subj ect with the viral or microbial infection as having a high risk of developing severe disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is similar to a reference or normal value of a subject without an infection; or diagnosing the subject with the viral or microbial infection as having a low risk of developing severe disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is greater than a reference or normal value.

[0066] In certain embodiments, methods further comprise providing particles coated with anti- IgAl antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal sample from the subject diagnosed with a viral or bacterial infection with the fluorescent particles coated with anti-IgAl antibodies that specifically bind with the anti-IgAl fluorescent particles providing IgAl anti-IFN- alpha autoantibody fluorescent particles in the sample; and analyzing the IgAl anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of IgAl anti-IFN-alpha autoantibodies in the nasal sample of the subject.

[0067] In certain embodiments, methods further comprise detecting elevated levels of INF-alpha in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN- alpha autoantibodies and does not produce IFN-alpha in the nasal or airway, then those subjects are at greater risk of developing severe disease.

[0068] In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of elevated levels of IgAl and IgA2 anti-IFN-alpha autoantibodies in the same nasal sample.

[0069] In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of elevated levels of IgAl or IgA2 anti-IFN-alpha autoantibodies in the same nasal sample.

[0070] In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of IgAl and / or IgA2 anti-IFN-alpha autoantibodies and simultaneous measurement of IFN-a and CXCL10 in the same nasal sample.

[0071] In certain embodiments, methods further comprise detecting elevated levels of CXCL10 in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and / or does not produce CXCL10 in the nasal or airway, then the subject is at greater risk of developing severe disease.

[0072] In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of IgAl and / or IgA2 anti-IFN-alpha autoantibodies and simultaneous measurement of IFN-a and CXCL10 in the same nasal sample.

[0073] In certain embodiments, the methods include the use of fluorescent beads that are used to analyze the presence of IgAl and / or IgA2 anti-IFN-alpha autoantibodies and simultaneous measurement of IFN-a and CXCL10 in the same nasal sample.

[0074] Disclosed herein are methods of diagnosing a subject for a risk of developing a coronaviral or other viral or microbial disease severity comprising: quantifying nasal or airway IgAl anti-IFN- alpha autoantibodies from a nasal or airway sample of a subject exhibiting symptoms of a coronavirus or other viral or microbial disease; determining the quantity of nasal or airway IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value of a subject without microbial disease or ii) greater than a reference or normal value of a subject without microbial disease; and diagnosing the subject exhibiting symptoms of a coronavirus or other viral or microbial disease as at high risk of developing severe coronavirus or other viral or microbial disease if quantifying nasal or airway IgAl anti-IFN-alpha autoantibodies from the nasal airway sample is similar to a reference or normal value of a subject without a microbial disease, i.e., of a low quantitative value; or diagnosing the subject exhibiting symptoms of microbial disease as a low risk of developing severe microbial disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal airway sample is greater than a reference or normal value of a subject without microbial disease.

[0075] In certain embodiments methods further comprises providing particles coated with anti- IgAl antibodies and particles conjugated to anti-Ig subclasses antibodies other than IgAl antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl and anti-Ig subclasses other than IgAl antibodies coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal, airway or other sample from the subject exhibiting symptoms of a coronavirus or other viral or microbial disease with a mixture of the fluorescent particles coated with anti-IgAl and anti-Ig subclass other than IgAl under conditions such that nasal IgAl anti-IFN-alpha autoantibodies in the nasal or airway sample specifically bind with the anti-IgAl fluorescent particles providing nasal or airway IgAl anti-IFN-alpha autoantibody fluorescent particles in the sample, and nasal or airway anti-Ig subclass other than IgAl anti-IFN-alpha autoantibodies in the sample specifically bind with the anti-Ig subclass other than IgAl fluorescent particles providing nasal or airway anti-Ig subclass other than IgAl anti-IFN-alpha autoantibody fluorescent particle in the sample; and analyzing the nasal or airway IgAl anti-IFN-alpha autoantibody fluorescent particles and the nasal or airway anti-Ig subclass other than IgAl anti-IFN-alpha autoantibodies fluorescent particles by flow cytometry providing a quantity of nasal or airway IgAl anti-IFN-alpha autoantibodies and nasal or airway anti-Ig subclass other than IgAl anti-IFN-alpha autoantibodies in the nasal or airway sample of the subject.

[0076] In certain embodiments, methods further comprise detecting elevated levels of INF-alpha in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN- alpha autoantibodies and does not produce IFN-alpha in the nasal or airway, then those subjects are at greater risk of developing severe disease.

[0077] In certain embodiments, methods further comprise detecting elevated levels of CXCL10 in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce CXCL10 in the nasal or airway, then the subject is at greater risk of developing severe disease.

[0078] In certain embodiments, the subject is exposed to an environment providing a high risk of contracting a coronaviral infection, e.g., medical professional, and the subject is not exhibiting symptoms of an infection.

[0079] In certain embodiments, the coronavirus is an epidemic coronavirus, pandemic coronavirus, SARS-CoV2, SARS-CoV-1, MERS-CoV, endemic human coronavirus, HCoV-229E, HCoV-OC43, HCoV-NL63, or HCoV-HKUl.

[0080] In certain embodiments, the viral infection is an influenza viral infection and the autoantibodies bind a hemagglutinin and / or neuraminidase.

[0081] In certain embodiments, this disclosure relates to methods of diagnosing a subject for a risk of developing severe influenza disease comprising: quantifying IgAl anti-IFN-alpha autoantibodies from a nasal sample of a subject diagnosed with an influenza infection; determining the quantity of nasal IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value or ii) greater than a reference or normal value; and diagnosing the subject with the influenza infection as having a high risk of developing severe influenza disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is similar to a reference or normal value of a subject without an influenza infection; or diagnosing the subject with the coronaviral infection as having a low risk of developing severe influenza disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is greater than a reference or normal value.

[0082] In certain embodiments, methods further comprise providing particles coated with anti- IgAl antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal sample from the subject diagnosed with an influenza infection with the fluorescent particles coated with anti-IgAl antibodies that specifically bind with the anti-IgAl fluorescent particles providing IgAl anti-IFN-alpha autoantibody fluorescent particles in the sample; analyzing the IgAl anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of IgAl anti-IFN-alpha autoantibodies in the nasal sample of the subject.

[0083] In certain embodiments, methods further comprise detecting elevated levels of INF-alpha in the nasal sample.

[0084] In certain embodiments, methods further comprise detecting elevated levels of CXCL10 in the nasal sample.

[0085] In certain embodiments, methods further comprise the nasal sample of the subject is used to perform PCR of an influenza strain in the sample.

[0086] In certain embodiments, methods further comprise using the IgAl anti-IFN-alpha autoantibody fluorescent particles that have an average diameter of about 2 to 4 micrometers or 1 to 5 micrometers.

[0087] In certain embodiments, methods further comprise using IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.5 pm, is exposed to a 561 nm wavelength, and a fluorescent signal is measured from 586 to 610 nm in wavelength.

[0088] In certain embodiments, methods further comprise using the IgAl anti-IFN-alpha autoantibody fluorescent particles that have an average diameter of about 2.8 pm, is exposed to a 488 nm wavelength, and a fluorescent signal is measured from 475 to 540 nm in wavelength.

[0089] In certain embodiments, methods further comprise recording a quantification and / or the risk of developing coronavirus, influenza, or other viral or microbial disease severity on a non- transitory computer readable medium.

[0090] In certain embodiments, the risk of developing an influenza disease severity is a high, low, medium, or quantitative number.

[0091] In certain embodiments, methods further comprise communicating the risk of developing a coronaviral, influenza, viral or other microbial (e.g. bacteria or parasite) disease severity to a medical professional or the subject.

[0092] In certain embodiments, methods further comprise administering an influenza drug to the subject if the subject is diagnosed with a high risk of severe disease. In certain embodiments, methods further comprise administering mechanical assisted oxygen to the airways and lungs of the subject if the subject is diagnosed with a high risk of severe disease.

[0093] In certain embodiments, the nasal or airway IgAl anti-IFN-alpha autoantibody fluorescent particles are coated with an antibody that specifically binding IgAl and the nasal or airway anti- Ig subclass other than IgAl anti-IFN-alpha autoantibody fluorescent particles are coated with an antibody that specifically binding IgAl and have an average diameter of about 2 to 4 micrometers or 1 to 5 micrometers not including agent coating the particles. In certain embodiments, the nasal or airway IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.5 pm, is exposed to a 561 nm wavelength, and a fluorescent signal is measured from 586 to 610 nm in wavelength. In certain embodiments, the nasal or airway anti-Ig subclass other than IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.8 pm, is exposed to a 488 nm wavelength, and a fluorescent signal is measured from 475 to 540 nm in wavelength.

[0094] In certain embodiments, the anti-Ig subclass other than IgAl is IgM, IgD, IgG, IgGl, IgG2, IgG3, IgG4, IgE, and IgA2.

[0095] In certain embodiments, methods disclosed herein further comprise recording a quantification and / or the risk of developing coronaviral, or other viral or microbial disease severity on a non-transitory computer readable medium. In certain embodiments, the risk of developing microbial disease severity is a high, low, medium, or quantitative number.

[0096] In certain embodiments, methods further comprise communicating the risk of developing a coronaviral, or other viral or microbial disease severity to a medical professional or the subject.

[0097] In certain embodiments, this disclosure relates to methods of diagnosing a subject for a risk of developing coronaviral or other viral or microbial disease severity comprising: quantifying IgG anti-IFN-alpha autoantibodies from a nasal or airway sample of a subject exhibiting symptoms of a coronaviral or other viral or microbial disease; determining the quantity of nasal or airway IgG anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value of a subject without coronaviral or other viral or microbial disease or ii) greater than a reference or normal value of a subject without coronaviral or other viral or microbial disease; and diagnosing the subject exhibiting symptoms of coronaviral or other viral or microbial disease as at high risk of developing severe coronaviral or other viral or microbial disease if quantifying IgG anti-IFN-alpha autoantibodies from the nasal or airway sample is higher to a reference or normal value of a subject without a coronaviral or other viral or microbial disease; or diagnosing the subject exhibiting symptoms of coronaviral or other viral or microbial disease as a low risk of developing severe coronaviral or other viral or microbial disease if quantifying IgG anti-IFN-alpha autoantibodies from the nasal or airway sample is similar to a reference or normal value of a subject without coronaviral or other viral or microbial disease.

[0098] In certain embodiments, methods further comprising providing particles coated with anti- IgG antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgG antibodies coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal or airway sample from the subject exhibiting symptoms of a coronavirus or other viral or microbial disease with the fluorescent particles coated with anti-IgG under conditions such that nasal or airway IgG anti- IFN-alpha autoantibodies in the nasal or airway sample specifically bind with the anti-IgG fluorescent particles providing airway IgG anti-IFN-alpha autoantibody fluorescent particles in the sample, and analyzing the airway IgG anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of nasal or airway IgG anti-IFN-alpha autoantibodies in the nasal or airway sample of the subject.

[0099] In certain embodiments, the microbial disease is a viral infection, coronaviral infection, an epidemic coronavirus, pandemic coronavirus, SARS-CoV2, SARS-CoV-1, MERS-CoV, endemic human coronavirus, HCoV-229E, HCoV-OC43, HCoV-NL63, or HCoV-HKUl.

[0100] In certain embodiments, methods include simultaneously measuring / quantifying human antibody isotypes and subclasses (IgM, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE) with multiantigen specificity in a single multiplexed assay.

[0101] In certain embodiments, the isotypes and subclasses are antibodies that bind SARS-CoV-2 variants and endemic human CoV-encoded proteins, including proteins from emerging virus mutants. In certain embodiments, it is contemplated that methods have superior sensitivity / dynamic range, have high dimensionality (e g., measuring 25, 30, 50, 100, 150, 192 or more parameters per assay), and high throughput. In certain embodiments, methods include measuring neutralizing antibodies observed in severe COVID- 19 patients, or antibody responses in any microbial infection or autoimmune antibodies present in an autoimmune disease. In certain embodiments, this disclosure relates to methods of detecting whether a sample comprises antibodies that are neutralizing or non-neutralizing by mixing the antibody bound coated fluorescent particles with tagging agents wherein if the tagging agent displace the antibodies bound to the fluorescent particle this indicates that the antibodies were non-neutralizing antibodies, and wherein if the tagging agent does not displace the antibodies this indicates that the antibodies are neutralizing antibodies.

[0102] In certain embodiments, this disclosure relates to a method of detecting a scatter pattern of antibody expression comprising: i) conjugating proteins to particles that contain a detectable scatter pattern such that individual and distinct proteins are coated on individual fluorescent particles with unique scatter pattern which are distinguishable by flow cytometry providing protein coated fluorescent particles; ii) contacting a sample from a subject at risk of, exhibiting symptoms of, or diagnosed with an infection or autoimmune disease with the protein coated fluorescent particles under conditions such that antibodies in the sample specifically bind with the proteins providing antibody bound coated fluorescent particles; iii) contacting the antibody bound coated fluorescent particles with agents that specifically bind antibodies of different isotypes providing agent bound isotype / subclass specific coated fluorescent particles; and iv) analyzing the agent bound isotype specific coated fluorescent particles by flow cytometry providing a scatter pattern of protein and isotype / subclass specific agent particles in the sample of the subject.

[0103] In certain embodiments, the method further comprises comparing the flow cytometry scatter pattern of protein and isotype / subclass specific agent particles to a normal pattern, reference pattern, or control pattern, wherein the subject is diagnosed with a low risk of death or severe disease if the scatter pattern of protein and isotype / subclass specific agent particles is the same or similar to the normal pattern or the reference pattern associated with low risk of death or severe disease or a control pattern; and wherein the subject is diagnosed with heightened risk of death or severe disease if the scatter pattern of protein and isotype / subclass specific agent particles is the same or similar to a reference pattern associated with high risk of death or severe disease.

[0104] In certain embodiments, this disclosure relates to a computer program that generates diagnostic predictions and drug treatment predictions using methods of artificial intelligent generated with data obtained using methods disclosed herein, e.g., trained with flow cytometry separation patterns. In certain embodiments, the input values are measured data generated using flow cytometry results such as measurements of specific antibody isotypes and subclasses, fluorescence at specific wavelengths, and forward and side scatter.

[0105] In certain embodiments, the medical value is the health, normal, risk, high or low severity of disease or death of a subject based on measurements of analytes in a sample from a subject.

[0106] In certain embodiments, the medical value is the likelihood of survival of a subject based on measurements of analytes in a sample from a subject wherein the subject is infected with a viral (e.g., coronaviral) or other microbial infection.

[0107] In certain embodiments, the subject has symptoms such as coughing, runny or stuffy nose, sore throat and / or fatigue.

[0108] In certain embodiments, the subject with sever coronaviral disease has developed longterm symptoms after initial illness, e.g., symptoms for more than two weeks or a month, often referred to as “Long COVID.”

[0109] In certain embodiments, method disclosed herein are performed on a subject diagnosed with a coronavirus with symptoms, long-COVID, or before any symptoms start, i.e. asymptomatic, e.g., tested positive before symptoms.

[0110] In certain embodiments the coronavirus is an Omicron variant such as JN.l, LP.8.1, XFC, XEC, LF.7.7.2, or LF.7.

[0111] In certain embodiments, the subject has upper respiratory tract symptoms (runny nose, sore throat, headache). In certain embodiments, the subject is older than 50, 60, or 65 year old or an immunocompromised patients may be at risk for more severe lung infections, hospitalization or death, e.g., HIV patient, chronic or long-term health conditions of serious organ disease heart disease, kidney disease, transplant patient, type 1 or type 2 diabetes, lung conditions, bronchitis, chronic obstructive pulmonary disease (COPD), asthma, sickle cell disease, cystic fibrosis, obesity, nursing home residents, obesity, pregnancy, diseases that create a weakened immune system (immunosuppressed), or other condition wherein treatment includes the administration of immunosuppressive drugs.

[0112] In certain embodiments, methods further comprise communicating the risk of developing a coronaviral disease severity to a medical professional or the subject. In certain embodiments, if the subject is diagnosed with a high risk of severe disease, methods further comprise administering a coronaviral drug and / or anti-inflammatory agent to the subject. In certain embodiments, the drug is ivermectin, tocilizumab, tocilizumab, nirmatrelvir / ritonavir, remdesivir, vilobelimab, anakinra, molnupiravir, pemivibart, and / or convalescent plasma. In certain embodiments, if the subject is diagnosed with a high risk of severe disease, methods further comprise administering mechanical assisted oxygen to the airways and lungs of the subject.

[0113] In certain embodiments, the anti-inflammatory agent is steroidal or non-steroidal antiinflammatory drug (NSAID), aspirin, acetaminophen, or a corticosteroid such dexamethasone.

[0114] In certain embodiments, this disclosure relates to a computer program that generates diagnostic predictions and drug treatment predictions using methods of artificial intelligent generated with data obtained using methods disclosed herein, e.g., trained with flow cytometry separation patterns.

[0115] In certain embodiments, the method comprises using an artificial neural network. In some embodiments, the method comprises a Bayesian network, a support vector machine or the like.

[0116] In certain embodiment, the method is based on a neural network, which was trained using a large amount of historic medical data, preferably from hospitalized patients diagnosed with an autoimmune disease, viral, coronaviral, or other microbial infection.

[0117] In certain embodiments, patient is diagnosed with high blood pressure, fatigue, shortness of breath, anxiety, depression, brain fog, joint pain, and / or chest pain, and optionally diabetes, stroke, heart rhythm abnormality, and / or blood clot in the lungs.

[0118] Preferably at least 100, more preferably at least 1000 datasets are used or were used to train the neural network.

[0119] In certain embodiment, the neural network is trained using data from a certain cluster of patients. Patients from a cluster may have one or several defined or pre-defined or selected or preselected characteristics or attributes in common. One cluster of patients, e.g., may only comprise patients of over 65 years of age, other clusters may exclusively contain virally or microbially infected patients, patients with diabetes, men only, women only, patients with a history of stroke and / or overweight patients according to body mass index, etc. Advantageously, providing a neural network trained on a relevant cluster of patients, recommendations of drug dosages may be improved over such neural networks, which were trained on an unselected set of patients.

[0120] In certain embodiments, the neural network is trained by the user, using a training software running on the device or on a network of devices. In some embodiments, the device is already trained and ready to be used without further training. In some embodiments, the device is partially trained and may optionally or must be trained further before use. In some embodiments, the method comprises the training of the neural network. In other embodiments, the method excludes the training of a neural network, but employs a neural network, which has already been trained.

[0121] In certain embodiments, the method provides a certain drug dosage to ascertain that a certain measurable diagnostic value remains inside a certain range or moves towards a certain range.

[0122] In certain embodiments, the method is adaptive. That is, during application of the method, measurement values are fed into the method to further improve recommendations. For example, a drug dosage is recommended with the aim to yield a certain diagnostic value level. After a certain time, said diagnostic value is measured and provided to the method such that future recommendations may be improved.

[0123] Artificial intelligence is generally the ability of a device or a method to perform tasks by employing intelligence. Artificial intelligence may comprise or consist of knowledge-based systems, interference engines, expert systems, neural networks, data mining systems, machine learning systems and combinations thereof.

[0124] The digital storage means is defined by the feature combination described herein. Accordingly, the digital storage means, in particular a hard disc drive, CD or DVD, has electronically readable control signals, which are able to interact with a programmable computer system such that a method will be executed.

[0125] The computer program product is defined by the feature combination described herein. Accordingly, in another aspect, the computer program product has a program code stored on a machine-readable data medium for executing a method when executing the program product on a computer.

[0126] A computer program product can be understood as, for example, a volatile signal, a computer program which may be stored on a storage device, an embedded system as a comprehensive system with a computer program (e.g., an electronic device with a computer program), a network of computer-implemented computer programs (e.g., a client-server system, a cloud computing system, etc.), or a computer on which a computer product is loaded, executed, saved, or developed.

[0127] The "term machine-readable data medium" as used herein denotes in certain embodiments a medium containing data or information, which is interpretable by software and / or hardware. The medium may be a data medium, like a disk, a CD, DVD, a USB stick, a flashcard, an SD card or the like.

[0128] A computer program can be understood as, for example, a physical, ready-for-distribution software product which comprises a computer program.

[0129] It also applies to the digital storage means, the computer program product and the computer program that all or some of the machine-executed steps of the method are prompted.

[0130] The computer program is defined by the feature combination as described herein. Accordingly, in another aspect, the computer program has a program code for the execution of a method when executing the program on a computer.

[0131] In certain embodiments, the at least one input value comprises a drug recommendation.

[0132] In certain embodiments, the method may use stored experiences and / or rules on how to use experience as well as current input values to arrive at a dosage recommendation.

[0133] The input values, which may be used by the method, may be recently acquired measurements, for examples results of a biomolecule, e.g., an antibody isotypes or subclass, or group of biomolecules as identified in blood tests. The input values may additionally include past test results such that the method takes into account a time course of one or several parameters.

[0134] Serendipitous discovery of anti-interferon-alpha (anti-IFN-alpha) autoantibodies in nasal swabs originally collected for PCR were correlated to disease severity and survival likelihood

[0135] In the process of collecting samples to perform coronaviral PCR of nasal swabs, i.e., originally for the purpose of determining specific coronaviral strains, experiments of the contents of the nasal swabs were also evaluated to determine presence of other cytokines and antibodies. Unexpectedly, it was discovered that in certain patients, high concentrations of anti-interferon- alpha (anti-IFN-alpha) autoantibodies were in nasal swabs despite substantially lower or undetectable systemic and / or saliva concentrations. It was also noticed that these patients typically recovered from infections without the need to be admitted to in intensive care units (ICU), whereas patient with low / undetectable anti-interferon-alpha (anti-IFN-alpha) autoantibodies in the nasal samples often progressed to severe disease requiring hospitalization. The presence of anti- interferon-alpha (anti-IFN-alpha) autoantibodies were further evaluated confirm the ability to predict disease severity for coronavirus and influenza virus. Experiments indicate that nasal samples with elevated concentrations of anti-interferon-alpha (anti-IFN-alpha) autoantibodies are correlated to a low likelihood of severity of symptoms of coronaviral and influenza disease and typically indicate the ability to recover without the need for mechanically assisted air / oxygen, wherein low levels of anti-interferon-alpha (anti-IFN-alpha) autoantibodies indicate a higher likelihood of severe disease and increase the risk of mortality.

[0136] Transient anti-interferon autoantibodies in the airways are associated with recovery from COVID-19

[0137] Preexisting anti-interferon-alpha (anti-IFN-alpha) autoantibodies in blood are associated with susceptibility to life-threatening COVID-19. Whether anti-IFN-alpha autoantibodies in the airways can be used to predict disease outcomes was evaluated. A multiparameter technology, was developed to quantify and profile the isotypes of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and anti-IFN-alpha antibodies in longitudinal samples collected over 20 months from the airways and blood of donors spanning mild to severe COVID- 19. It was discovered that nasal IgAl anti-IFN-alpha autoantibodies were induced after infection onset in more than 70% of mild and moderate COVID- 19 cases and were associated with robust anti- SARS-CoV-2 immunity, fewer symptoms, and efficient recovery. Nasal anti-IFN-alpha autoantibodies followed the peak of host IFN-alpha production and waned with disease recovery, revealing a regulated balance between IFN-alpha and anti-IFN-alpha response. In contrast, systemic IgGl anti-IFN-alpha autoantibodies appeared later and were detected only in a subset of patients with elevated systemic inflammation and worsening symptoms.

[0138] Experiments were performed to determine whether a regulated balance among host IFN- alpha, anti-IFN-alpha autoantibodies, and anti-SARS-CoV-2 antibodies in the airway mucosa is necessary for efficient recovery during infection and whether dysregulation of this balance in the airways is detrimental, leading to life-threatening COVID-19. A flow cytometry-based bead assay to detect antigen-specific antibody isotypes (FlowBEAT™) was used to determine the longitudinal dynamics of anti-SARS-CoV-2 and anti-IFN-alpha autoantibodies in the airway, the site of infection, and in matching blood, revealing their contribution to the progression of COVID-19 spanning from disease onset to full recovery.

[0139] The nasal epithelium is the initial site of SARS-CoV-2 infection. As such, antiviral immune responses locally in the nasal mucosa can determine disease recovery or progression to severe COVID-19. IgG is mainly produced in the blood, while IgA (both IgAl and IgA2) is produced in mucosa such as the nasal cavity. Experiments indicate that transient autoantibodies against type I IFNs (anti-IFN-alpha) in the nasal mucosa are a common feature of the host immune response to SARS-CoV- 2 infection and are associated with efficient recovery from mild and moderate disease. Mild-and moderate-recovered patients who produced nasal IgAl but not systemic IgGl anti-IFN- alpha showed fewer symptoms, less systemic inflammation, and increased anti-SARS-CoV-2 humoral immunity in the airways. In contrast, patients who progressed directly to systemic IgGl anti-IFN-alpha without detectable nasal IgAl showed worsened symptoms (particularly shortness of breath), increased systemic inflammation, and a switch in anti-SARS-CoV-2 antibody isotypes from IgGl to the inflammation-associated IgG3. Thus, the high prevalence (>70%) of nasal IgA anti-IFN-alpha autoantibodies in mild-and moderate-recovered patients suggests that autoreactivity to type I IFN at the nasal site of infection is an intrinsic aspect of host-protective antiviral immunity and not a precondition to severe COVID-19 .

[0140] Experiments indicate a direct association between nasal anti-type I IFN and disease. Type I IFN is an antiviral cytokine. However, its role in protection and recovery from COVID-19 and other coronavirus infections was uncertain. Prior reports in animal models of coronavirus infection suggest that type I IFN is dispensable or detrimental to resolving the disease. One report suggests that mice that lack the IFN-alphaP receptor (IFNAR- / -) are protected from lethal infection with SARS-CoV without affecting viral load. See Channappanavar, Cell Host Microbe 19, 181-193 (2016). Similarly, a mouse model of SARS-CoV-2 infection shows that type I IFN is not required for viral clearance. See Israelow et al. Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling. J. Exp. Med. 217, e20201241 (2020).

[0141] Although it is not intended that embodiments of this disclosure be limited by any particular mechanism, experiments reported herein support the idea that a SARS-CoV-2 infection induces early and transient anti-IFN-alpha autoantibodies to counteract the detrimental effects of excessive IFN-alpha secretion in the airways. Conclusions drawn from these studies infer that anti-IFN- alpha autoantibodies are preexisting rather than infection induced on the basis of their findings showing no detectable anti-IFN-alpha in the blood of outpatients with mild COVID-19 measured at disease onset, whereas systemic anti-IFN-alpha was detected in more than 10% of patients hospitalized with COVID- 19. Patients with mild COVID- 19 develop systemic anti-IFN-alpha only later, more than 2 weeks after infection. Hospitalized patients with systemic anti-IFN-alpha may develop their infection-induced autoantibodies before being admitted to the ICU. Therefore, by considering tissue-specific immune responses (transient nasal IgAl versus persistent blood IgGl), finding herein provide a framework to understand the immunopathology of COVID-19 in different tissues, challenging the notion that anti-IFN-alpha autoantibodies are universally pathologic. Although autoantibodies are often seen as pathologic, the prevalence of anti-IFN-alpha in more than 70% of infected individuals and its association with efficient recovery suggest a common mechanism of host antiviral immunity instead of a break in B cell tolerance. Nasal and blood anti-IFN-alpha did not enhance exogenous IFN-alpha signaling or CXCL10, a readout for IFN signaling. Moreover, anti-IFN-alpha correlated with a decrease in IFN signaling and CXCL10 in blood but not nasal samples.

[0142] FlowBEAT™ reveals distinct anti-SARS-CoV-2 and anti-type I IFN autoantibody responses across tissues and disease states

[0143] To quantify antibody responses, including isotype usage and antigen specificity across COVID-19 severity states, a multiparameter assay was developed (FlowBEAT™) to measure various antibody parameters per sample, including eight human antibody isotypes [immunoglobulin G1 (IgGl), IgG2, IgG3, IgG4, IgAl, IgA2, IgE, and IgM] against a panel of up to 22 host and viral antigens, including host type I IFNs (anti-IFN-alpha and anti-IFN-co) and SARS-CoV-2 proteins, including anti-spike protein receptor binding domain (RBD), anti-spike protein subunits SI and S2, and structural proteins (anti-nucleocapsid, anti-membrane, and antienvelope) and nonstructural proteins (NSPs) (anti-NSPs), including open reading frame (ORF) proteins (anti-ORF). Bovine serum albumin (BSA)-coated control beads were used to measure nonspecific background signal (noise) and establish the lower limits of detection of the assay.

[0144] FlowBEAT1Mwas applied to paired longitudinal samples from the airway [nasal swabs and endotracheal aspirates (ETAs)] and blood (serum or plasma) of patients with COVID-19 before and after vaccination. Donors were recruited 125 spanning from pre-pandemic infection-naive (n = 36), mild (n = 34), moderate (n = 32), and severe life-threatening (n = 23) COVID-19 patients. Outpatient samples were collected longitudinally from 1 to 97 days after onset with additional post-recovery samples collected for more than 2 years. Samples were collected from inpatients with severe COVID-19 as available, which spanned days 7 to 48 after onset, a window included within the timing of the outpatient cohort. The nasal swabs and blood were analyzed from outpatients with COVID- 19, whose disease severity (mild versus moderate) was on the basis of the total number of assessed symptoms and the time needed to recover from those symptoms. The ETA and blood from inpatients with severe COVID-19 who were hospitalized in the intensive care unit (ICU) under mechanical ventilation were analyzed.

[0145] The experiments revealed that the amount and diversity of antibody responses increased with disease severity, represented as peak antibody signal during acute infection. As the disease severity increased from mild to severe COVID-19, additional antibody isotypes and specificities against SARS-CoV- 2 NSPs and ORF proteins and host IFNs were observed distinguishing abroad breadth of antibody response against SARS-CoV- 2 and host type I IFNs across tissues and disease states.

[0146] Airway-specific antibody isotypes and specificities against SARS-CoV-2 proteins distinguish mild, moderate, and severe disease

[0147] The isotypes and specificities of antibodies against SARS-CoV-2 proteins were assessed longitudinally across disease states. The nasal mucosa and blood starting at the time of disease onset were sampled, determined as the date of polymerase chain reaction (PCR)-confirmed infection, and through recovery and subsequent mRNA vaccination. In outpatients with mild and moderate COVID-19 who recovered from the disease, IgGl, IgG3, and IgAl were the predominant anti-spike protein antibody isotypes in both nasal mucosa and blood, reaching their respective peaks before 30 days after onset. Although most antibody isotypes were maintained for greater than 3 months after onset in blood, they quickly waned in the nasal mucosa after disease recovery, except for IgAl, which was still detectable in more than 60% of mild-and moderate-recovered donors 3 months after onset. Nasal IgAl, IgE, and IgM isotypes of anti-nucleocapsid protein predated the detection of anti-spike protein by at least 1 week, likely representing preexisting immunity to nucleocapsid epitopes conserved between SARS-CoV-2 and endemic common cold coronaviruses.

[0148] In contrast with the relative stability of isotype usage, particularly that of IgGl, IgG3, and IgAl, throughout SARS-CoV-2 infection and recovery, mRNA vaccination induced de novo systemic and mucosal IgG4 and systemic IgG2 after two or more vaccine doses (Fig. 2). Moreover, intramuscular mRNA vaccination boosted distal nasal IgAl and IgGl anti-spike protein antibodies but not anti-nucleocapsid protein antibodies, supporting a role for vaccination in promoting mucosal sterilizing immunity. Airway-and blood-exclusive antibody isotypes and specificities (per-patient maximum signals during the peak of infection) were associated with COVID-19 severity. In the airway, the production of IgA2, often related to hyperinflammatory responses in the mucosa, increased with disease severity and was detected at the highest levels within ETAs of patients with severe COVID- 19. In blood, whereas IgM was comparable across disease states, an increase in the production of IgGl and IgG4 anti-spike protein was associated with an increase in COVID-19 severity. High IgG3 in the blood, which can suppress type I IFN , distinguished patients with moderate to severe disease. For example, whereas 61% of moderate cases developed systemic IgG3 anti-spike protein, only 17% of mild cases had detectable systemic IgG3 anti-spike protein during acute infection (<90 days after onset).

[0149] A nasal IgAl antibody signature against SARS-CoV-2 NSPs and ORF proteins (anti-NSP signature was identified. It emerged within 2 weeks of infection. Subsequently, a fraction of patients (25% of those with mild and 46% of those with moderate CO VID-19) went on to develop a matching IgGl anti-NSP signature in the blood. This sequential progression from the nasal IgAl to systemic IgGl anti-NSP was linked to an increased concentration of systemic C-reactive protein (CRP, a clinical marker of inflammation) at the peak of infection, independent of viral load. Thus, FlowBEAT™ identified IgA2, IgG3, and IgG4 as features associated with COVID- 19 severity, revealed an IgG2 and IgG4 signature induced by repeated vaccination, and identified an initial IgAl anti-NSP response restricted to the airway site of infection that later progressed to a systemic IgGl response primarily in patients with increased disease severity.

[0150] Anti-IFN-alpha autoantibodies are induced after SARS-CoV- 2 infection of the airways and peak after local production of host IFN-alpha

[0151] To reveal the longitudinal dynamics between anti-SARS-CoV-2 antibodies and autoantibodies against type I IFN (anti-IFN-alpha and anti-IFN-co), 36 patients were longitudinally assessed with mild and moderate COVID-19 for whom matched nasal and blood samples were collected throughout infection, recovery, and subsequent mRNA vaccination. To quantify anti-IFN autoantibodies, assay beads were coated with the IFN-alpha (IFN-alpha2a) and IFN-OJ proteins. In addition, other IFN-alpha subtypes, IFN-alpha5 and IFN-alphal4 were included. To distinguish the background from the antigen-specific signal, BSA-coated control beads were included in every multiplexed assay to calculate nonspecific antibody binding. Signals above the BSA background were considered positive and specificities of low anti-IFN signals (signals above but near the BSA background) were confirmed the by serial dilution of samples with positive signals.

[0152] Experiments were performed to determine whether anti-IFN autoantibodies (anti-IFN- alpha and anti-IFN-w) could be detected locally in the airways throughout COVID- 19 progression. A robust IgAl anti-IFN-alpha autoantibody response was induced shortly after infection, frequently emerging within 2 weeks after onset in the nasal mucosa (anti-IFN-alpha autoantibodies were detected in 72% or 26 of 36 mild and moderate cases of COVID-19). Consistent with the anti-SARS-CoV-2 antibody isotype response, nasal anti-IFN was dominated by IgAl in mild and moderate COVID-19, whereas IgA2, often associated with mucosal hyperinflammation, was detected at lower signals measured as median fluorescence intensity (MFI). These new-onset nasal IgAl anti-IFN-alpha autoantibodies were transient and waned as patients recovered from symptomatic disease. The transient IgAl anti-IFN-alpha recognized all three IFN-alpha subtypes (IFN-alpha2a, IFN-alpha5, and IFN-alphal4) with a strong positive correlation, whereas systemic IgGl poorly recognized anti-IFN-alpha5, suggesting a systemic response unlinked from the nasal mucosa. A similar pattern of transient production of anti-IFN-co was observed in the same donors but at a lower FlowBEAT™ MFI signal. In contrast with anti-IFN-alpha, other autoantibodies, including the canonically autoreactive antibodies of the VH4-34 Ig gene family, which is increased in the blood of hospitalized patients with COVID-19, remained stable in the airways throughout the disease course, revealing a transient property of the autoantibodies against type I IFN in COVID-19.

[0153] The initial IgAl anti-IFN-alpha response was limited to the nasal mucosa, and fewer donors progressed to a systemic response in the blood. Only 36% of the patients (13 of 36) developed blood anti-IFN-alpha, which appeared more than 2 weeks after onset. This systemic autoantibody response was dominated by the IgGl isotype, which was rarely detected in the airways. Unlike nasal IgAl, systemic IgGl persisted for more than 2 months in some patients. They were more frequently detected in donors with moderate (42%) as compared with mild (14%) disease. In contrast with SARS-CoV-2 infection, mRNA vaccination did not induce anti-IFN- alpha autoantibodies, indicating that anti-IFN-alpha autoantibodies are a specific response to SARS-CoV-2 infection. Experiments were performed to determine whether the anti-IFN-alpha response was associated with viral load by assessing the same nasal swabs for SARS-CoV-2 copy numbers. Patients who produced detectable anti-IFN-alpha autoantibodies experienced significantly greater peak viral load than those who never produced nasal anti-IFN-alpha. The temporal separation between peak viral load and anti-IFN-alpha induction confirmed that nasal anti-IFN-alpha was induced after viral exposure and not preexisting and that the association of anti-IFN-alpha with elevated viral load further suggests that this is a viral-induced autoantibody response.

[0154] Experiments were performed to determine whether viral-induced anti-IFN-alpha autoantibodies were associated with local secretion of IFN-alpha. Host IFN-alpha was longitudinally measured in the same matched nasal swab and serum samples. Peak nasal IFN-alpha cytokine was detected within the first week of infection, waning after 2 weeks. Only the patients who produced high nasal IFN-alpha early after infection developed nasal anti- IFN-alpha autoantibodies . Longitudinally, the peak production of nasal IFN-alpha preceded the anti-IFN- alpha autoantibody response, which peaked 2 weeks after onset, when IFN-alpha waned. A similar pattern of IFN-alpha and anti-IFN-alpha dynamics was observed in the blood.

[0155] Experiments were performed to determine whether the viral-induced anti-IFN-alpha autoantibodies could neutralize the activity of IFN-alpha signaling using a type I IFN-reporter human embryonic kidney (HEK) 293 cell line optimized using exogenous IFN-alpha2a and monoclonal anti-IFN-alpha2a in healthy control samples. Patient serum or plasma containing anti- IFN-alpha autoantibodies neutralized exogenous IFN-alpha signaling in vitro. Unlike blood samples, nasal mucosal samples neutralized exogenous IFN-alpha even without detectable anti- IFN-alpha, suggesting a robust mechanism to control IFN-alpha signaling in the nasal mucosa.

[0156] To determine whether nasal anti-IFN-alpha autoantibodies can contribute to the neutralization potential of nasal samples, a cell-based assay was repeated using low-throughput optimized conditions to obtain a higher dynamic range near the lower limit of detection of the assay. Tested were a subset of nasal swabs containing a range of IgA anti-IFN-alpha titers in the presence of exogenous IFN-alpha (60 activity U / ml). Although a decrease in IFN-alpha signal correlated with anti-IFN-alpha was not observed. In blood, high titers of IgGl anti-IFN-alpha showed a negative correlation with IFN-alpha signaling, further supporting that anti-IFN-alpha can neutralize IFN-alpha. To further validate whether the production of anti-IFN-alpha can neutralize IFN signaling, C-X-C motif chemokine ligand 10 [CXCL10, also known as IFN-y- induced protein-10 (IP-10)] was measured longitudinally in the same samples. CXCL10 is a reliable marker for IFN signaling in the nasal mucosa of patients with COVID- 19. Serum or plasma anti-IFN-alpha was significantly associated with reduced CXCL10.

[0157] Transient IgAl anti-IFN-alpha autoantibodies in the nasal mucosa are associated with fewer symptoms, more anti-SARS-CoV-2 antibodies, and full recovery

[0158] Experiments were performed to determine whether anti-IFN-alpha autoantibodies could be associated with symptomatology or other clinical features in patients with mild and moderate disease. Experiments indicated that nasal autoantibody response was unlinked from the blood given that there was no direct correlation between nasal and blood anti-IFN-alpha. Patients were grouped into three categories on the basis of the presence or absence of nasal and blood anti-IFN- alpha (nasal, blood-only producers, and nonproducers). Between these groups, no statistical differences were found by sex, age, or body mass index, other than those males produced the highest titers of blood IgGl anti-IFN-alpha. However, a significant association was identified between anti-IFN-alpha autoantibodies and disease features, including shortness of breath, sleep disturbance, balance issues, and chills.

[0159] Patients who developed systemic IgGl anti-IFN-alpha (blood-only producers) reported persistent shortness of breath and other relevant symptoms. In contrast with the systemic IgGl producers, nasal IgAl anti-IFN-alpha autoantibody producers showed significantly fewer symptoms, including less shortness of breath. COVID-19 treatment guidelines consider shortness of breath to be a symptom associated with disease severity and worse prognosis, likely reflecting viral spread to the lower respiratory tract. Consistent with this, patients who developed systemic IgGl anti-IFN-alpha and shortness of breath had elevated systemic CRP.

[0160] Experiments were performed to determine whether nasal anti-IFN-alpha autoantibodies were associated with an increased humoral immune response against SARS-CoV- 2 in the mucosal site of infection. A positive linear correlation was found between nasal IgAl and IgA2 anti-IFN- alpha autoantibodies and antibodies against SARS-CoV-2 proteins, including an anti-NSP and ORF protein signature, showing that nasal anti-IFN-alpha production is linked to enhanced humoral immunity against SARS-CoV-2 in the upper airway. In contrast, patients who did not develop anti-IFN-alpha (nonproducers) had fewer anti-SARS-CoV- 2 antibodies in the nasal mucosa. When compared with nasal IgAl producers, patients who developed systemic IgGl anti- IFN-alpha (blood-only producers) had higher anti-SARS-CoV-2 antibodies in the blood but not in the upper airway. Thus, nasal IgAl anti-IFN-alpha is associated with robust anti-SARS-CoV-2 immunity in the nose.

[0161] Hospitalized patients show elevated anti-IFN-alpha in the airways associated with uncontrolled IFN-alpha, higher viral load, and lower anti-SARS-CoV-2 antibody titers

[0162] To determine whether the coordinated balance between host IFN-alpha and anti-IFN-alpha identified in mild-and moderate-recovered patients is disrupted in severe COVID- 19, FlowBEAT™ was performed on ETAs (airway mucosa) and peripheral blood from 23 hospitalized and unvaccinated patients admitted to the ICU with life-threatening severe COVID-19. IgAl and IgA2 anti-IFN-alpha autoantibodies were detected in the airway mucosa (ETAs) of 86% of patients with severe COVID-19, and the remaining 14% also had detectable anti-IFN-alpha but of a different antibody isotype. In contrast, only 43% of patients with severe COVID- 19 developed detectable systemic IgGl anti-IFN-alpha. In contrast with outpatients with mild and moderate CO VID-19, sustained IFN-alpha production was detected in the ETAs of hospitalized patients that positively correlated with IgAl and IgA2 anti-IFN-alpha autoantibodies, revealing a dysregulated IFN-alpha / anti-IFN-alpha response at the airway site of infection. Moreover, patients with severe COVID- 19 produced more hyperinflammati on-associated IgA2 subclass anti-IFN-alpha, consistent with sustained hyperinflammation in the airways Similarly, the anti-SARS-CoV-2 response also switched from the IgAl to the hyperinflammation-associated IgA2 in the airway. Given that nasal swabs and ETAs are sampled from different anatomic locations (upper and lower airways), these differences in the IgA subclass may also reflect differences in humoral responses at each mucosal site. Consistent with the findings in mild and moderate disease, airway IgA anti- IFN-alpha autoantibodies in severe disease were associated with increased viral load suggesting an ongoing antiviral immune response in the hospitalized patients. Airway anti-IFN-alpha was positively associated with local (ETA) proinflammatory cytokines, including those that promote myeloid recruitment and activation, such as IFN-y, IFN-X1, CXCL10, CXCL11, C-C motif chemokine ligand 19 (CCL19), tumor necrosis factor-alpha (TNF-alpha), and TNF-related apoptosis-inducing ligand (TRAIL). However, whereas airway IgA anti-IFN-alpha correlated with enhanced anti-SARS-CoV-2 humoral immunity in patients with mild and moderate COVID-19, this correlation no longer persisted in the airways of severe patients, suggesting weaker airway humoral immunity as a feature of severe disease.

[0163] Plasma and serum processing

[0164] Samples were collected from all study participants, regardless of COVID- 19 diagnosis or severity. To obtain serum, whole peripheral blood was collected in tubes and allowed to clot, and supernatant serum was isolated and cryopreserved. For plasma collection, whole blood was collected in EDTA tubes, and plasma was isolated by centrifugation at 400g for 10 min at 4°C followed by removal of the supernatant. To further precipitate platelets, contaminating cells, or remaining debris the isolated plasma and serum samples were centrifuged at 4000g for 10 min at 4°C. Samples were distributed in aliquots and cryopreserved at -80°C. Th

[0165] Nasal swab processing

[0166] Samples were collected with flocked nasal swabs from both nostrils and transferred to 3 ml of viral preservation medium. Swabs (in media) were vortexed to liberate collected antibodies into media. Samples were further mechanically dissociated using a syringe and 25-gauge needle to disrupt aggregates of mucins and other respiratory secretions. Samples were centrifuged at 2000g to precipitate non-soluble aggregates. Processing of samples from SARS-CoV-2 positive donors.

[0167] ETA processing

[0168] Samples were collected by health care providers in the ICU from either endotracheal intubation or tracheostomy. ETA samples were transferred to a US Department of Agriculture- approved BSL3 containment facility at Emory for further processing. To disrupt mucin aggregates, ETA was mixed 1:1 with a 50 mM EDTA solution (final concentration 25 mM EDTA) in custom RPMI 1640 medium deficient in biotin, 1-glutamine, phenol red, riboflavin, and sodium bicarbonate (def RPMI 1640) and dissociated using a syringe. Samples were centrifuged at 250g, and supernatants were cryopreserved at -80°C. Before removal from BSL3 containment, ETA supernatants were ultraviolet (UV) inactivated. Antigen preparation and conjugation

[0169] SARS-CoV- 2 structural proteins and NSPs, type I IFN proteins, and the 9G4 antibody clone to detect VH4-34 autoantibodies were either produced in house or obtained from commercial sources. Antigens, including a purified BSA control antigen, were biotin conjugated using N- hydroxy-succinimide-biotin reagents at a 20-fold molar excess. Antigens were loaded onto streptavidin-coated beads at an empirically determined saturation point in 50 pl of phosphate- buffered saline (PBS) + 0.05% BSA+ 0.2% Tween 20 (PBS-BSA) and allowed to bind for 20 min on ice before the excess (uncaptured) antigen was removed by washing with additional PBS-BSA. The antigen-bead saturation point for the SARS-CoV- 2 RBD was experimentally determined, SI subunit, and S2 subunit, as well as for the IFN-alpha2a and IFN-co antigens. All were found to have a saturation point of 2 pmol per 50,000 beads in 50 pl of PBS-BSA. The capacity of saturated beads to capture antigen-specific antibodies was confirmed by flow cytometry using serial dilution of mouse monoclonal antibodies. Antigen-specific binding was revealed by a secondary stain with goat polyclonal antibodies fluorescently conjugated with phycoerythrin or Alexa Fluor 488™. Each antigen-specific antibody had a maximum signal output at least 1000-fold greater than the unstained control, which decreases linearly with serial dilution.

[0170] Fluorescent detection antibodies

[0171] Monoclonals anti-IgGl and anti-IgA2 were pre-conjugated, and anti-IgE and anti-IgM were pre-conjugated. Monoclonals anti-IgG2, anti-IgG3, anti-IgG4, and anti-IgAl were purchased unconjugated from a commercial supplier and conjugated using fluorescent conjugation kits following the protocol of the manufacturer. Each lot of antibodies were individually titrated to identify the dilutions used for the assay.

[0172] FlowBEAT™ assays

[0173] After thawing, samples were centrifuged at 2000g for 10 min at 4°C to precipitate any remaining aggregates. Serum or plasma samples were diluted to a final concentration of 1 : 125 with PBS-BSA in a reaction mix containing beads coated with antigen at an empirically determined saturation point. Airway samples were diluted to a final concentration of 1:2. Samples and beads were incubated on ice in the dark for 30 min, mixing at least every 15 min to prevent beads from settling. Beads were washed with PBS-BSA and resuspended before fluorescent antibody staining. Antibody staining was performed by mixing equal volumes of resuspended beads and a master mix containing the eight fluorescently conjugated monoclonal antibodies specific to IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, and IgM. The optimal concentration of each antibody species was empirically determined by titration. The reaction was incubated on ice in the dark for 30 min, mixing at least every 15 min. Stained beads were washed with PBS-BSA and resuspended in l x PBS. Samples were fixed in a 1 :6 lysing solution, which inactivates SARS-CoV- 2. During fixing, samples were kept in the dark at room temperature for 20 min, mixing at least every 10 min. A final wash with PBS-BSA was performed, and beads were resuspended in PBS-BSA. All samples were analyzed within 24 hours of staining using a five-laser Cytek Aurora™ flow cytometer. To standardize FlowJAM™ batches, a daily standard plasma sample with strong reactivity to SARS- CoV-2 spike protein subunits were established. The standard positive and a sample-free control were included in all FlowBEAT™ assays to confirm the performance of the assay and control for potential variations. Most samples were analyzed twice on different days using freshly prepared antigen-coated beads. The patient sample repeats and the daily standard calibration values showed high concordance. IFN neutralization assays HEK-Blue IFN-alpha / p cells, commercially available HEK cell line engineered to report on type I IFN activity by secreted embryonic alkaline phosphatase (SEAP), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) enriched with 10% fetal bovine serum, 1% penicillin-streptomycin, and selective antibiotics included with the HEK-Blue™ cells (Blasticidin, Zeocin™, andNormocin™). Cells were seeded at 50,000 cells per well in 96-well plates and allowed to adhere for up to 4 hours (80% confluency) at 37°C in a humidified atmosphere containing 5% CO2 using medium free from selection antibiotics. Medium was gently aspirated and replaced by donor samples in DMEM containing IFN-alpha2a (60 activity U / ml) per well. Nasal swabs were incubated at a final concentration of 50%, and plasma or serum was incubated at a final concentration of 20% for 1 hour, after which samples were gently aspirated and replaced with antibiotic-free DMEM. To detect SEAP activity, conditioned medium was collected after overnight incubation. Medium (20 pl; from a 200-pl total volume per well) was added to 180 pl of QUANTI-Blue™ solution. This mixture was incubated for an additional 1.5 hours under the same conditions to allow for the development of the colorimetric reaction. The optical density of each well was measured at 620 nm using a spectrophotometer to quantify the IFN-induced activity. Measurements of IFN-free (standard negative) and sample-free, IFN- containing (standard positive) conditions were used to define the range of each assay. Cell viability and density were assessed using a cell counter that reveals live and dead cells.

[0174] Cytokine quantification

[0175] The concentrations of human cytokines in sera, plasma samples, nasal swabs, and ETAs were measured using a Meso Scale Discovery U-plex™ custom multiplex assay kit. An additional 10 analytes were measured IFN-alpha2a, IFN-y, IL-15, IL-29 / IFN-X1, CXCL10 / IP-10, monocyte chemoattractant protein- 1 / CCL2, macrophage-derived chemokine, macrophage inflammatory protein-30, stromal cell-derived factor- la / CXCL 12a, and TRAIL) following the protocol of the manufacturer in plasma samples, nasal swabs, and ETAs. These samples were prepared following UVC-inactivation.

[0176] Flow cytometry-based Just-in-time Antibody and cytokine Measurements (FlowJAM™) processes clinical leftovers nasal samples for prediction infection severity

[0177] This method provides information from immediate immune response in the nose to predict disease outcomes and informing healthcare decisions. As a ”just-in-time” test, it can detect the nasal immune response immediately after COVID-19 infection. FlowJAM™ repurposes clinical waste samples (leftover PCR+ swabs) and designed to identify subject with a risk of developing acutely infected patients.

[0178] Specific antibody measurements can be obtained from antigen-coated bead captures to antigen-specific nasal (auto)antibodies. It is contemplated that one can use antibody coated bead disclosed herein in methods disclosed herein to further provide quantitative cytokine measurements wherein antibody-coated bead captures markers of immune response such as CXCL10 and type 1 INF / INF alpha. In certain embodiments, the assay has the benefit of providing a direct measurement of assay noise a nonsense-protein bead measures background antibody binding (background noise-BSA). Another advantage is that the methods are able to utilize PCR in combination with the disclosed processed using the same nasal swab samples (leftover swabs after PCR testing).

[0179] By measuring IFN-a (Type I IFN) and CXCL10 “together” with autoantibodies against IFN-a, increased assay sensitivity was observed for detecting patients protected against symptomatic COVID-19. If the patient does not produce nasal autoantibodies AND does not produce IFN-a or CXCL10, then those patients are at greater risk of developing symptomatic COVID-19. Therefore, the FlowBEAT™ / FlowJAM™ assays simultaneously measure these 3 molecules in nasal swabs or other samples. If one finds detectable levels of all 3, it increases the confidence that those patients will recover better (without symptoms or severe symptoms) than those who did not express 1, 2, or all 3 of these molecules. If one does not find any of these 3 molecules, the patient will likely develop symptomatic COVID-19.

Claims

CLAIMS1. A method of diagnosing a subj ect for a risk of developing severe viral or microbial disease comprising: quantifying IgAl anti-IFN-alpha autoantibodies from a nasal sample of a subject diagnosed with a viral or microbial infection; determining the quantity of nasal IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value or ii) greater than a reference or normal value; and diagnosing the subject with the viral or microbial infection as having a high risk of developing severe disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is similar to a reference or normal value of a subject without an infection; or diagnosing the subject with the viral or microbial infection as having a low risk of developing severe disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is greater than a reference or normal value.

2. The method of claim 1 further comprising providing particles coated with anti-IgAl antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal sample from the subject diagnosed with a viral or microbial infection with the fluorescent particles coated with anti-IgAl antibodies that specifically bind with the anti- IgAl fluorescent particles providing IgAl anti-IFN-alpha autoantibody fluorescent particles in the sample; and analyzing the IgAl anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of IgAl anti-IFN-alpha autoantibodies in the nasal sample of the subject.

3. The method of claim 1, wherein the method further comprises detecting elevated levels of INF-alpha in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce IFN-alpha in the nasal or airway, then those subjects are at greater risk of developing severe disease.

4. The method of any of claims 1 -3, wherein the method further comprises detecting elevated levels of CXCL10 in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce CXCL10 in the nasal or airway, then the subject is at greater risk of developing severe disease.

5. A method of diagnosing a subject for a risk of developing severe coronavirus disease or long COVID comprising: quantifying IgAl anti-IFN-alpha autoantibodies from a nasal sample of a subject diagnosed with a coronaviral infection; determining the quantity of nasal IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value or ii) greater than a reference or normal value; and diagnosing the subject with the coronaviral infection as having a high risk of developing severe microbial disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is similar to a reference or normal value of a subject without a coronaviral infection; or diagnosing the subject with the coronaviral infection as having a low risk of developing severe coronaviral disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is greater than a reference or normal value.

6. The method of claim 5 further comprising providing particles coated with anti-IgAl antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal sample from the subject diagnosed with a coronaviral infection with the fluorescent particles coated with anti-IgAl antibodies that specifically bind with the anti-IgAl fluorescent particles providing IgAl anti-IFN-alpha autoantibody fluorescent particles in the sample; analyzing the IgAl anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of IgAl anti-IFN-alpha autoantibodies in the nasal sample of the subject.

7. The method of claim 5, wherein the method further comprises detecting elevated levels of INF-alpha in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce IFN-alpha in the nasal or airway, then those subjects are at greater risk of developing severe coronaviral disease.

8. The method of any of claims 5-7, wherein the method further comprises detecting elevated levels of CXCL10 in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce CXCL10 in the nasal or airway, then the subject is at greater risk of developing severe coronaviral disease.

9. The method of claim 5, wherein the nasal sample of the subject is used to perform PCR of a coronavirus strain or other viral strain in the sample.

10. The method of claim 6, wherein the IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2 to 4 micrometers or 1 to 5 micrometers.

11. The method of claim 6, wherein IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.5 pm, is exposed to a 561 nm wavelength, and a fluorescent signal is measured from 586 to 610 nm in wavelength.

12. The method of claim 6, wherein the IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.8 pm, is exposed to a 488 nm wavelength, and a fluorescent signal is measured from 475 to 540 nm in wavelength.

13. The method of any of claims 5-12 further comprising recording a quantification and / or the risk of developing coronaviral disease severity on a non-transitory computer readable medium.

14. The method of claim 13, wherein the risk of developing a coronaviral disease severity is a high, low, medium, or quantitative number.

15. The method of claim 13, further comprising communicating the risk of developing a coronaviral disease severity to a medical professional or the subject.

16. The method of claim 13, wherein if the subject is diagnosed with a high risk of severe disease, then administering a coronaviral drug to the subject.

17. The method of claim 13, wherein if the subject is diagnosed with a high risk of severe disease, further administering mechanical assisted oxygen to the airways and lungs of the subject.

18. A method of diagnosing a subject for a risk of developing severe influenza disease comprising: quantifying IgAl anti-IFN-alpha autoantibodies from a nasal sample of a subject diagnosed with an influenza infection; determining the quantity of nasal IgAl anti-IFN-alpha autoantibodies to be either: i) similar to a reference or normal value or ii) greater than a reference or normal value; and diagnosing the subject with the influenza infection as having a high risk of developing severe influenza disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is similar to a reference or normal value of a subject without an influenza infection; or diagnosing the subject with the coronaviral infection as having a low risk of developing severe influenza disease if quantifying nasal IgAl anti-IFN-alpha autoantibodies from the nasal sample is greater than a reference or normal value.

19. The method of claim 18 further comprising providing particles coated with anti-IgAl antibodies, wherein the particles contain detectable fluorescent signatures providing individual and distinct anti-IgAl coated on individual particles with unique fluorescent signatures which are distinguishable by flow cytometry; contacting the nasal sample from the subject diagnosed with an influenza infection with the fluorescent particles coated with anti-IgAl antibodies that specifically bind with the anti-IgAl fluorescent particles providing IgAl anti-IFN-alpha autoantibody fluorescent particles in the sample;analyzing the IgAl anti-IFN-alpha autoantibody fluorescent particles by flow cytometry providing a quantity of IgAl anti-IFN-alpha autoantibodies in the nasal sample of the subject.

20. The method of claim 18 wherein the method further comprises detecting elevated levels of INF-alpha in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce IFN-alpha in the nasal or airway, then those subjects are at greater risk of developing severe influenza disease.

21. The method of any of claims 18-20, wherein the method further comprises detecting elevated levels of CXCL10 in the nasal and / or airway sample, wherein if the subject does not produce nasal IgAl anti-IFN-alpha autoantibodies and does not produce CXCL10 in the nasal or airway, then the subject is at greater risk of developing severe influenza disease.

22. The method of claim 18, wherein the nasal sample of the subject is used to perform PCR of an influenza strain in the sample.

23. The method of claim 19, wherein the IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2 to 4 micrometers or 1 to 5 micrometers.

24. The method of claim 19, wherein IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.5 pm, is exposed to a 561 nm wavelength, and a fluorescent signal is measured from 586 to 610 nm in wavelength.

25. The method of claim 19, wherein the IgAl anti-IFN-alpha autoantibody fluorescent particles have an average diameter of about 2.8 pm, is exposed to a 488 nm wavelength, and a fluorescent signal is measured from 475 to 540 nm in wavelength.

26. The method of any of claims 18-25 further comprising recording a quantification and / or the risk of developing influenza disease severity on a non-transitory computer readable medium.

27. The method of claim 26, wherein the risk of developing an influenza disease severity is a high, low, medium, or quantitative number.

28. The method of claim 26, further comprising communicating the risk of developing an influenza disease severity to a medical professional or the subject.

29. The method of claim 28, wherein if the subject is diagnosed with a high risk of severe disease, then administering an influenza drug to the subject.

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