WO2022165264A1

WO2022165264A1 - Compositions, methods and kits for analyzing and treating virally infected subjects using galectin-3 as a biomarker

Info

Publication number: WO2022165264A1
Application number: PCT/US2022/014432
Authority: WO
Inventors: Christene A. HUANG; Nalu NAVARRO-ALVAREZ
Original assignee: The Regents Of The University Of Colorado, A Body Corporate; Instituto Nacional De Ciencias Medicas Y Nutricion Salvador Zubiran
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2022-08-04

Abstract

Embodiments of the instant disclosure relate to novel methods and compositions for diagnosing and/or treating a respiratory condition. In certain embodiments, methods disclosed herein can be used to diagnose severity of the condition in a subject by analyzing a sample from a subject for concentration of at least one biomarker including, but not limited to, galectin-3, C-reactive protein (CRP), albumin, or the combination thereof. In certain embodiments, methods disclosed herein can be used to diagnose and then treat a viral-related condition having an adverse respiratory component by analyzing a sample from a subject for concentration of at least one biomarker including galectin-3, C-reactive protein (CRP), albumin, or combination thereof and intervening with a treatment for the condition based on the concentration of the at least one biomarker. In some embodiments, the viral-related condition is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Description

COMPOSITIONS, METHODS AND KITS FOR ANALYZING AND TREATING VIRALLY INFECTED SUBJECTS USING GALECTIN-3 AS A BIOMARKER

PRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63/143,513, filed January 29, 2021. This provisional application is incorporated by reference in its entirety for all purposes.

FIELD

[0002] Embodiments of the instant disclosure relate to novel methods and compositions for diagnosing and/or treating a respiratory disease or condition. In certain embodiments, methods disclosed herein can diagnose severity of a respiratory disease or condition in a subject by analyzing a sample from a subject for concentration of at least one biomarker including, but not limited to, galectin-3 (Gal-3), C-reactive protein (CRP), albumin, or the combination thereof. BACKGROUND

[0003] Respiratory-related conditions are a leading cause of death and disability globally. Prevention, control and cure of these conditions and promotion of respiratory health require diagnostics that effectively predict patient outcome and guide care givers in providing effective treatments. Respiratory infections, such as viral infections, lead to millions of deaths annually. [0004] COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) infection has afflicted tens of millions of people in a worldwide pandemic, straining health care systems across the world. Severe COVID-19 is associated with a systemic hyperinflammatory response characterized by high levels of circulating cytokines and chemokines and substantial lung infiltration of innate immune cells that can lead to respiratory distress such as acute respiratory distress syndrome (ARDS), multi-organ failure and death. Early intervention in COVID-19-related conditions is needed to reduce respiratory-related side-effects of this infection.

SUMMARY

[0005] Embodiments of the instant disclosure relate to novel methods and compositions for diagnosing and/or treating conditions having adverse respiratory components. In certain embodiments, methods disclosed herein can be used to diagnose onset of, or severity of, a respiratory condition in a subject. In some embodiments, methods disclosed herein can be used for diagnosing onset of or severity of a respiratory condition in a subject including one or more of the following: (a) analyzing one or more samples from a subject having an infection for concentration of at least one biomarker including, but not limited to, galectin-3, C-reactive protein (CRP), albumin, or the combination thereof; and (b) diagnosing the subject as developing or having a severe respiratory condition. In certain embodiments, diagnosing the subject as developing or having a severe respiratory condition includes analyzing the concentration of at least one biomarker including galectin-3, C-reactive protein (CRP), albumin, or the combination thereof and if the concentration of galectin-3, C-reactive protein (CRP), albumin, or the combination thereof in the sample is increased and/or decreased in order to diagnose respiratory involvement of the subject. In accordance with these embodiments, diagnosing the subject as developing or having a severe respiratory condition includes analyzing the concentration of Gal- 3 and diagnosing the subject as developing or having a severe respiratory condition when Gal-3 is elevated in the subject to or above a certain level.

[0006] In other embodiments, the instant applications relate to novel methods and compositions for diagnosing and/or treating conditions having adverse respiratory components. In certain embodiments, methods disclosed herein can be used to diagnose onset of, or severity of, a respiratory condition in a subject. In some embodiments, methods disclosed herein can be used for diagnosing onset of or severity of a respiratory condition in a subject including one or more of the following: (a) analyzing one or more samples from a subject having an infection for concentration of at least one biomarker including, but not limited to, galectin-3, C-reactive protein (CRP), albumin, or the combination thereof; (b) comparing the concentration of the at least one biomarker including galectin-3, C-reactive protein (CRP), albumin, or the combination thereof in the sample to at least one control sample, where the at least one control sample is from a subject having the same or similar condition not having respiratory affects and/or from a healthy subject; and (c) diagnosing the subject as developing or having a severe respiratory condition. In certain embodiments, diagnosing the subject as developing or having a severe respiratory condition includes analyzing the concentration of at least one biomarker including galectin-3, C-reactive protein (CRP), albumin, or the combination thereof and if the concentration of galectin-3, C-reactive protein (CRP), albumin, or the combination thereof in the sample is increased and/or decreased compared to the control sample from a subject having the same or similar condition not having respiratory affects and/or from a healthy subject. In accordance with these embodiments, diagnosing the subject as developing or having a severe respiratory condition includes analyzing the concentration of Gal-3 and diagnosing the subject as developing or having a severe respiratory condition when Gal-3 is elevated in the subject.

[0007] In some embodiments, the subject has a viral infection or other condition having or at risk of developing a respiratory condition. In certain embodiments, the subject has a SARS or for example, a COVID-19 infection or mutant viral infection thereof (e.g., delta variant or Omicron variant).

[0008] In certain embodiments, a sample from a subject having an infection can include, but is not limited to, whole blood, blood serum, blood plasma, saliva, nasal swab, sputum, urine, feces or other body fluid or excrement or a combination thereof. In other embodiments, more than one sample type is obtained from a subject for comparison and improved diagnosis. In some embodiments, one or more sample obtained from the subject is obtained within 24 hours, within 2 days, within 3 days, within 4 days, within 5 days, within a week, within a couple of weeks or more from the date of suspected or actual exposure to a virus. In some embodiments, one or more sample obtained from the subject is obtained within 24 hours, within 2 days, within 3 days, within 4 days, within 5 days, within a week, within a couple of weeks or more from the date of suspected or actual exposure to SARS or CO VID.

[0009] In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition if the concentration of galectin-3 in the sample is increased (e.g., for example reaches or is above a threshold amount) or increased compared to the control sample. In accordance with these embodiments, a subject can be diagnosed as having a severe respiratory disease or condition if the concentration of galectin-3 in the sample is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40% or more or at least 1-fold or more or when compared to the control sample (e.g., healthy, or infected control subject). In some embodiments, the biomarker analyzed in the at least one sample is galectin-3 and the concentration for determining the severity of the potential or existence of severe respiratory conditions is at least 25 ng/ml; at least 30 ng/ml; at least 35 ng/ml; at least 40 ng/ml or more. In certain embodiments, a subject is newly admitted with an infection or a subject that has been under mechanical respiratory control. In other embodiments, a subject having gal-3 concentration of 25 ng/ml or greater in at least one sample from the subject can indicate an adverse clinical outcome and potential for death. In other embodiments, the subject can be diagnosed with having or likelihood of developing acute respiratory syndrome (ARDS) when the concentration of Gal-3 in the sample is about 25 ng/ml or greater or about 30 ng/ml or greater or about 30.99 ng/ml or greater.

[0010] In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition if the concentration of CRP in the sample is increased (e.g., for example reaches or is above a threshold amount) or in other embodiments, increased compared to one or more control sample. In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition if the concentration of CRP in the sample is increased by at least 1-fold compared to the control sample (e.g., healthy, or infected control subject).

[0011] In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition if the concentration of albumin in the sample is decreased (e.g., for example reaches or is below a threshold amount) or increased compared and in other embodiments, compared to at least one control sample. In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition if the concentration of albumin in the sample is decreased by at least 1-fold (e.g., healthy, or infected control subject). In other embodiments, samples disclosed herein from a subject to be diagnosed can be compared to one or more control samples as described herein.

[0012] In some embodiments, methods disclosed herein can further include any one of the following: analyzing a CT scan image taken of a subject having an infection; comparing the CT scan image to a control image, wherein the control image is from a subject having an infection and diagnosed as not having a severe respiratory disease or condition; and diagnosing the subject as having a severe respiratory disease or condition if the CT scan image shows lung damage. In other embodiments, methods disclosed herein can further include any one of the following: analyzing a CT scan image taken of a subject having an infection; comparing the CT scan image to a control image, wherein the control image is from a subject having an infection and diagnosed as not having a severe respiratory disease or condition; and diagnosing the subject as having a severe respiratory disease or condition if the CT scan image shows lung damage compared to the control image.

[0013] In certain embodiments, methods disclosed herein can be used for diagnosing and/or treating a severe respiratory disease or condition in a subject. In some embodiments, methods for diagnosing and/or treating a condition having a severe respiratory component or side effect in a subject can include one or more of the following: (a) analyzing one or more sample from a subject having an infection for concentration of at least one biomarker including galectin-3, C- reactive protein (CRP), albumin, or the combination thereof; (b) diagnosing the subject as having a severe respiratory disease or condition if the concentration of galectin-3, C-reactive protein (CRP), albumin, or the combination thereof in the sample is increased and/or decreased compared to the control sample; and (c) administering at least one treatment for a severe respiratory disease or condition to the subject diagnosed as having a severe respiratory disease or condition.

[0014] In certain embodiments, methods disclosed herein can be used for diagnosing and/or treating a severe respiratory disease or condition in a subject. In some embodiments, methods for diagnosing and/or treating a condition having a severe respiratory component or side effect in a subject can include one or more of the following: (a) analyzing one or more sample from a subject having an infection for concentration of at least one biomarker including galectin-3, C- reactive protein (CRP), albumin, or the combination thereof; (b) comparing the concentration of the at least one biomarker including galectin-3, C-reactive protein (CRP), albumin, or the combination thereof in the sample to a control sample, wherein the control sample is from a subject having an infection diagnosed as not having a severe respiratory disease or condition or a healthy control; (c) diagnosing the subject as having a severe respiratory disease or condition if the concentration of galectin-3, C-reactive protein (CRP), albumin, or the combination thereof in the sample is increased and/or decreased compared to the control sample; and (d) administering at least one treatment for a severe respiratory disease or condition to the subject diagnosed as having a severe respiratory disease or condition.

[0015] In some embodiments, methods of treating a severe respiratory disease or condition disclosed herein can include, but is not limited to, administering to the subject a composition including, but not limited to, ACE2 recombinant proteins, ACE2 antibodies, ACE2 inhibitors, remdesivir, favipiravir, merimepodib, lopinavir, ritonavir, prednisone, methylprednisolone, hydrocortisone, dexamethasone, anti-inflammatory drugs, convalescent plasma, amlodipine, ivermectin, losartan, famotidine, monoclonal antibodies, polyclonal antibodies, antibiotics, albuterol, invasive mechanical ventilation, or a combination thereof. In other embodiments, a subject in need of such a treatment can be treated by providing oxygen or other respiratory mechanically administered intervention. In some embodiments, combination treatments can be administered to the subject that include treating the subject with an agent and providing oxygen to the subject, for example. In yet other embodiment, Gal-3 expression or levels can be treated or controlled in the subject using any method known in the art.

[0016] In certain embodiments, kits for diagnosing severity of a respiratory disease or condition in a subject are contemplated. In some embodiments, kits for diagnosing severity of a respiratory disease or condition in a subject can include: (a) at least one protein biomarker array or biomarker binding agent, where the protein biomarker array can include one or more probe suitable for binding to the one or more protein biomarkers selected from albumin, CRP, galectin- 3, or a combination thereof; and (b) at least one detection agent, wherein the least one detection agent can detecting binding of the binding agent to the one or more biomarkers in order to assess concentration of the biomarker.

[0017] In some embodiments, kits disclosed herein can include at least one reference standard wherein the reference standard encompasses one or more biomarkers selected from albumin, CRP, galectin-3, or a combination thereof representative of a normal, healthy subject. In other embodiments, kits disclosed herein can include, but are not limited to, protein biomarker arrays or biomarker binding agents having the capacity to detect about 1.0 to about 300 ng/mL galectin-3 in a sample, and/or about 1.0 to about 300 ng/mL CRP in a sample, and/or about 1.0 to about 20 g/dL albumin, or a combination thereof in a sample. In some embodiments, samples of use in the kits disclosed herein can include, but are not limited to, whole blood, blood serum, blood plasma, saliva, nasal sample, urine, feces or a combination thereof collected from a human subject. In some embodiments, kits herein can further include one or more containers, one or more reagents, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figs. 1A-1C illustrate examples of galectin-3 serum levels in virally infected patients (e.g. CO VID-19) compared to controls in accordance with certain embodiments of the present disclosure.

[0019] Figs. 2A-2F illustrate examples of galectin-3 and CRP biomarkers as indicators of severe outcome in moderate (2 A and 2D) and graphic representations of critical COVID-19 patients (2B, 2C, 2E and 2F) in accordance with certain embodiments of the present disclosure. [0020] Figs. 3A-3F illustrate examples of galectin-3 correlation with different inflammatory markers in CO VID-19 patients in accordance with certain embodiments of the present disclosure. [0021] Figs. 4A-4D illustrate graphic examples of galectin-3, albumin and CRP as independent predictors of severe outcome in COVID-19 patients in accordance with certain embodiments of the present disclosure.

[0022] Fig. 5 illustrates an example of galectin-3 levels that correlate critical versus moderate patients having a viral infection in accordance with certain embodiments of the present disclosure.

[0023] Figs. 6A-6C illustrates examples of smoothing splines of galectin-3 (a), CRP (b) and albumin (c) in accordance with certain embodiments of the present disclosure.

DEFINITIONS

[0024] Terms, unless specifically defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.

[0025] Unless otherwise indicated, all numbers expressing quantities of agents and/or compounds, properties such as molecular weights, reaction conditions, and as disclosed herein are contemplated as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that can vary from about 10% to about 15% plus and/or minus depending upon the desired properties sought as disclosed herein. Numerical values as represented herein inherently contain standard deviations that necessarily result from the errors found in the numerical value's testing measurements.

[0026] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject can be a human.

[0027] As used herein, "treatment," "therapy" and/or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient can be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder, or condition. As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, a symptom of the disease or disorder, or the predisposition toward the disease or disorder.

[0028] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder, or physiological condition, relative to the time and/or degree of onset or severity in the absence of intervention.

[0029] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0030] Alleviating a target disease, condition or disorder disclosed herein includes delaying the development or progression of the disease, condition or disorder or reducing severity of a side effect or severity of the condition or prolonging survival of the subject. Alleviating the disease or prolonging survival does not necessarily require curative results. As used therein, "delaying" the development of a target disease, condition, or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0031] “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that can be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.

[0032] The term “analyte,” as used herein, can refer to the substance to be detected, which can be present in a sample from a subject. The analyte can be any substance having a naturally occurring specific binding partner or for which a specific binding partner can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding partners in an immunoassay. One example of an analyte as described herein is an endogenous antigen, including but not limited to galectin-3, which is an antigen that can be assessed as a measure of, or measure of risk of developing, for example, at least one respiratory disease and/or respiratory condition (e.g. COVID-19 or SARS or variant or mutant thereof)

[0033] As used interchangeably herein, the terms “epitope”, “epitopes” or “epitopes of interest” can refer to a site(s) on any molecule that is recognized and is capable of binding to a complementary site(s) on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope can be a polypeptide, protein, hapten, carbohydrate antigen (such as, but not limited to, glycolipids, glycoproteins or lipopolysaccharides) or polysaccharide and its specific binding partner, can be, but is not limited to, an antibody, which can be an autoantibody. Typically an epitope is contained within a larger antigenic fragment (i.e., region or fragment capable of binding an antibody) and refers to the precise residues known to contact the specific binding partner. An antigenic fragment can contain more than one epitope.

[0034] As used herein, the terms “specific binding”, “specificity” and “specifically binding”, characterize the interaction between two molecules having the ability to selectively react with one another as a pair (e.g., an antigen and antibody). The phrase “specifically binds to” refers for example to the ability of an antibody to specifically bind to its target antigen (e.g., an endogenous antigen such as galectin-3), while not specifically bind to other entities. Antibodies or antibody fragments that specifically bind to an analyte can be identified, for example, by diagnostic immunoassays (e.g., radioimmunoassays (“RIA”) and enzyme-linked immunosorbent as says (“ELISAs”) (See, for example, BGM Galectin-3 assay (BG Medicine, Inc) and ARCHITECT Galectin-3 kit (Abbott Laboratories)) or other techniques known to those of skill in the art. The term “specifically binds” indicates that the binding preference (e.g., affinity) for the target molecule/sequence is at least 2-fold, more preferably at least 5-fold, and most preferably at least 10- or 20-fold over a non- specific target molecule (e.g. a randomly generated molecule lacking the specifically recognized site(s)).

[0035] As used herein the term “detectable label” can refer to any moiety that generates a measurable signal via optical, electrical, or other physical indication of a change of state of a molecule or molecules coupled to the moiety. Such physical indicators encompass spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, and chemical means, such as but not limited to fluorescence, chemifluorescence, chemiluminescence, and the like. As used with reference to a labeled detection agent, a “direct label” is a detectable label that is attached, by any means, to the detection agent. As used with reference to a labeled detection agent, an “indirect label” is a detectable label that specifically binds the detection agent. Thus, an indirect label includes a moiety that is the specific binding partner of a moiety of the detection agent. Biotin and avidin are examples of such moieties that are employed, for example, by contacting a biotinylated antibody with labeled avidin to produce an indirectly labeled antibody. An indicator reagent can be used to contact a detectable label to produce a detectable signal. Thus, for example, in conventional enzyme labeling, an antibody labeled with an enzyme can be contacted with a substrate (the indicator reagent) to produce a detectable signal, such as a colored reaction product.

[0036] As used herein, the term “sample” generally refers to a biological material being tested for and/or suspected of containing a target analyte, /.< ., an analyte of interest, e.g., galectin-3. The biological material can be derived from any biological source but preferably is a biological fluid likely to contain the target analyte. Examples of biological materials can include, but are not limited to, stool, whole blood, serum, plasma, red blood cells, platelets, bronchial lavage, bone marrow aspirate, pleural effusion, interstitial fluid, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, as well as tumor tissue or any other bodily constituent or any tissue culture supernatant that could contain the analyte of interest. Samples herein can be obtained by routine procedures such as but not limited to venipuncture, tissue biopsy including needle biopsy, swab, wipe, and fluid collection. Samples herein are obtained from an animal, preferably a mammal, and more preferably a human. The sample can be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment can include preparing plasma from blood, diluting viscous fluids and so forth. Methods of pretreatment can also involve centrifugation, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc. If such methods of pretreatment are employed with respect to the test sample, such pretreatment methods are such that the target analyte remains in the test sample at a concentration proportional to that in an untreated test sample (e.g., namely, a test sample that is not subjected to any such pretreatment method(s)).

[0037] As used herein, the term “ROC” means “receiver operating characteristic”. A ROC analysis can be used to evaluate the diagnostic performance, or predictive ability, of a test or a method of analysis. A ROC graph is a plot of sensitivity and specificity of a test at various thresholds or cut-off values. Each point on an ROC curve represents the sensitivity and its respective specificity. A threshold value can be selected based on an ROC curve to identify a point where sensitivity and specificity both have acceptable values, and this value can be used in applying the test for diagnostic purposes. If specificity only is optimized, then the test will be less likely to generate a false positive (diagnosis of the disease in more subjects who do not have the disease) at the cost of an increased likelihood that some cases of disease will not be identified (e.g., false negatives). If sensitivity is only optimized, the test will be more likely to identify most or all of the subjects with the disease but will also diagnose the disease in more subjects who do not have the disease (e.g., false positives). A user can modify the parameters, and therefore select an ROC threshold value suitable for a given clinical situation, in ways that will be readily understood by those skilled in the art.

[0038] In certain embodiments, another useful feature of the ROC curve is an area under the curve (AUC) value, which quantifies the overall ability of the test to discriminate between different sample properties, for example, to discriminate between those subjects with a severe COVID-19 outcome (e.g., elevated galectin-3 levels compared to control(s)) and those with a non-severe COVID-19 outcome (e.g., reduced galectin-3 levels). A test that is no better at identifying true positives than random chance can generate a ROC curve with an AUC of about 0.5 or at 0.5. A test having perfect specificity and sensitivity (e.g., generating no false positives and no false negatives) will have an AUC of about 1.00 or 1.00.

[0039] As used herein, the term “sensitivity” can refer to percentage of truly positive observations which is classified as such by a test and indicates the proportion or percentage or number of subjects correctly identified as having a severe respiratory condition. In other words, sensitivity is equal to (true positive result)/[(true positive result)+(false negative result)].

[0040] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. DETAILED DESCRIPTION OF THE INVENTION

[0041] In the following sections, certain exemplary compositions and methods are described in order to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times, and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0042] In some embodiments, compositions methods and kits disclosed herein can be used to diagnose respiratory-related conditions in a subject having an infection or other health condition. Respiratory conditions can include, but are not limited to, temporary or acute inflammatory conditions (e.g., caused by an environmental insult, such as exposure to an irritant) and temporary or acute infectious conditions (e.g., caused by exposure to a pathogen) that affects the lungs and/or other parts of the respiratory system. Respiratory diseases or conditions (e.g., chronic airway diseases and pulmonary diseases) can include, but are not limited to, long-term or chronic diseases or conditions with underlying inflammation/irritation, such as asthma, airway hyperresponsiveness, seasonal allergic allergy, brochiectasis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary parenchyl inflammatory conditions, and the like. In some embodiments, compositions and methods disclosed herein can diagnose the severity of at least one respiratory- related condition in a subject. In other embodiments, methods disclosed herein can be used to select a subject having or suspected of having at least one respiratory-related condition or an infection (e.g., viral infection) for one or more therapies to treat the condition. In other embodiments, compositions and methods disclosed herein can be used in monitoring efficacy of one or more therapies of a respiratory -related condition in a subject by measuring levels of biomarkers. In other embodiments, compositions and methods disclosed herein can be used in monitoring efficacy of one or more therapies of a respiratory-related condition in a subject by measuring levels of biomarkers compared to controls.

[0043] In certain embodiments, compositions methods and kits disclosed herein can be used to diagnose respiratory-related conditions in a subject having an infection or other health condition. Respiratory conditions can include, but are not limited to, temporary or acute inflammatory conditions (e.g. caused by an environmental insult, such as exposure to an irritant) and temporary or acute infectious conditions (e.g. caused by exposure to a pathogen) that affects the lungs and/or other parts of the respiratory system. Respiratory diseases or conditions (e.g., chronic airway diseases and pulmonary diseases) can include, but are not limited to, long-term or chronic diseases or conditions with underlying inflammation/irritation, such as asthma, airway hyperresponsiveness, seasonal allergic allergy, brochiectasis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary parenchyl inflammatory conditions, and the like. In some embodiments, methods disclosed herein can diagnose the severity of at least one respiratory -related condition in a subject. In other embodiments, methods disclosed herein can be used to select a subject having or suspected of having at least one respiratory-related condition or an infection (e.g., viral infection) for one or more therapies to treat the condition. In some embodiments, methods disclosed herein can be used in monitoring efficacy of one or more therapies of a respiratory- related condition in a subject by measuring levels of biomarkers and assessing concentration of the one or more biomarkers in the sample. In some embodiments, methods disclosed herein can be used in monitoring efficacy of one or more therapies of a respiratory -related condition in a subject by measuring levels of biomarkers compared to controls.

[0044] In some embodiments, methods of diagnosing at least one respiratory-related condition can include determining the absence, presence, relative amount, over- or underrepresentation, or fold-change of one or more biomarkers contemplated herein wherein the absence, presence, relative amount, over- or underrepresentation of the one or more biomarkers correlates with the absence or presence of at least one respiratory disease and/or respiratory condition or severity of at least one respiratory disease and/or respiratory condition in a subject. In certain embodiments, a biomarker profile of expression can include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more biomarkers. In some embodiments, biomarkers disclosed herein can be a protein, a polypeptide, a peptide fragment, a nucleic acid, an mRNA, microRNA or a combination thereof. In other embodiments, a sample having the one or more biomarkers contemplated herein can be, for example, one or more of, an exhaled breath condensate, sputum, bronchoalveolar lavage (BAL) fluid, nasal lavage, bronchial or nasal biopsy, epithelial brushings, whole blood, serum, plasma, lymph fluid, cerebrospinal fluid, saliva, urine, mucus, nasal sample, urine, excrement or the like. [0045] In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of one or more biomarkers including, but not limited to, Gal-3, CRP and/or albumin. In certain embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of galectin-3 in the sample as being increased or elevated. In certain embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of galectin-3 in the sample as being increased or elevated compared to at least one control sample (e.g., a control having an infection but not a condition or healthy control). In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of CRP in the sample as being increased. In some embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of CRP in the sample as being increased compared to at least one control sample (e.g., a control having an infection but not a condition or healthy control). In other embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of albumin in the sample as being decreased. In other embodiments, a subject can be diagnosed as having a severe respiratory disease or condition based on the concentration of albumin in the sample as being decreased compared to at least one control sample (e.g., a control having an infection but not a condition or healthy control).

[0046] In some embodiments, the biomarker analyzed in the at least one sample includes, but is not limited to, galectin-3 and the concentration for determining the severity of the potential or existence of severe respiratory conditions is at least 25 ng/ml; at least 30 ng/ml; at least 35 ng/ml; at least 40 ng/ml or more. In certain embodiments, a subject is newly admitted with an infection or a subject that has been under mechanical respiratory control. In other embodiments, a subject having gal-3 concentration of 25 ng/ml or greater in at least one sample from the subject can indicate an adverse clinical outcome and potential for death. In other embodiments, the subject can be diagnosed with having or likelihood of developing acute respiratory syndrome (ARDS) when the concentration of Gal-3 in the sample is about 25 ng/ml or greater or about 30 ng/ml or greater or about 30.99 ng/ml or greater. In certain embodiments, control samples can be used to compare Gal-3 levels in at least one control sample compared to a subject having an infection and having a Gal-3 concentration of at least 25 ng/ml in a sample obtained from the subject. [0047] In some embodiments, methods disclosed herein can include monitoring efficacy of a treatment. In certain embodiments, methods of monitoring efficacy of a treatment can include, but is not limited to, obtaining a first sample from a subject suspected of having at least one respiratory disease and/or respiratory condition at the commencement of treatment, which can represent a suitable base level sample, and a second, third, fourth, or more sample at certain time intervals during treatment, which can be compared to the first sample. In some embodiments, the second, third, fourth, or more sample can be compared to the base level in a first sample or a sample obtained from normal (healthy or un-infected) subjects. In certain embodiments, methods disclosed herein that demonstrate a change in biomarker profile closer to a biomarker profile at baseline could indicate that a given treatment is efficacious and should or can be continued. In some embodiments, methods disclosed herein that show a biomarker profile that does not significantly change in the second, third, fourth, etc. sample when compared to the first sample could indicate that the treatment regimen is not efficacious, and the treatment should be altered or stopped. In other embodiments, subsequent sampling demonstrating an improvement can assist a health professional in assessing length of treatment to clearance or near clearance of a respiratory condition. In some embodiments, at least one sample is analyzed for Gal-3 concentration and the subject is treated or the treatments are adjusted based on Gal-3 concentration. In other embodiments, at least one sample is analyzed for Gal-3 concentration and the subject is treated or the treatments are adjusted based on elevated Gal-3 concentrations of about 30 ng/ml or more in the at least one sample. In accordance with these embodiments, the subject can receive an adjusted treatment based on the level of Gal-3 alone or in combination with analysis of other biomarkers. In certain embodiments, a subject having an elevated Gal-3 concentration in the at least one sample can be treated with an agent to treat or reduce the onset of a respiratory condition or provide mechanical lung aids or the like. In other embodiments, the subject can be treated with an agent to reduce Gal-3 alone or in combination with other treatments.

[0048] In certain embodiments, a subject can be diagnosed with having at least one respiratory -related condition based on results obtained from a biomarker array contacted with a sample collected from the subject, wherein the biomarkers' absence, presence and/or concentration (e.g. under- or overrepresentation relative to a suitable control sample) in the sample are determined according to the methods described herein, where the biomarker array or biomarker binding agent can include, but is not limited to, one or more, two or more, three or more, four or more, five or more biomarkers. In some embodiments, the biomarker array used herein can have one or more, two or more, three or more, four or more, or five or more biomarkers including, but not limited to, albumin, ferritin, D-Dimer, C-reactive protein (CRP), lactate dehydrogenase (LDH), neutrophil count, galectin-3, or a combination thereof. In other embodiments, a subject can be diagnosed with having at least one respiratory-related condition in need of intervention when expression of one or more biomarkers is increased or decreased by at least 1-fold, at least 2-fold, at least 3 -fold, or at least 4-fold or at least reduced by about 10% or about 20%, or about 30% or more when compared to a suitable control profile.

[0049] In other embodiments, a subject can be diagnosed as having a respiratory-condition related to CO VID-19 or mutant thereof infection when one or more biomarkers including, but not limited to, albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof are increased or decreased as disclosed herein. In some embodiments, when biomarkers of ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof are increased and/or if albumin is decreased, a respiratory-related condition is diagnosed in the subject. In certain embodiments, a subject can be diagnosed with having COVID-19 or mutant or derivative thereof or a respiratory-related condition related to COVID-19 or other viral infection when expression or concentrations of a biomarker including, but not limited to, albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof is increased or decreased by at least 1-fold, at least -fold 2, at least 3 -fold, or at least 4-fold or at least reduced by about 10% or about 20%, or about 30% or more. In certain embodiments, a suitable control sample can be used for comparison.

[0050] In some embodiments, a subject can be diagnosed as having severe COVID-19 or mutant or derivative thereof, infection including a respiratory-related condition using the methods disclosed herein. As used herein, a subject having “severe COVID-19” can include those subjects who are most likely to require drastic intervention including but not limited to, intermittent or continuous mechanical ventilation (IMV) and/or in-hospital death. As used herein, COVID-19 patients can be classified as either critical (>50% lung damage) or moderate (<50% of lung damage) based on computerized tomography (CT). In some embodiments, a subject contemplated herein can be a critical COVID-19 subject diagnosed as having non-severe COVID-19 using the methods disclosed herein, wherein the subject has >50% lung damage but is not likely to have a severe outcome. In other embodiments, a subject herein can be a critical COVID-19 subject diagnosed as having severe COVID-19 using the methods disclosed herein, wherein the subject has >50% lung damage but is likely to have a severe outcome including IMV and/or in-hospital death. In yet other embodiments, a subject herein can be a moderate COVID-19 subject diagnosed as having non-severe COVID-19 using the methods disclosed herein, wherein the subject has <50% lung damage but is not likely to have a severe outcome. In some embodiments, a subject herein can be a moderate COVID-19 subject diagnosed as having severe COVID-19 using the methods disclosed herein, wherein the subject has <50% lung damage but is likely to have a severe outcome including IMV and/or in-hospital death. In certain embodiments, methods disclosed herein can be used to diagnose a subject having or suspected of having COVID-19 prior to or after classifying the subject by CT scan. In some embodiments, methods disclosed herein can be used to diagnose a subject having or suspected of having COVID-19 without classifying the subject by CT scan. In some embodiments, probability of death of the patient having excessive Gal-3 expression or other marker disclosed herein in a subject’s samples can be used in pandemic situations in order to assess chance of recovery of the subject or chance of survival of the subject compared to other subjects.

[0051] In some embodiments, a subject can be diagnosed as having a severe viral infection, for example, a severe CO VID-19 or mutant thereof when one or more biomarkers including, but not limited to, albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof are increased or decreased when compared to a suitable control sample. In some embodiments, a subject can be diagnosed as having a severe COVID-19 infection if ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof are increased and/or if albumin is decreased. In certain embodiments, a suitable control sample can be used for comparison. In certain embodiments, a subject can be diagnosed with having a severe COVID-19 infection when expression of albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof is increased or decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40% of at least about 1-fold, at least about 2-fold, at least about 3 -fold, or at least about 4-fold. In certain embodiments, a suitable control sample can be used for comparison (e.g., healthy subject or infected subject not having a severe COVID-19 infection). In some embodiments, a subject can be diagnosed with having a severe CO VID-19 or mutant thereof infection when expression of ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40% of at least about 1-fold, at least about 2-fold, at least about 3-fold, or at least about 4-fold and/or when expression of albumin is decreased by at least 1-fold, at least about 2-fold, at least 3 -fold, or at least 4-fold. In certain embodiments, a suitable control sample can be used for comparison (e.g., healthy subject or infected subject not having a severe COVID-19 infection). In some embodiments, a subject can be diagnosed with having a severe CO VID-19 or mutant thereof infection when expression of CRP, galectin-3 or a combination thereof is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40% of at least about 1-fold, at least about 2-fold, at least about 3 -fold, or at least about 4-fold and/or when expression of albumin is decreased by at least 1-fold, at least 2-fold, at least 3 -fold, or at least 4-fold. In certain embodiments, a suitable control sample can be used for comparison (e.g., healthy subject or infected subject not having a severe CO VID-19 infection). In some embodiments, a subject can be diagnosed with having a severe COVID-19 or mutant thereof infection when expression of galectin-3 is increased by at least 1-fold, at least 2-fold, at least 3 -fold, or at least 4-fold. In certain embodiments, a suitable control sample can be used for comparison e.g., healthy subject or infected subject not having a severe COVID-19 infection). [0052] In some embodiments, at least one of an increase of Gal-3 and CRP expression and/or protein levels and a decrease of albumin expression and/or protein levels in a sample collected from a subject herein can discriminate between severe COVID-19 and less-severe COVID-19 respiratory related conditions with at least about a 50% to about a 99% probability; or at least about a 55% to about a 95%; or at least about a 75%, or at least about a 80%, or at least about a 90%, or at least about a 95%, or at least about a 99% probability. In other embodiments, at least one of an increase of Gal-3 and CRP expression and/or protein levels and a decrease of albumin expression and/or protein levels in a sample collected from a subject herein can discriminate between severe COVID-19 and less-severe COVID-19 respiratory related conditions with at least about a 84% probability. In certain embodiments, at least one of an increase of Gal-3 and CRP expression and/or protein levels and a decrease of albumin expression and/or protein levels in a sample collected from a subject herein can discriminate between severe COVID-19 and less-severe COVID-19 respiratory related conditions with at least about a 84% probability independent of whether the subject’s condition has not been classified by CT scan, the subject has been classified as having moderate COVID-19 by CT scan, or the subject has been classified as having critical COVID-19 by CT scan as disclosed herein.

[0053] Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has afflicted tens of millions of people in a worldwide pandemic, straining health care systems across the world. Prognostic biomarkers are needed that can identify high-risk patients to improve clinical management and allow appropriate allocation of healthcare resources. Moreover, the lack of current curative therapies emphasizes the need to get a better understanding of the pathophysiological process behind SARS-CoV-2 infection and its long-term consequences for the development of targeted therapeutic strategies.

[0054] Severe COVID-19 is associated with a systemic hyperinflammatory response characterized by high levels of circulating cytokines and chemokines and substantial lung infiltration of innate immune cells that can lead to acute respiratory distress syndrome (ARDS), multi-organ failure and death. Among the inflammatory cytokines are those associated with the activation of monocyte/macrophages such as Interleukin 6 (IL-6), Tumor necrosis factor (TNF), and the CC-chemokine ligand 2 (CCL2).

[0055] Studies have shown that those inflammatory cytokines can contribute to the recruitment of additional inflammatory cells that not only aggravate lung damage but can also lead to pulmonary fibrosis. Subsets of M2 macrophages expressing profibrogenic genes have been found in the bronchoalveolar lavage of COVID-19 patients, reflecting that the pathological process of SARS-CoV-2 infection not only involves an acute inflammatory response in the lungs, but can also be associated with fibrotic complications. Embodiments disclosed herein are designed to provide an early diagnosis and intervention in order to avoid complications from severe respiratory infections, long term effects and even death of a subject having these conditions.

[0056] Galectin-3 is a 29-35 kDa B-galactoside binding lectin first identified in macrophages. It plays an important role as a driver and amplifier of the pro-inflammatory response by promoting the release of several cytokines including IL-6 and TNF-a, which are some of the major cytokines present in severe COVID-19 patients. High levels of galectin-3 are known to drive neutrophil infiltration and the release of pro-inflammatory cytokines, contributing to acute airway inflammation. In addition, studies have shown that endogenous galectin-3 can enhance the effects of viral infection by promoting host inflammatory responses. [0057] In some embodiments, it was found that levels of serum galectin-3 upon hospital admission indicates a subject that is at high-risk of progressing to a severe COVID-19 outcome resulting in invasive mechanical ventilation (IMV) and/or death. As disclosed herein, galectin-3 levels were correlated with clinical and inflammatory laboratory markers. In certain methods, COVID-19 patients were diagnosed as either critical (>50% lung damage) or moderate (<50% of lung damage) based on computerized tomography (CT). In some embodiments, it was demonstrated that elevated serum galectin-3 was significantly higher in critically-ill subjects compared to moderate ones and surprisingly, was also found to be an independent predictor of severe outcome regardless of the percentage of lung involvement in the subject. In other embodiments, it was demonstrated that galectin-3 was a useful prognostic biomarker in COVID- 19 patients to provide early identification of subjects at high risk of severe illness and to provide guidance on resource allocation.

[0058] In other embodiments, relative levels of the one or more biomarkers disclosed herein can be determined in a sample collected from a subject contemplated herein (e.g., COVID-19 infected or other viral infected subject) using detection methods well known in the art. Useful detection methods or “assays” can include, but are not limited to, immunoassays, mass spectroscopy, PCR, DNA arrays, and restriction fragment length polymorphism (RFLP) analysis. In some embodiments, examples of immunoassays suitable for use herein can be enzyme immune assay (EIA), enzyme-linked immunosorbent assays (ELISAs), enzyme multiplied immunoassay (EMIT), radio-immunoassays (RIA), radioimmune precipitation assays (RIP A), Farr assay, immunobead capture assays, Western blotting, dot blotting, gel-shift assays, flow cytometry (fluorescent activated cell sorting (FACS)), immunofluore scent microscopy, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), fluorescence polarization immunoassay (FPIA), fluorescence recovery /localization after photobleaching (FRAP/FLAP), and combinations thereof. Generally, immunoassays used herein can involve contacting a sample with a capturing agent (e.g. an antibody or antibody fragment) capable of interacting with a recognition site (e.g. an antigen) present on a biomarker under conditions effective to allow the formation of immunocomplexes. In some embodiments, immunoassays herein can further include one or more steps wherein the capturing agent is bound to or is capable of binding to a solid support (e.g., tube, well, bead, or cell) to capture the biomarker protein of interest from a sample, optionally combined with a method of detecting the biomarker protein or capturing agent specific for the biomarker protein on the support. Non-limiting examples of such immunoassays include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), flow cytometry, protein array, multiplexed bead assay, and magnetic capture. In some embodiments, immunoassays disclosed herein can employ one or more detection agents. In some embodiments, detection agents herein can be specific for a biomarker disclosed herein. In some embodiments, immunoassays disclosed herein can employ a labeled detection agent, such as a labeled antibody or a labeled antigen. Detectable labels suitable for use herein can include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Non-limiting examples of detection labels include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA), chemiluminescent compounds such as acridinium (e.g., acridinium-9-carboxamide), phenanthridinium, dioxetanes, luminol and the like, radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), catalysts such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40-80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0059] In certain embodiments, one or more of the biomarkers disclosed herein can be detected using a protein array. As used herein, protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The assays can be highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). In some embodiments, a protein array used herein can include one or more probes suitable for detection of protein biomarkers, for example, antibodies (e.g., monoclonal and/or polyclonal antibodies) or antibody fragments, specific ligands, hetero- or homodimerization protein partners, fusion proteins or fragments thereof. In some embodiments, methods for determining the expression of protein biomarkers using a protein biomarker array can include one or more of the immunoassays disclosed herein. In some embodiments, methods disclosed herein can be used to detect the level of protein of biomarkers contemplated herein in at least one sample from a subject infected with a virus compared to at least one control sample in order to diagnose the condition of the infected subject.

[0060] In some embodiments, the present disclosure provides for methods that include analysis using protein biomarker arrays suitable to detect presence, absence or levels of biomarkers disclosed herein. In certain embodiments, a suitable protein biomarker array can include one or more biomarkers that indicate at least one respiratory disease and/or respiratory condition with reasonably high confidence as disclosed herein. In other embodiments, the degree of confidence required can be selected according to standard practices or can exceed standard practices. Generally, the higher the number of biomarkers on an array for a given indication, the higher the degree of confidence that a given indication is present. However, certain biomarkers when combined in low numbers can be fully sufficient to indicate an underlying condition, while others may need to be combined in larger numbers to confer the same degree of confidence. High confidence biomarkers might be those that change more significantly than others (e.g. by a factor of 3, 4, 5, or more), are more abundantly or more selectively expressed, or can be expressed more consistently among different subjects and/or different conditions. Other factors that can determine a high confidence biomarker can include relative high affinity interactions between the capturing agent or the visualizing agent and the biomarker, relative ease of isolation of the biomarker from the sample, relative stability of the biomarker, and the like, when compared to other biomarkers. One of skill in the art can determine the necessary and sufficient number of biomarkers on an array using only routine optimization.

[0061] In certain embodiments, protein biomarker arrays can further include at least one or more reference standards. As used herein, a “reference standard” used in the arrays disclosed herein can refer to one or more probes suitable for detection of one or more biomarkers for which clinical outcome, positive or negative, is known. In some embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a normal, healthy subject. In other embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a normal, healthy subject. In yet other embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a subject having at least one respiratory disease and/or respiratory condition. In some embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a subject having COVID-19. In certain embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a subject having mild COVID-19, moderate COVID-19, critical COVID-19, or a combination thereof. In other embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a subject having non-severe COVID-19, severe COVID-19, or a combination thereof. In some embodiments, protein biomarker arrays herein can include one or more reference standards toward one or more biomarkers representative of a subject having a COVID-19 clinical outcome selected from IMV, death, or a combination thereof.

[0062] In some embodiments, protein biomarker arrays disclosed herein can include, but are not limited to, one or more probes suitable for detection of one or more protein biomarkers selected from albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3, or a combination thereof. In some embodiments, protein biomarker arrays disclosed herein can have one or more probes suitable for detection of one or more protein biomarkers including, but not limited to, albumin, CRP, galectin-3, or a combination thereof. In certain embodiments, protein biomarker arrays disclosed herein can include one or more probes suitable for detection of the protein biomarkers albumin, CRP, and galectin-3. In some embodiments, protein biomarker arrays disclosed herein can include one or more probes suitable for detection of the protein biomarker galectin-3.

[0063] In some embodiments, protein biomarker arrays disclosed herein can detect at least about 1.0 to about 300 ng/mL of a biomarker in a sample. In certain embodiments, protein biomarker arrays disclosed herein can detect at least about 1.0, or at least about 10, or at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 100, or at least about 150, or at least about 200, or at least about 250, or at least about 300 ng/mL or more of Galectin-3 in a sample. In some embodiments, protein biomarker arrays disclosed herein can detect at least about 1.0 to about 200 ng/mL, or at least about 1.0, or at least about 10, or at least about 50, or at least about 100, or at least about 150, or at least about 200 ng/mL of CRP in a sample. In some embodiments, protein biomarker arrays disclosed herein can detect at least about 1.0 to 20 g/dL or at least about 1.0, or at least about 5, or at least about 10, or at least about 15, or at least about 20 g/dL albumin in a sample. [0064] In some embodiments, protein biomarker arrays disclosed herein can be a quantitative enzyme-linked immunosorbent assays (ELISA) or other quantitative assay known in the art. In certain embodiments, protein biomarker arrays disclosed herein can be chemiluminescent microparticle immunoassays. In other embodiments, protein biomarker arrays disclosed herein can be modified using commercially available assays. In some embodiments, protein biomarker arrays disclosed herein can be modified from one or more commercially available ARCHITECT™ chemiluminescent microparticle immunoassays (e.g., Fujirebio Diagnostics, Inc; Abbott Laboratories). In some embodiments, protein biomarker arrays disclosed herein can be modified from the commercially available BGM galectin-3 ELISA (BG Medicine, Inc.). [0065] In certain embodiments, the present disclosure can include methods for identifying or classifying a subject as a candidate for further diagnostic testing and/or for therapeutic intervention, for example, immediate therapeutic intervention. In some embodiments, methods disclosed herein can be used for identifying or classifying a subject as having at least one respiratory disease and/or respiratory condition due to an infection (e.g., COVID-19 or mutant or variant thereof) as a candidate for further diagnostic testing and/or for therapeutic intervention. In other embodiments, methods include detecting and/or quantifying any one of the biomarkers toward at least one respiratory disease and/or respiratory condition due to an infection (e.g., COVID-19 or mutant or variant thereof) as disclosed herein. In some embodiments, methods disclosed herein can be used for identifying or classifying a subject having at least one respiratory disease and/or respiratory condition due to an infection (e.g., COVID-19 or mutant or variant thereof) as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying the concentration of albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof in a sample obtained from a subject, and identifying or classifying the subject as a candidate for further diagnostic testing and/or therapeutic intervention when the subject has a decreased and/or increased concentration of one or more biomarkers in the samples. In certain embodiments, a suitable control sample can be used for comparison (e.g., healthy subject or infected subject not having a severe COVID-19 infection).

[0066] In some embodiments, methods disclosed herein can be used for identifying or classifying a subject having a COVID-19 infection or mutant thereof as a candidate for further diagnostic testing and/or for therapeutic intervention for example, by detecting and quantifying the concentration of albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof in a sample obtained from the subject and identifying or classifying the subject as a candidate for further diagnostic testing and/or therapeutic intervention when the subject has a decreased and/or increased concentration of one or more biomarkers as disclosed herein in the sample compared to a control sample. In some embodiments, methods disclosed herein can be used for identifying or classifying a subject having COVID-19 infection or mutant thereof as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying the concentration of albumin, CRP, galectin-3 or a combination thereof in at least one sample obtained from a subject, and identifying or classifying the subject as a candidate for further diagnostic testing and/or therapeutic intervention when the subject has a decreased concentration of albumin and/or an increased concentration of one or more of CRP and galectin-3 in the at least one sample compared to a control sample as detailed herein. In certain embodiments, methods disclosed herein can be used for identifying or classifying a subject having COVID-19 infection or mutant thereof as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying concentration of galectin-3 in at least one sample obtained from a subject, and identifying or classifying the subject as a candidate further diagnostic testing and/or therapeutic intervention when the subject has an increased or elevated concentrations of galectin-3 in the at least one sample. In certain embodiments, a suitable control sample can be used for comparison (e.g., healthy subject or infected subject not having a severe COVID-19 infection).

[0067] In certain embodiments, compositions, combination compositions and methods disclosed herein can be used for identifying or classifying a subject having moderate and/or severe COVID-19 infection. In some embodiments, compositions, combination compositions and methods disclosed herein can be used for identifying or classifying a subject having moderate and/or severe COVID-19 infection as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying the concentration of one or more biomarker. In accordance with these embodiments, a biomarker can include one or more of albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof. In other embodiments, one or more biomarker analyzed in at least one sample obtained from a subject can be used for identifying or classifying the subject as a candidate for further diagnostic testing and/or therapeutic intervention when the subject has a decreased and/or increased concentration of one or more biomarkers in the sample. Alternatively, when the subject has a decreased and/or increased concentration of one or more biomarkers in the sample compared to a control sample as detailed herein. In some embodiments, methods disclosed herein can be used for identifying or classifying a subject having moderate and/or severe COVID-19 infection as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying the concentration of albumin, CRP, galectin-3 or a combination thereof. In accordance with these embodiments, a sample obtained from a subject contemplated herein can be used for identifying or classifying the subject as a candidate further diagnostic testing and/or therapeutic intervention when the subject has a decreased concentration of albumin and/or an increased concentration of one or more of CRP and galectin-3 in the sample. Alternatively, when the subject has a decreased and/or increased concentration of one or more biomarkers in the sample compared to a control sample as detailed herein. In some embodiments, methods disclosed herein can be used for identifying or classifying a subject having moderate and/or severe COVID-19 infection as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying the concentration of galectin-3 in at least one sample obtained from a subject. In accordance with these embodiments, a sample obtained from a subject contemplated herein can be used for identifying or classifying the subject as a candidate further diagnostic testing and/or therapeutic intervention when the subject has increased concentration of galectin-3 in the at least one sample. Alternatively, when the subject has a increased concentration Gal-3 in the sample as detailed herein.

[0068] In some embodiments, the biomarker analyzed in the at least one sample is galectin-3 and the concentration for determining the severity of the potential or existence of severe respiratory conditions is at least 25 ng/ml; at least 30 ng/ml; at least 35 ng/ml; at least 40 ng/ml or more. In certain embodiments, a subject is newly admitted with an infection or a subject that has been under mechanical respiratory control. In other embodiments, a subject having gal-3 concentration of 25 ng/ml or greater in at least one sample from the subject can indicate an adverse clinical outcome and potential for death. In other embodiments, the subject can be diagnosed with having or likelihood of developing acute respiratory syndrome (ARDS) when the concentration of Gal-3 in the sample is about 25 ng/ml or greater or about 30 ng/ml or greater or about 30.99 ng/ml or greater. [0069] In some embodiments, methods disclosed herein can be used for identifying or classifying a subject having severe, less-severe, or moderate COVID-19 infection as a candidate for further diagnostic testing and/or for therapeutic intervention can include detecting and quantifying the concentration of albumin, ferritin, D-Dimer, CRP, LDH, neutrophil count, galectin-3 or a combination thereof in at least one sample obtained from a subject. In some embodiments, compositions, combination compositions and methods disclosed herein can include detecting and quantifying the concentration of one or more biomarker and identifying or classifying the subject as a candidate for further diagnostic testing and/or therapeutic intervention when the subject has a decreased and/or increased concentration of one or more biomarkers or when compared to a control sample. In other embodiments, the concentration of albumin, CRP, galectin-3 or a combination thereof can be analyzed in a sample and identifying or classifying the subject as a candidate further diagnostic testing and/or therapeutic intervention when the subject has a decreased concentration of albumin and/or an increased concentration of one or more or CRP and galectin-3 in the sample. Alternatively, when the subject has a decreased concentration of albumin and/or an increased concentration of one or more or CRP and galectin- 3 in the sample. In certain embodiments, the subject samples can be compared to at least one negative or positive control sample.

[0070] In certain embodiments, compositions contemplated herein include detections agents for detecting levels or concentrations of at least two biomarkers including galectin-3 (Gal-3), C- reactive protein (CRP), and albumin in a biological sample. In certain embodiments, the combination composition includes commercially available detection agents such as chemicals or antibodies. In other embodiments, the composition comprises a composition as a component for a kit for analyzing at least one sample from a subject contemplated herein. In certain embodiments, the composition includes detection agents for at least Gal-3 such as a chemical detection agent or an antibody, antibody fragment or monoclonal antibody or other Gal-3 detection agent. In certain embodiments, the detection agent is capable of rapidly detecting Gal-3 in any sample contemplated herein for quick diagnosis.

[0071] In some embodiments, methods disclosed herein can be used for diagnosing or selecting a subject for therapy or monitoring efficacy of therapy of acute exacerbations of respiratory diseases (e.g., a chronic airway disease and a pulmonary disease). In accordance with these embodiments, a respiratory disease or condition can include but is not limited to, asthma, airway hyperresponsiveness, seasonal allergic allergy, bronchiectasis, chronic bronchitis, emphysema, chronic obstructive pulmonary disease, cystic fibrosis, and the like. In some embodiments, methods disclosed herein can be used for diagnosing, selecting a subject for therapy, or monitoring efficacy of therapy of acute respiratory distress syndrome (ARDS). [0072] In some embodiments, methods disclosed herein can be used for diagnosing, selecting a subject for therapy, or monitoring efficacy of therapy of a respiratory -related condition, e.g. pulmonary parenchymal inflammatory/fibrotic, respiratory distress conditions.

[0073] In some embodiments, methods disclosed herein can be used for diagnosing, selecting a subject for therapy, or monitoring efficacy of therapy of a respiratory disease or respiratory condition associated with a pathogenic infectious (e.g., viral or bacterial) of the respiratory tract. In other embodiments, respiratory diseases and/or respiratory conditions subject to the methods disclosed herein can include inflammatory conditions (e.g., caused by an environmental insult, such as exposure to an irritant), infectious conditions (e.g., caused by exposure to a pathogen), or a combination thereof. In some embodiments, respiratory diseases and/or respiratory conditions subject to the methods disclosed herein can be a respiratory tract infection caused by one or more parasites, one or more fungi, one or more bacteria, one or more viruses, or a combination thereof. In some embodiments, parasites causing a respiratory tract infection herein can include Toxoplasma gondii, Strongyloides stercoralis, and the like. In some embodiments, fungi causing a respiratory tract infection herein can include Histoplasma capsulatum, Cryptococcus neoformans, Pneumocvstis jiroveci, Coccidioides immitis, and the like. In some embodiments, bacteria causing a respiratory tract infection herein can include Streptococcus pneumoniae, which is commonly referred to as pneumococcus, Staphylococcus aureus, Burkholderis ssp., Streptococcus agalactiae, Haemophilus influenzae. Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens, Mycobacterium tuberculosis, Bordetella pertussis, and the like. In some embodiments, viruses causing a respiratory tract infection herein can include influenza virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, adenovirus, metapneumovirus, coxsackie virus, echo virus, coronavirus, herpes virus, cytomegalovirus, and the like. In some embodiments, viruses causing a respiratory tract infection herein can include coronavirus subtypes including 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome, or MERS), SARS-CoV (the beta coronavirus that causes severe acute respiratory syndrome, or SARS), SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID- 19), or a combination thereof. In certain embodiments, respiratory diseases and/or respiratory conditions subject to the methods disclosed herein can include the coronavirus infection caused by COVID-19 or mutant or derivative thereof.

[0074] In some embodiments, therapeutic intervention of use to treat a subject having a moderate to severe viral-related respiratory condition disclosed herein can include, but is not limited to, administering a composition including, but not limited to, ACE2 recombinant proteins, ACE2 antibodies, and/or ACE2 inhibitors. In some embodiments, other therapeutic interventions of use herein can include, but are not limited to, one or more antiviral agents including, but not limited to, remdesivir, favipiravir, merimepodib, lopinavir, ritonavir, and the like. In certain embodiments, other therapeutic intervention of use in methods of treating a respiratory condition disclosed herein can include, but is not limited to, administering one or more corticosteroids including, but not limited to, prednisone, methylprednisolone, hydrocortisone, dexamethasone, and the like to a subject in need thereof. In some embodiments, therapeutic intervention of use in methods of treating a respiratory condition disclosed herein can include, but is not limited to, administering to the subject, one or more of anti-inflammatory agents, convalescent plasma, amlodipine, ivermectin, losartan, famotidine, monoclonal antibodies, polyclonal antibodies, antibiotics, and albuterol. In some embodiments, other diagnostic methods of use in methods disclosed herein can include, but are not limited to, methods for diagnostic imaging, including but not limited to, CT scans, MRI scans, and X-rays. In some embodiments, further diagnostic testing and/or for therapeutic intervention for use herein can be placement on invasive mechanical ventilation.

[0075] In certain embodiments, kits are disclosed of use for assaying one or more samples from a subject for one or more biomarkers for presence, absence, and/or concentration levels of biomarkers contemplated herein. Kits according to the present disclosure can include one or more reagents useful for practicing one or more immunoassays according to the present disclosure. In some embodiments, a kit can include a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as admixture where the compatibility of the reagents will allow. In some embodiments, kits herein can also include other material(s), which can be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.

[0076] In some embodiments, kits disclosed herein can include one or more first or capture antibodies, each of which binds to at least one epitope on the target biomarker (e.g., galectin-3), and one or more second or detection antibodies, each of which binds to at least one epitope on the target biomarker (e.g., galectin-3), that is different from any epitope to which any of the capture antibodies bind, and further instructions for detecting or quantifying the biomarker. [0077] In certain embodiments, kits disclosed herein can include a solid phase and a capture agent affixed to the solid phase, wherein the capture agent is an antibody specific for the biomarker (e.g., galectin-3) being assessed in the sample. In some embodiments, a solid phase can be a material such as a magnetic or paramagnetic particle including a microparticle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a quartz crystal, a film, a filter paper, a disc, or a chip.

[0078] In certain embodiments, where kits disclosed herein are to be used for conducting sandwich immunoassays, the kits can additionally include at least one labeled antibody as a detection reagent. In some embodiments, a kit can include at least one direct label, which can be an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin. In other embodiments, kits according to the present disclosure can instead or in addition also include at least one indirect label. In some embodiments, if the label employed generally includes an indicator reagent to produce a detectable signal, a kit herein can include one or more suitable indicator reagents. In some embodiments, kits herein can contain one or more polyclonal or non-human monoclonal antibodies against the target biomarker (e.g., galectin-3), including for example mouse monoclonal antibodies, and these can be used as capture and/or detection antibodies.

[0079] The kits of this invention include suitable packaging. Suitable packaging includes, but is not limited to, vials, assay trays, tubes, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some embodiments, a kit has a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-Galectin- 3 antibody as those described herein.

[0080] In some embodiments, kits herein can optionally provide additional components such as buffers and interpretive information. Typically, a kit can include a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture including contents of the kits described above. In certain embodiments, a Biomerieux Diagnostics (Vidas Galectin-3), Abeam™, Thermofisher™, Ray Biotech™, RND Systems™ Rockland™ or other commercially available assay or molecule can be used and adapted for assessing concentration of biomarkers disclosed herein. In certain embodiments, threshold concentrations of biomarkers contemplated herein can be used in order to assess low, intermediate or high need for intervention and/or continued respiratory intervention in a subject. In some embodiments, kits can be used as disclosed herein for a more personalized diagnosis and treatment regimen based on concentrations of the biomarkers contemplated herein. In other embodiments, Gal-3 detection kits are contemplated where monoclonal antibody detection directed to binding one or more Gal-3 epitopes can be used to compare Gal-3 levels in control versus experimental samples.

EXAMPLES

[0001] The following examples are included to illustrate certain embodiments and are not considered limiting to the instant disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function in the practice of the claimed methods, compositions, and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes can be made in some embodiments and examples which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1. Demographic, clinical and laboratory characteristics

[0081] In this exemplary method, 156 patients with real-time reverse transcriptase- polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection and CT findings were enrolled in the study, of which 107 (68.6%) were male and 49 (31.4%) were female. According to the CT scan images, pulmonary affection was greater than 50% in 94 patients (critical patients) and less than 50% in 62 (moderate patients). Critical patients were significantly older (54.89 ± 11.79 vs. 48.21 ± 14.30, p<0.01) and the majority were male (71 [66.4%] vs. 36 [33.6%], p=0.02). The principal comorbidities among our cohort were obesity (44.2%), hypertension (30.1%) and diabetes (21.2%), all of which had been diagnosed prior to hospital admission. Alcohol consumption was higher in critical patients (p<0.04).

[0082] Critical patients had a significantly longer hospital stay (9 [6 - 18] vs. 6 [4 - 8], p<0.0001) and more often required IMV (44 [46.8%] vs. 7 [11.3%], p<0.0001), both of which reflected a superior need of hospital care. The overall mortality rate was 13.5% (n=21), and critical patients had the highest mortality rate (18 [19.1%] vs. 3 [4 .8%], p=0.02). A total of 54 (34.6%) patients developed a severe outcome, including IMV and/or in-hospital death, and the majority of these patients were critical (47 [50%] vs. 7 [11.3 %], pO.OOOl) (Table 1).

Table 1. Demographic and clinical characteristics

[0083] Laboratory data obtained upon hospital admission are summarized in Table 2. While glucose levels were slightly elevated in critical patients (135.5 mg/dL [111.38 - 166.38] vs.

116.0 mg/dL [106.0 - 152.33], p=0.12), the platelet count was similar in both groups (231.0 K/uL [194.17 - 318.5] vs. 226.0 K/uL [187.0 - 261.0], p=0.12) for which no significant difference was found in either laboratory parameter between those diagnosed as critical or moderate based on CT findings. However, albumin and triglycerides were statistically different between critical and moderate patients. Albumin levels were significantly lower (3.54 ± 0.49 g/dL vs. 4.07 ± 0.42, p<0.0001) in critical patients compared to moderate patients while triglycerides were significantly higher (166.0 mg/dL [119.5 - 207.0] vs. 131.0 mg/dL [106.0 - 181.0], p=0.04).

Table 2. Laboratory data characteristics

[0084] In these exemplary methods, it was demonstrated that the inflammatory markers ferritin, D-dimer and C-reactive protein (CRP) were all significantly elevated (p<0.0001) upon hospital admission in critical patients (671.95 ng/mL [333.55- 1164.58] vs. 279.25 ng/mL [166.7 - 549.89]; 895.5 ng/mL [535.3 - 1718.63] vs. 514.5 ng/mL [382.5 - 779.63]; and 19.04 mg/dL [9.35 - 26.85] vs. 6.78 mg/dL [2.86 - 14.12], respectively), presumably reflecting a greater inflammatory state due to more severe lung damage. The total lymphocyte count was significantly lower in critical patients (0.68 X10³/pL [0.46 - 0.89] vs. 0.98 X10³/pL [0.72 - 1.40], p<0.0001). The neutrophil count was significantly higher in patients characterized as critical based on CT upon admission (9.03 X10³/pL [5.82 - 12.88] vs. 4.49 X10³/pL [3.18 vs. 6.75], p<0.0001). Both lymphocyte and neutrophil findings are reflected in the NLR ratio, which was higher in critical patients (14.01 [7.67 - 24.74] vs. 5.12 [2.85 - 8.16], p<0.0001) (Table 2).

Example 2. Galectin-3 serum levels were higher in COVID-19 patients with a severe outcome

[0085] In another exemplary method, circulating levels of galectin-3 were measured in sera from COVID-19 patients using an enzyme-linked immunosorbent assay (ELISA). It was observed that COVID-19 patients upon hospital admission had significantly elevated circulating levels of galectin-3 when compared to age-matched pre-pandemic healthy subjects (28.77 ng/mL [IQR 17.52 - 42.04] vs. 9.65 ng/mL [IQR 8.27 - 14.71], p<0.0001) (Table 3; Fig. 1A).

Table 3. Odds ratios from the multivariable logistic regression model for severe outcome

[0086] Figs. 1A-1C represent galectin-3 serum levels in COVID-19 patients, a, Galectin-3 circulating levels upon hospital admission of COVID-19 patients (n=156) and age-matched healthy pre-pandemic controls (n=10). b, Galectin-3 is associated with COVID-19 severity, critical patients (n=94) presented significantly higher levels than moderate patients (n=62). c, Severe outcomes in COVID-19 patients were associated with elevated levels of galectin-3. Data in a and c are shown as median with IQR, data in b as mean ± SD. ***p < 0.001, ****p < 0.0001; two-tailed Mann-Whitney U test or two-tailed t-test. Samples were assessed in duplicate in ELISA assays.

[0087] Critical patients had significantly higher levels of galectin-3 when compared to moderate patients (35.91 ± 19.37 ng/mL vs. 25.0 ± 14.85 ng/mL, p<0.001) (Fig. IB). Patients who developed a severe outcome, including IMV and/or in-hospital death, had significantly higher galectin-3 levels than those with a non-severe outcome, (41.17 ng/mL [IQR 29.71 - 52.25] vs. 23.76 ng/mL [IQR 15.78 - 33.97], p<0.0001) (Fig. 1C). When outcomes were analyzed according to the initial disease state, galectin-3 levels were significantly higher in those with a severe outcome (39.21 ng/mL [IQR 28.01 - 50.85] vs. 26.60 ng/mL [IQR 20.22 - 42.59, p<0.01 in critical patients and 50.03 ± 11.88 vs. 21.81 ± 11.91 ng/mL, p<0.0001, in moderate patients) regardless of their initial diagnose by CT (Fig. 2A). Galectin-3 not only predicted an adverse outcome in critical patients but was able to even more accurately identify patients within the moderate group likely to progress in disease severity (AUC 0.66 vs. 0.95) (Figs. 2B and 2C). CRP levels were analyzed between critical and moderate patients according to outcome. Similar to findings with galectin-3, CRP was found to be higher in those with severe outcome regardless of the initial disease state (21.90 ± 9.56 mg/dL vs. 15.25 ± 9.50, p<0.01 in critical patients and 17.80 mg/dL [IQR 15.36 - 21.30] vs. 6.49 mg/dL [IQR 2.70 - 12.66], p<0.01, in moderate patients) (Fig. 2D). When comparing the predictive ability of CRP for a severe outcome in critical and moderate patients, CRP performed very similar to galectin-3 when the critical group was analyzed (AUC=0.70 vs. AUC=0.66, respectively) (Fig. 2E). However, galectin-3 had additional power and significance in predicting severe outcomes in the moderate group compared to CRP (AUC=0.95, p<0.0001, and AUC=0.87, p<0.01, respectively) (Fig. 2F).

[0088] Figs. 2A-2F represent galectin-3 and CRP as predictors of severe outcome in moderate and critical COVID-19 patients, a, significantly higher galectin-3 levels associate with severe outcome in both critical and moderate groups b, c, Receiver-operating characteristic (ROC) curves show that galectin-3 has significant predictive power for severe outcome in critical and moderate patients, d, Similarly, C-reactive protein (CRP) levels are elevated in critical or moderate patients with severe outcome, e, f, Predictive power is also found with CRP when ROC curves for severe outcome are plotted in both groups. Data in a and d are shown as median with IQR. **p < 0.01, ****p < 0.0001; two-tailed Mann-Whitney U test.

Example 3. Galectin-3 correlated with other inflammatory biomarkers

[0089] In yet another exemplary method, given that elevated galectin-3 levels were observed in patients who progressed to a severe outcome, and considering galectin-3 ’s contribution to the inflammatory response, correlations with inflammatory parameters in COVID-19 infections using the spearman correlation coefficient, in accordance with the nonnormal distribution of the data were carried out. Spearman correlations show significant associations between galectin-3 and commonly measured inflammatory markers in SARS-CoV-2 infected patients, a, C-reactive protein (CRP), b, ferritin, c, D-dimer, d, albumin, e, neutrophil count and f, lactate dehydrogenase (LDH). It was observed that galectin-3 protein levels correlated positively with CRP (r=0.42, p<0.0001), neutrophil count (r=0.39, p<0.0001), D- dimer (r=0.19, p=0.02), ferritin (r=0.30, p<0.001), LDH (r=0.36, p<0.0001); but a negative correlation was observed measuring albumin levels (r= -0.35, p<0.0001) (see for example, Figs.

3A-3F)

Example 4. Galectin-3, CRP and albumin levels were independent predictors of severe outcome in COVID-19 patients

[0090] To assess the discriminative power of galectin-3 as a predictor of severe outcome, ROC curves were plotted. Galectin-3 levels were able to discriminate between critical and moderate patients with an AUC of 0.69 (95% CI 0.61 - 0.78, p<0.0001), with a cut-point of 30.59 ng/mL (57.45% sensitivity, 75.81% specificity and 2.37 likelihood ratio (LR) (Fig. 5). Galectin-3 also discriminated well between those with severe and non-severe outcome, with an AUC of 0.75 (95% CI 0.67 - 0.84, p<0.0001), with a cut-point of 30.99 ng/mL (74.07% sensitivity, 73.53% specificity, and 2.79 LR) (Fig. 4A). Receiver-operating characteristic curves (ROCs) of the independent predictors for the prediction of binary outcomes (severe/non-severe) using a, galectin-3, b, CRP, c, albumin and d, the predicted probabilities of galectin-3, CRP and albumin when used together. Values for AUC, sensitivity (SE), specificity (SP), likelihood ratio (LR) and cut-off values are shown, with 95% Cis in parentheses.

[0091] Fig. 5 Illustrates that galectin-3 associates with CT findings upon hospital admission as it shows power discriminating between critical patients (>50% of lung affection) and moderate patients (<50% of lung affection). Values for AUC, sensitivity (SE), specificity (SP), likelihood ratio (LR) and cut-off values are shown, with 95% Cis in parentheses.

[0092] In another exemplary method, a forward- stepwise logistic regression analysis was performed to identify independent demographic and laboratory parameters that strongly correlated with a severe outcome, and thus with disease progression (e.g., IMV and/or death). A smoothing spline of galectin-3 showed a non-linear relationship with severe outcome; therefore, a Youden’s J statistic was used to determine the ideal binary cut-point of galectin-3 for classifying severe outcomes (Fig. 6A). The initial model included age, gender, comorbidities, and inflammatory parameters. The final model selected via the stepwise procedure included galectin-3 (odds ratio [OR] 3.89 [95% CI 1.50 - 10.10], p<0.01), CRP (OR 1.05 [95% CI 1.00 - 1.11], p=0.04), and low albumin levels upon admission (OR 0.24 [95% CI 0.09 - 0.65], p<0.01) when regressed on severe outcome (IMV and/or death; Table 3). Of note, CRP and albumin were entered as continuous variables according to their smoothing splines which showed linear relationships with severe outcome (Fig. 6B and 6C). The equation fitted by this logistic regression model was the following, where p = P(Y = 1): log^^= 3.0 + [1.359 * binary galectin-3] + [0.052 * CRP] - [1.421 * albumin], where galectin-3 was coded as binary (less than 30.99 ng/mL with 0 and above 30.99 ng/mL with 1), and CRP and albumin as continuous variables. The obtained values were transformed into predicted probabilities with the formula exp

[0093] Figs. 6A-6C represent that galectin-3 demonstrates that a non-linear relationship with the patients’ outcomes (severe=l and non-severe=0), for which it is used as a binary variable in the logistic regression analysis. b,c, Smoothing splines of CRP (b) and albumin (c), showing a linear relationship with outcome, using both as continuous variables in further analysis. Data in a,b,c are computed with 4 knots.

[0094] In another example, potential for using binary cut-points of CRP and albumin were explored to classify severe outcomes in subject studied. CRP had an AUC of 0.76 (95% CI 0.68 - 0.85, p<0.0001) at a cut-point of 14.04 mg/dL (78.85% sensitivity, 67.02% specificity and 2.39 LR), while albumin had an AUC of 0.73 (95% CI 0.65 - 0.82, p<0.0001) with a 3.74 g/dL cutpoint (78.43% sensitivity, 62.11% specificity and 2.07 LR) (see for example, Figs. 4B and 4C). The combination of galectin-3, CRP and albumin were analyzed to further assess severe outcomes in CO VID-19 patients than any of the markers on their own. To determine this information, the predicted probabilities for this combination of values were computed and plotted in a ROC curve (see for example, Fig. 4D). The AUC demonstrated an enhanced ability to classify severe outcomes compared to assessing each biomarker value independently 0.84 (95% CI 0.77 - 0.91, p<0.0001). This is a significant improvement over using the biomarkers alone but the biomarkers alone each provided the ability to classify a subject having COVID-19. [0095] In certain exemplary methods, the hyperinflammatory state in COVID-19 patients and its relationship with galectin-3 was observed in these studies, as higher levels of this lectin upon admission were found in critical patients with lung affection greater than 50%. In certain examples, it was demonstrated that galectin-3 levels above 30.59 ng/mL discriminated between patients with critical or moderate disease with a high specificity. Acute respiratory distress syndrome (ARDS) is characterized by a diffuse alveolar damage in the lung, caused by severe inflammatory processes. The high levels of galectin-3 found in these COVID-19 patients might be an indication of the possible role of galectin-3 in the pathophysiology of ARDS and might be a reflex of the excessive inflammatory response associated with ARDS in these patients as well as any condition that results in ARDS. It was found that galectin-3 possessed power as an independent predictor of severe outcome when adjusting for age, gender, comorbidities and other inflammatory parameters. ARDS in COVID-19 leads to more severe outcomes than ARDS due to other causes. With a general mortality of 26%-61.5% in those admitted to the intensive care unit, and significantly higher in those requiring IMV (65.7% to 94%). This data indicates that in certain examples, values greater than 30.99 ng/mL have a high sensitivity and specificity to predict an adverse clinical course with the possibility of needing invasive mechanical ventilation (IMV) and/or death. Galectin-3 was not only able to classify a severe outcome in critical patients, but, more importantly, was able to identify severe outcomes in moderate patients (AUC= 0.95).

[0096] While IMV is intended to minimize the progression of lung injury, it can also induce or aggravate lung damage and in the long-run may contribute to lung fibrosis. Chronic pulmonary fibrosis has been observed in recovered COVID-19 patients. Galectin-3 could provide an important biomarker for severe COVID-19 with potential for involvement in the direct pathophysiological process of the underlying disease.

[0097] In other examples, the relationship between galectin-3 and C-reactive protein (CRP) was assessed. CRP was identified as an independent predictor of severe outcome and had a positive correlation with galectin-3. This novel association between galectin-3 and CRP has not been reported in viral infection, much less in CO VID-19 but it suggests the utility of this molecule in detecting the inflammatory state of patients upon hospital arrival. As both CRP and galectin-3 were identified as independent predictors. The non-linear relationship of galectin-3, observed in a smoothing spline, demonstrated that higher levels of this lectin were a common characteristic of patients at high-risk of progressing to a severe COVID-19 outcome. Furthermore, although a slope could be interpreted by healthcare providers for continuous predictors, these methods simplify testing by using a binary cut-point of galectin-3 level to classify patients as low or high risk for severe outcomes.

[0098] In other studies, it was demonstrated that hypoalbuminemia was/is a common characteristic among critically ill patients and demonstrated a correlation between albumin and the systemic inflammation in COVID-19 patients, as a negative correlation was found with galectin-3, which contributes to the release of pro-inflammatory cytokines. Albumin is an important biomarker that reflects the inflammatory state, as its production is decreased due to higher levels of IL-6.

[0099] In these studies, as galectin-3 was found to reflect the hyperinflammatory state of patients, its predictive ability together with CRP and albumin was tested. Results revealed that when used jointly, severe outcomes can be more accurately classified upon hospital admission (AUC=0.84), thus providing clinicians more resources to efficiently identify patients with higher odds of adverse progression. Given that galectin-3 is highly correlated with the severity of the disease, as demonstrated when patients were first classified based of CT findings, these experiments support using galectin-3 in the initial screening of COVID-19 patients, alone or together with CRP and albumin. The assessment of this panel upon admission will help identify patients at high-risk of disease progression as galectin-3 was found to be associated with the patients’ clinical state. Therapies that decrease inflammatory response would be expected to reduce galectin-3 levels during the clinical course of the patient and influence outcomes. METHODS

Study design and groups

[0100] 156 patients admitted between April and October 2020 for the hospitalization of patients with COVID-19 were prospectively included according to the following criteria: laboratory confirmed COVID-19 by RT-PCR, CT and clinical characteristics consistent with COVID-19. Those who presented with respiratory symptoms but had a negative RT-PCR test result were excluded from the analyses. Patients were divided into two groups based on the pulmonary affection on a CT. Moderate patients: those with <50% of lung damage. Critical patients: those with >50% of lung damage. This study was approved by our hospital’s Research Ethics Committee (No. GAS-3385-20-21-1) and complied with the provisions of the Declaration of Helsinki. Informed written consent was obtained from all patients prior to blood sample collection.

Primary outcome definition

[0101] Patients who required IMV and/or died during hospitalization were categorized as having a severe outcome. Patients who recovered and were discharged without needing IMV were categorized as having a non-severe outcome. Data collection

[0102] Clinical and laboratory data were extracted from the electronic medical records including: Demographics (age, gender, comorbidities), clinical (days of hospital stay), radiological (chest CT findings), laboratory and patient outcome data (need for IMV and/or death). Laboratory data included arterial blood gas, complete blood count, triglycerides, albumin, lactate dehydrogenase, liver enzymes, coagulation tests (D-dimer, INR) and inflammation- related parameters (CRP and ferritin).

Sample collection and Galectin-3 levels measurement [0103] Blood samples were collected upon hospital admission from all 156 patients. Samples were centrifuged at 3,000 rpm for 10 min, and serum was aliquoted and stored at -70°C until further analysis. Galectin-3 was measured in the serum samples using an ELISA according to methods known in the art. All samples were evaluated in duplicate. The inter-assay coefficient of variation was 8.52% and the intra-assay 5.34%.

Statistical analysis

[0104] Data are expressed as frequencies for categorical variables and as mean with standard deviation (SD) or median with interquartile range (IQR) for continuous variables according to their distributions. Student’s t-tests or Mann-Whitney U tests were used for univariate statistical comparisons, while correlation analyses were performed with Spearman's correlation coefficient for pairs of continuous variables. To determine the prognostic ability of galectin-3 and inflammatory markers for the primary outcome, receiver operating characteristic (ROC) curves were plotted, and cut-point values were chosen as those with the highest Youden’s J statistic. Independent predictors of the primary outcome were determined using multivariable logistic regression analysis (forward-stepwise selection). This analysis included variables with a p value <0.20 in bivariate analyses; goodness of the fit was assessed with the Hosmer-Lemeshow test. The combined power of the identified independent predictors was evaluated with a ROC curve using the model selected by the stepwise logistic regression procedure. Statistical analyses were performed with SPSS (version 24.0, SPSS Inc., Chicago, IL, USA) and GraphPad Prism (version 8.00, GraphPad Software, La Jolla, CA, USA). A selected alpha level of 0.05 indicated statistical significance.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the COMPOSITIONS and METHODS have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variation can be applied to the COMPOSITIONS and METHODS and in the steps or in the sequence of steps of the METHODS described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing severity of a respiratory condition in a subject, the method comprising:

(a) analyzing at least one sample from a subject having an infection for concentration of at least one biomarker comprising galectin-3, C-reactive protein (CRP), albumin, or a combination thereof; and

(b) diagnosing the subject as having a severe respiratory condition based on the concentration of the at least one biomarker comprising galectin-3 (Gal-3), C-reactive protein (CRP), albumin, or the combination thereof in the at least one sample.

2. The method according to claim 1, wherein the infection comprises a viral infection.

3. The method according to either claim 1 or claim 2, wherein the infection comprises a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a derivative or a mutant infection thereof.

4. The method according to claim 1, wherein the biomarker comprises Gal-3 and the subject is diagnosed as having a severe respiratory condition if the concentration of Gal-3 in the at least one sample is increased by about 1.5 times or greater.

5. The method according to claim 1, wherein the biomarker comprises Gal-3 and the subject is diagnosed as having a severe respiratory condition if the concentration of Gal-3 in the at least one sample is at least about 25.00 ng/ml or more.

6. The method according to claim 1, wherein the biomarker comprises Gal-3 and the subject is diagnosed as having a severe respiratory condition if the concentration of Gal-3 in the at least one sample is at least about 30.00 ng/ml or more.

7. The method according to any one of claims 1-5, wherein the biomarker comprises Gal-3 and the subject is diagnosed as having a severe respiratory condition if the concentration of Gal- 3 in the at least one sample is increased by at least 1-fold compared to the at least one control sample.

8. The method according to claim 1, wherein the biomarker comprises CRP and the subject is diagnosed as having a severe respiratory condition if the concentration of CRP in the at least one sample is increased.

9. The method according to any one of claims 1-8, wherein the biomarker comprises CRP and the subject is diagnosed as having a severe respiratory condition if the concentration of CRP in the at least one sample is increased by at least 1-fold compared to the at least one control sample.

10. The method according to claim 1, wherein the biomarker comprises albumin and the subject is diagnosed as having a severe respiratory condition if the concentration of albumin in the at least one sample is decreased.

11. The method according to any one of claims 1-10, wherein the biomarker comprises albumin and the subject is diagnosed as having a severe respiratory condition if the concentration of albumin in the at least one sample is decreased by at least 1-fold compared to the at least one control sample.

12. The method according to any one of claims 1-11, wherein the at least one sample from a subject having an infection comprises whole blood, blood serum, blood plasma, saliva, sputum, mucus, a nasal swab, urine, or a combination thereof.

13. The method according to any one of claims 1-12, further comprising treating the subject for the respiratory condition.

14. The method according to any one of claims 1-13, wherein the subject does not have symptoms or signs of respiratory complications.

15. The method according to claim 5 or 6, wherein the subject comprises a subject having Gal-3 concentration of at least 30.99 ng/mL and diagnosing the subject as having high probability of having at least one of having a severe respiratory condition and an adverse clinical course.

16. The method according to claim 15, wherein the subject is diagnosed with the probability of requiring respiratory intervention.

17. A method for diagnosing and treating a severe respiratory condition in a subject, the method comprising:

(a) analyzing at least one sample from a subject having an infection for concentration of at least one biomarker comprising galectin-3, C-reactive protein (CRP), albumin, or a combination thereof;

(b) diagnosing the subject as having a severe respiratory condition based on the concentration of the at least one biomarker comprising galectin-3 (Gal-3), C-reactive protein (CRP), albumin, or the combination thereof in the sample; and

(c) treating the subject having the severe respiratory condition.

18. The method according to claim 17, wherein treating the subject comprises at least one of administering at least one treatment for the severe respiratory condition and inhibiting Gal-3 expression in the subject.

19. The method according to claim 17 or 18, wherein the infection comprises a viral infection.

20. The method according to either claim 19, wherein the infection comprises a SARS-CoV- 2 or a derivative or a mutant infection thereof.

21. The method according to claim 17, wherein the biomarker comprises Gal-3 and the subject is diagnosed and treated for a severe respiratory condition if the concentration of Gal-3 in the at least one sample is increased by at least about 1.5 times or at least about 1-fold.

22. The method according to any one of claims 17-21, wherein the biomarker comprises CRP and the subject is diagnosed and treated for a severe respiratory condition if the concentration of CRP in the at least one sample is increased by at least about 1-fold.

23. The method according to any one of claims 17-22, wherein the biomarker comprises albumin and the subject is diagnosed and treated for a severe respiratory condition if the concentration of albumin in the at least one sample is decreased by at least about 1-fold.

24. The method according to any one of claims 17-23, wherein treating the subject having the severe respiratory condition comprises providing a composition comprising one or more of ACE2 recombinant proteins, ACE2 antibodies, ACE2 inhibitors, remdesivir, favipiravir, merimepodib, lopinavir, ritonavir, prednisone, methylprednisolone, hydrocortisone, dexamethasone, anti-inflammatory drugs, convalescent plasma, amlodipine, ivermectin, losartan, famotidine, monoclonal antibodies, polyclonal antibodies, antibiotics, albuterol, invasive mechanical ventilation, or a combination thereof.

25. The method according to any one of claims 17-24, wherein treating the subject having the severe respiratory condition comprises providing mechanical intervention for assisting breathing in the subj ect.

26. The method according to claim 17, wherein the subject is suspected of developing Acute respiratory distress syndrome (ARDS) and treating the subject reduces onset or progression of ARDS.

27. A kit for diagnosing severity of a respiratory condition in a subject having an infection, the kit comprising:

(a) at least one biomarker array or biomarker detection system, wherein the biomarker array comprises one or more probes or indicators for binding to the one or more biomarkers comprising albumin, CRP, galectin-3, or a combination thereof; (b) at least one detection agent for detecting the binding agent for measuring concentration of the one or more biomarker in a sample from a subject; and

(c) at least one container.

28. The kit according to claim 27, further comprising at least one reference standard wherein the reference standard comprises one or more biomarkers selected from albumin, CRP, galectin- 3, or a combination thereof representative of a normal, healthy subject.

29. The kit according to claim 27 or claim 28, wherein the at least one biomarker array or detection system detects at least one of 1.0 to 300 ng/mL galectin-3 in a sample, 1.0 to 200 ng/mL CRP in a sample, 1.0 to 20 g/dL albumin in a sample, or a combination thereof.

30. The kit according to any one of claims 27-29, further comprising a chemiluminescent agent for measuring concentration of the one or more biomarkers.

31. The kit according to claim 27, wherein the kit further comprises at least one control from a subject having a severe respiratory infection.