CROSS-REFERENCE TO RELATED APPLICATION

[0001] This PCT Application claims benefit to U.S. Provisional Application No. 63/396,085, filed August 8, 2022, entitled “Defensin as a Protein Biomarker for Distinguishing Bacterial and Viral Infections”, the entirety of which is incorporated herein by reference.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (S1647178111_sequence listing.xml; Size: 7,095 bytes; and Date of Creation: August 8, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

[0003] The capability of healthcare providers to make treatment deciding diagnostic determinations for a subject (e g., patient) within a time frame of an office visit can lead to improved patient outcomes and lessening of unnecessary suffering resulting from incorrect therapeutic intervention. In addition to the individual patient benefit, there is also an overall benefit to society from diminished pressure on healthcare systems and/or pressures of developing antibiotic resistance due to improper antibiotic treatment. The Center for Disease Control and Prevention (CDC) estimates that antibiotic resistance in the United States alone affects more than two million people with a direct health cost up to $20 billion. Treating viral infections with antibiotics is, frequently, medically unnecessary, and may contribute to the development of antibiotic resistance.

SUMMARY

[0004] The present invention is directed to overcoming the above-mentioned challenges and others related to infection assessment and disease stratification, such as for distinguishing between viral and bacterial infections using a defensin protein. In various aspects, the defensin protein in various processed forms can be used as a biomarker to reliably categorize an infection as being either viral or bacterial, which can lead to improved patient outcomes and alleviate antibiotic resistance pressures. In some aspects, the defensin protein can be used as a biomarker to discriminate infection types based on the detection of up-regulated expression levels or concentrations of at least one processed form of the protein.

[0005] Various aspects of the present disclosure are directed to an apparatus comprising a support configured to receive a biological sample and a plurality of reagents, and the plurality of reagents. The plurality of reagents including: a plurality of first agents configured to bind to a pre-active peptide portion of a defensin protein in the biological sample; and a plurality of second agents that exhibit a detectable signal and are configured to bind to at least one of: the pre-active peptide portion; or respective ones of the plurality of first agents. Wherein binding between different respective ones of the plurality of first agents, the pre-active peptide portion, and the plurality of second agents on the support is configured to generate the detectable signal which is indicative of an expression level of the pre-active peptide portion in the biological sample.

[0006] In some aspects, the expression level of the pre-active peptide portion differentiates between a viral infection and a bacterial infection. In some aspects, the viral infection is associated with an increase in concentration of the pre-active peptide portion as compared to a threshold level. In some aspects, the bacterial infection is associated with a minimal or without an increase in concentration of the pre-active peptide portion as compared to a threshold level.

[0007] In some aspects, the plurality of the first agents include a first subset of first agents configured to bind to the pre-active peptide portion and a second subset of first agents configured to bind to an active peptide portion of the defensin protein in the biological sample. And, the plurality of second agents include: a first subset of the second agents configured to bind to at least one of the pre-active peptide portion or the first subset of the first agents; and a second subset of the second agents configured to bind to at least one of active peptide portion or the second subset of first agents. And wherein binding between different respective ones of the plurality of first agents, the pre-active peptide portion, the active peptide portion, and the plurality of second agents is configured to generate the detectable signal which is indicative of an expression level of the pre-active peptide portion and/or an expression level of the active peptide portion in the biological sample.

[0008] In some aspects, the detectable signal comprises a first detectable signal and a second detectable signal, and the first subset of the second agents exhibit the first detectable signal and the second subset of the second agents exhibit the second detectable signal. Wherein binding between respective ones of the plurality of first agents, the pre-active peptide portion, and respective ones the plurality of second agents is configured to generate the first detectable signal which is indicative of the expression level of the pre-active peptide portion in the biological sample, and binding between respective ones of the plurality of first agents, the active peptide portion, and respective other ones of the plurality of second agents is configured to generate the second detectable signal which is indicative of the expression level of the active peptide portion in the biological sample.

[0009] In some aspects, the pre-active peptide portion comprises an amino acid region including at least a portion of amino acids 39-64 of the defensin protein. In some aspects, the active peptide portion comprises an amino acid region including at least a portion of ammo acids 65-94 of the defensin protein.

[0010] In some aspects, the defensin protein is selected from the group consisting of: neutrophil defensin 1; neutrophil defensin 2; neutrophil defensin 3; and a combination thereof. In some aspects, the defensin protein is human neutrophil defensin 1 and 2 (DEF1/2).

[0011] In some aspects, the plurality of first agents further comprise respective first agents configured to bind to a plurality of additional target analytes selected from the group consisting of: calprotectin (CALP); protein SI00-A12 (SI00A12); neutrophil gelantinase-associated lipocalin (NGAL); vascular cell adhesion protein 1 (VCAM-1); golgi membrane protein 1 (GOLM 1); macrophage colony stimulating factor 1 (CSF-1); procalcitonin (PCT); tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL); interferon gamma-induced protein 10 (IP 10); C-reactive protein (CRP); Beta- 2-microglobulin (B2M); myxovirus resistance protein A (MxA); interferon (IFN)- stimulated gene 15 (ISG15); urokinase plasminogen activator (UP A) fermitin family homolog 3 (URP2); and a combination thereof. [0012] Various aspects are directed to a kit comprising a support, a plurality of first agents, and a plurality of second agents that exhibit a detectable signal. Wherein respective ones of the plurality of first agents are configured to bind to a pre-active peptide portion of a defensin protein in a biological sample, and other respective ones of the plurality of first agents are configured to bind to an active peptide portion (e.g., at least a portion of the active peptide or active form) of the defensin protein in the biological sample. And, with respective ones of the plurality of second agents configured to bind to at least one of: the pre-active peptide portion or the active peptide portion; or the respective ones or other respective ones of the plurality of first agents. Wherein the support is configured to receive the biological sample, the plurality of first agents, and the plurality of second agents, and in response to binding between different respective ones of the plurality of first agents, the pre-active peptide portion, the active peptide portion, and the plurality of second agents on the support, cause generation of the detectable signal which is indicative of an expression level of the pre-active peptide portion and/or an expression level of the active peptide portion in the biological sample. [0013] In some aspects, the expression level of the pre-active peptide portion and the expression level of the active peptide portion differentiates between a viral infection and a bacterial infection based on threshold levels associated with anon-infected biological sample.

[0014] In some aspects, the threshold levels comprise a first threshold level and a second threshold level. The viral infection being associated with an increase in concentration of the active peptide portion as compared to the second threshold level and an increase in concentration of the pre-active peptide portion as compared to the first threshold level, and the bacterial infection being associated with an increase in concentration of the active peptide portion as compared to the second threshold level and without an increase or with a minimal increase in concentration of the pre-active peptide portion as compared to the first threshold level.

[0015] In some aspects, the pre-active peptide portion comprises an amino acid region including at least a portion of amino acids 39-64 of the defensin protein, and the active peptide portion comprises an amino acid region including at least a portion of amino acids 65-94 of the defensin protein. [0016] In some aspects, the plurality of second agents are disposed on the support or disposed in a fluid configured to be placed on the support.

[0017] Various aspects are directed to a method comprising exposing a biological sample to a plurality of first agents, wherein: respective ones of the plurality of first agents are configured to bind to a pre-active peptide portion of a defensin protein present in the biological sample; and other respective ones of the plurality of first agents are configured to bind to an active peptide portion (e.g., at least a portion of the active peptide or active form) of the defensin protein present in the biological sample. The method further comprises causing binding of respective ones of a plurality of second agents that exhibit a detectable signal to at least one of: the pre-active peptide portion or the active peptide portion; or the respective ones or other respective ones of the plurality of first agents. Wherein binding between the plurality of first agents, the pre-active peptide portion, the active peptide portion, and the plurality of second agents generates the detectable signal which is indicative of an expression level of the pre-active peptide portion and/or an expression level of the active peptide portion in the biological sample. The method further comprises differentiating between a viral infection and a bacterial infection in the biological sample based on the expression level of the pre-active peptide portion and/or the expression level of the active peptide portion.

[0018] In some aspects, differentiating between the viral infection and bacterial infection comprises comparing the expression levels of the pre-active peptide portion and the active peptide portion, which are indicative of relative quantities of the pre- active peptide portion and the active peptide portion in the biological sample, to threshold levels associated with a non-infected biological sample.

[0019] In some aspects, the threshold levels comprise a first threshold level and a second threshold level, wherein: the viral infection is associated with an increase in concentration of the active peptide portion as compared to the second threshold level and an increase in concentration of the pre-active peptide portion as compared to the first threshold level; and the bacterial infection is associated with an increase in concentration of the active peptide portion as to the second threshold level and without an increase or with a minimal increase in concentration of the pre-active peptide portion as compared to the first threshold level. [0020] In some aspects, the pre-active peptide portion comprises an amino acid sequence having at least 70 percent identity to SEQ ID NO: 1 or 3 or a portion thereof, and the active peptide portion comprises an amino acid sequence having at least 70 percent identity to SEQ ID NO: 2 or 5 or a portion thereof.

[0021] In some aspects, the method further includes using the expression levels of the pre-active and active peptide portions to monitor treatment of a patient associated with the biological sample for cancer and/or to monitor a cancer model system for development of a cancer therapeutic.

[0022] Various aspects are directed to a method comprising exposing a biological sample to a plurality of first agents, wherein respective ones of the plurality of first agents are configured to bind to a pre-active peptide portion of a defensin protein present in the biological sample; and other respective ones of the plurality of first agents are configured to bind to an active peptide portion of the defensin protein present in the biological sample. The method further including causing binding of respective ones of a plurality of second agents that exhibit a detectable signal to at least one of: the pre- active peptide portion or the active peptide portion; or the respective ones or other respective ones of the plurality of first agents. Wherein binding between the plurality of first agents, the pre-active peptide portion, the active peptide portion, and the plurality of second agents generates the detectable signal which is indicative of an expression level of the pre-active peptide portion and/or an expression level of the active peptide portion in the biological sample.

[0023] In some aspects, the method further includes using the expression levels of the pre-active and active peptide portions to monitor treatment of a patient associated with the biological sample for an autoimmune disease and/or to monitor an autoimmune disease model system for development of an autoimmune disease therapeutic.

[0024] In some aspects, the method further includes using the expression levels of the pre-active and active peptide portions to stratify a disease status of a cancer patient associated with the biological sample and/or to determine an optimal treatment for the cancer patient.

[0025] In some aspects, the method further includes using the expression levels of the pre-active and active peptide portions to stratify a disease status of an autoimmune disease patient associated with the biological sample and/or to determine an optimal treatment for the autoimmune disease patient.

[0026] In some aspects, the method further includes using the expression levels of the pre-active and active peptide portions to monitor treatment of a patient with a bacterial infection or a viral infection, the patient being associated with the biological sample, and/or to monitor an infectious disease model system for development of an anti- infective therapeutic.

BRIEF DESCRIPTION OF THE DRAWINGS

Various example embodiments can be more completely understood in consideration of the following detailed description in connection with the accompanying drawings, in which:

[0027] FIG. 1 illustrates an example method for using a pre-active peptide portion of a defensin protein for infection assessment, in accordance with the present disclosure. [0028] FIG. 2 illustrates an example of a pre-active peptide portion and an active peptide portion of a defensin protein, in accordance with the present disclosure.

[0029] FIGs. 3A-3B illustrate example methods for using a defensin protein for differentiating between viral and bacterial infections, in accordance with the present disclosure.

[0030] FIGs. 4A-4B illustrate example apparatuses and/or kits for using a pre-active peptide portion of a defensin protein for infection assessment, in accordance with the present disclosure.

[0031] FIGs. 5A-5D illustrate example supports for determining a presence of a pre- active peptide portion and/or an active peptide portion of a defensin protein, in accordance with the present disclosure.

[0032] FIGs. 6A-6B illustrate example assays for determining a presence of a pre-active peptide portion and/or an active peptide portion of a defensin protein, in accordance with the present disclosure.

[0033] FIGs. 7A-7C illustrate example levels of expression of a pre-active peptide portion of a defensin protein in a viral infected sample and a bacterial infected sample, in accordance with the present disclosure. [0034] FIGs. 8A-8C illustrate further example levels of expression of a pre-active peptide portion of a defensin protein in a viral infected sample and a bacterial infected sample, in accordance with the present disclosure.

[0035] FIGs. 9A-9C illustrate example levels of expression of an active peptide portion of a defensin protein in a viral infected sample and a bacterial infected sample, in accordance with the present disclosure.

[0036] FIG. 10 is a graph showing enzyme-linked immunosorbent assay (ELISA) measurements for alpha-defensin 1 (DEFA), in accordance with the present disclosure. [0037] FIG. 11 is a graph showing a receiver operator characteristic (ROC) curve of DEFA, in accordance with the present disclosure.

[0038] FIG. 12 is a graph showing the ROC of DEFA in a two-biomarker pannel, in accordance with the present disclosure.

DETAILED DESCRIPTION

[0039] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is show n by way of illustration specific examples in which the disclosure can be practiced. It is to be understood that other examples can be utilized, and various changes can be made without departing from the scope of the disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the disclosure is defined by the appended claims. It is to be understood that features of the various examples described herein can be combined, in part or whole, with each other, unless specifically noted otherwise.

[0040] To properly treat subjects that exhibit symptoms of infection, a differential diagnosis can be made to determine whether the infection is of viral origin or bacterial origin. Bacterial infections are typically effectively treated with antibiotic therapy, while viral infections are frequently self-limiting and can only need palliative care or antiviral therapy. There is increasing awareness among healthcare providers that bacterial antibiotic-resistance is an emerging threat to human health. It is believed that the inappropriate use of antibiotics, for example with viral infections, can contribute to the development of antibiotic resistance by the bacteria or the subject. How ever, if a subject’s symptoms get worse before considering the possibility of a bacterial infection, this may lead to patient harm and/or death.

[0041] Embodiments of the present disclosure are directed to use of a defensin protein as a biomarker to assess for an infection being viral or bacterial. For example, up- regulated concentrations of variable processed forms of the defensin protein can be used to discriminate between a viral infection and a bacterial infection. In various embodiments, the defensin protein can be assessed in a test that can be conducted during a clinical visit, such that a differential diagnosis can be made quickly. Distinguishing between viral and bacterial infections can increase the proper use of antibiotics to mitigate risk of antibiotic resistance for viral infections and improving patient outcome for bacterial infections by more quickly allowing for antibiotic treatment.

[0042] Defensin proteins are a family of antimicrobial and cytotoxic peptides involved in host defense. When cells synthesize defensin proteins, cells initially produce a pre- pro-active form of the peptide and through further processing, in which sections of the peptide are cleaved off, produce various other forms such as the mature form and the active form of the defensin protein. The active form of the defensin protein can be used as a biomarker for both viral infections and bacterial infections as a subject can exhibit increased expression of the active form for either infection type. In various embodiments, the mature form of the defensin protein can be used to discriminate between bacterial and viral infections as a subject may exhibit an increased expression of the pre-active peptide portion of the mature form for a viral infection and may not exhibit an increase, or can exhibit a minimal increase, in expression of the pre-active peptide portion of the mature form for a bacterial infection.

[0043] In some embodiments, a peptide from a mature form of a defensin protein, herein sometimes referred to as a “pre-active peptide portion”, and/or a peptide from an active form of the defensin protein, herein sometimes referred to as a “active peptide portion”, are used for rapid characterization and/or discrimination between types of infections, such as between viral and bacterial infections. The pre-active peptide portion and active peptide portion are from separate regions of the defensin protein. In some experimental embodiments, as described herein, it has been shown that in isolated peripheral blood mononuclear cells (PBMCs) for subjects with viral infection, both the pre-active peptide portion and the active peptide portion are elevated as compared to cells from a noninfected or a healthy subject. It has further been shown that for subjects with a bacterial infection, only the active peptide portion is elevated as compared to cells from the noninfected or healthy subject. Embodiments of the present disclosure are directed to apparatuses, devices, kits, and methods of using the peptide portions for targeting the different expression levels of the pre-active peptide portion and/or active peptide portion of the defensin protein for rapid characterization of bacterial and viral infections, which provides for both early diagnosis and managing treatment.

[0044] In some embodiments, the defensin protein can include defensin 1, 2, and/or 3 (e.g., neutrophil defensin 1, 2, or 3) which can be used alone or in combination with other target analytes (e.g., biomarkers) for assessing viral and bacterial infections. In some embodiments, defensin 1, 2, or 3 can be used in combination with at least one of the following target analytes: calprotectin (CALP), protein S100-A12 (S100A12), neutrophil gelantinase-associated lipocalin (NGAL), vascular cell adhesion protein 1 (VCAM-1), golgi membrane protein 1 (GOLM 1), macrophage colony stimulating factor 1 (CSF-1), procalcitonin (PCT), tumor necrosis factor (TNF)-related apoptosis- mducing ligand (TRAIL), interferon gamma-induced protein 10 (1P10), C-reactive protein (CRP), Beta-2-microglobulin (B2M), myxovirus resistance protein A (MxA), interferon (IFN)-stimulated gene 15 (ISG15), urokinase plasminogen activator (UP A), and fermitin family homolog 3 (URP2).

[0045] Turning to the figures, FIG. 1 illustrates an example method for using a preactive peptide portion of a defensin protein for infection assessment, in accordance with the present disclosure. The method 100 can be implemented using various apparatuses, devices, and/or kits, such as those illustrated herein by at least FIGs. 4A-6B. An example apparatus can include hardware components for performing a photometric scan combined with processing circuitry to digitize detector signals for software analysis, although embodiments are not so limited. In other examples, the apparatus and/or kit can include reagents and a (solid) support for forming an assay. The assay can be used to test for the presence of the pre-active peptide portion and/or active portion of the defensin protein in a biological sample without the use of optical scanning and/or processing circuitry, such as a color-indicating, visibly-readable lateral flow test and/or ELISA test In various embodiments, the assay may be quantitative, as further described below.

[0046] As illustrated by FIG 1, the method 100 can include obtaining a biological sample 102 from a subject, such as a human. A subject, as used herein, refers to or includes a living organism. Embodiments are not limited to a blood sample being obtained from a human, and the blood sample can be previously obtained and/or can be obtained from other organisms (e.g., other vertebrates, such as horses, goats, sheep, dogs, pigs, cats, cattle, rodents, reptiles, fish, and birds). The biological sample 102 can include urine, sputum, pus, saliva, cerebrospinal, synovial and interstitial fluid samples or other types of samples.

[0047] At 104, the method 100 can include exposing the biological sample 102 to a plurality of first agents. Exposing the biological sample 102 to the plurality of first agents can cause a physical interaction between the biological sample 102 and at least one of the plurality of first agents, as further described herein.

[0048] At least a portion of the plurality of first agents can be specific to a pre-active peptide portion (e.g., of the mature form) of a defensin protein. For example, the plurality of first agents can be configured to bind to the pre-active peptide portion of the defensin protein present in the biological sample 102. In some embodiments, as further illustrated herein, a first subset of the plurality of first agents are specific to the pre- active peptide portion and a second subset of the plurality of first agents are specific to an active peptide portion (e.g., of an active form) of the defensin protein present in the biological sample 102. The plurality of first agents can be used to form an assay for the pre-active peptide portion (and/or the active peptide portion) and used as an indicator of infection.

[0049] In some embodiments, the plurality of first agents can include antibodies that bind specifically (e.g., with high affinity) to the pre-active peptide portion (or, optionally, the active peptide portion) of the defensin protein. In some embodiments, the plurality of first agents (e.g., antibodies) are immobilized on or irreversibly conjugated to a support (e.g., a solid substrate), such as a glass slide or plastic microtiter plates. As used herein, a support includes or refers to a material or medium which provides a surface for a reaction to occur. The support can be solid or porous, in some embodiments, and can be formed of a variety of material, such as glass, polymers, metals, paper, and other material. Example supports include glass slides, plastic microtiter plates, microwell arrays, among others. In some embodiments, the biological sample 102 is immobilized to the support and exposed to the first agents. Biological samples that exhibit the pre-active peptide portion can bind to at least a portion of the plurality of first agents.

[0050] In various embodiments, the defensin protein can include neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, or a combination thereof. In some embodiments, the defensin protein is human neutrophil defensin 1 and 2 (DEF1/2). The pre-active peptide portion can comprise an amino acid region including at least a portion of amino acids 39-64 of the defensin protein. In some embodiments, the active peptide portion can comprise an amino acid region including at least a portion of amino acids 65-94 of the defensin protein. In some embodiments, the pre-active peptide portion can comprise an amino acid region including amino acids 39-64 of the defensin protein and/or the active peptide portion can comprise an amino acid region including amino acids 65-94 of the defensin protein. In some embodiments, the pre-active peptide portion and active peptide portion can be from the same defensin protein. In other embodiments, the pre-active peptide portion and active peptide portion can be from different defensin proteins. In various embodiments, at least some (or all) of the plurality of first agents can bind to amino acids sequences having at least 70 percent (%), at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100 % identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5, and/or a portion thereof. For example, the pre-active peptide portion can comprise an amino acid sequence having at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NOs: 1 and/or 3, and/or a portion thereof. The active peptide portion can comprise an amino acid sequence having at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NOs: 2 and/or 5, and/or a portion thereof.

[0051] In some embodiments, the defensin protein can be used to assess for infection along with additional target analytes (e g., biomarkers). In some such embodiments, the plurality of first agents include a volume of a first agent specific to each target analyte of a set of target analytes. For example, the plurality of first agents can include aliquots of each of a plurality of different first agents, where each volume of a different first agent is specific to a different target analyte of the set. Further embodiments can include assays or tests targeting more than two different target analytes, such as targeting two to ten or more biomarkers. Example target analytes include the pre-active peptide portion of the defensin protein, the active peptide portion of the defensin protein, CALP, S100A12, NGAL, VCAM-1, GOLM 1, CSF-1, PCT, TRAIL, IP10, CRP, B2M, MxA, ISG15, UP A, and URP2.

[0052] At 106, the method 100 can include causing binding between respective ones of a plurality of second agents to at least one of the pre-active peptide portion or respective ones of the plurality of first agents. The second agents can exhibit a detectable signal, such as a colorant, a fluorescent, an enz matic signal, a magnetic signal, a radioactive signal, or an otherwise include a detectable molecular tag such as biotin or a molecule possessing a unique mass or isotope. In some embodiments, the second agents can include or be a label that exhibits the detectable signal or can be activated to exhibit the detectable signal via a reaction with a substrate. As used herein, a label includes or refers to a molecule, a complex, or other compound, which exhibits, or may be modified via reaction to exhibit, a detectable signal. Example labels include antibodies, particles, or other molecules or compounds which are labeled, such as with a colorant (e.g., dye, fluorescent) or other detectable signal. A detectable signal includes or refers to a signal which may be detected on its own, after a reaction, and/or responsive to accumulation. In some embodiments, the label can be pre-bound to the first agents prior to exposing the biological sample 102 to the first agents. In other embodiments, the second agents can include an antibody or other molecule and the label, which bind to either the respective first agent or another epitope or region associated with the pre-active peptide portion (or other target analyte) before or after the biological sample 102 is exposed to the first agents.

[0053] Accordingly and in different embodiments, the plurality of first agents can be capture agents and/or detection agents, and the plurality of second agents can form capture agents and/or detection agents with the plurality of first agents (e g., first agents being bound to a label) and/or can be detection agents (e.g., a labeled-second antibody). An agent includes or refer to a molecule, complex, or other compound. Example agents include a label, an antibody, an anti-antibody, a partial antibody, an enzyme, and/or other affinity molecules exhibiting specific binding to the target molecule or compound. In some embodiments, an agent can include a synthetic molecule, complex or other compound, such as a non-natural molecule that acts like an antibody. A capture agent includes or refers to an agent that binds to the target analyte (e.g., molecule or compound of interest) and effectively captures the target analyte. A detection agent includes or refers to an agent that binds to the target analyte or first agent bound to the target analyte and which includes a label exhibiting a detectable signal. In some embodiments, the detectable signal is detectable after accumulating on a region of a support. For example, in such embodiments, in response to the accumulation, the detectable signal is above a detection threshold, and/or in response to activation of the signal via reaction with a substrate, the detectable signal can be detected. A substrate includes or refers to a molecule or complex that reacts with another molecule (e.g., enzyme, a catalyst, an inhibitor) to generate a detectable signal as a product, such as an optical signal. Example substrates include a fluorophore or pairs of fluorophores, , 3,3',5,5'-tetramethylbenzidine (TMB), 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), 3,3 '-Diaminobenzidine (DAB), luminol, homovanillic acid, 2,4- dinitrophenyl phosphate (DNP), among others.

[0054] As with the first agents, in some embodiments, a first subset of the plurality of second agents can bind to the pre-active peptide portion (or first agents configured to bind to the pre-active peptide portion) and a second subset of the plurality of second agents can bind to an active peptide portion of the defensin protein (or first agents configured to bind to the active peptide portion). Similarly, in some embodiments, the defensin protein can be used to assess for infection along with additional target analytes. In some such embodiments, the plurality of second agents include a volume of a second agent specific to each target analyte of a set of target analytes or to each first agent specific to each target analyte of the set of target analytes.

[0055] At 108, the method can include assessing for the presence of the pre-active peptide portion of the defensin protein. The assessment can include detecting the detectable signal which is generated responsive to binding between different respective ones of the plurality of first agents, the pre-active peptide portion, and the plurality of second agents. As noted above, in some embodiments, the detectable signal can be activated using a substrate and/or can accumulate such that the detectable signal is above the detection threshold and can be detected.

[0056] The detectable signal can be indicative of an expression level of the pre-active peptide portion in the biological sample 102. In various embodiments, the expression level of the pre-active peptide portion can be used to assess for an infection, such as differentiating between a viral infection and a bacterial infection. For example, a viral infection can be associated with an increase in expression level (e.g., concentration) of the pre-active peptide portion as compared to a threshold level and/or the bacterial infection can be associated with a minimal or without an increase in expression level (e.g., concentration) of the pre-active peptide portion as compared to the threshold level. [0057] In some embodiments, and as noted above, additional target analytes can be used in order to increase the accuracy, sensitivity, and/or speed of differentiating between viral and bacterial infections. In some embodiments, both the pre-active peptide portion and the active peptide portion can be detected in the biological sample 102, which can be used to increase accuracy, sensitivity, and/or speed in verifying a bacterial infection. For example, with a bacterial infection, a detectable signal for the pre-active peptide portion may not be generated and/or may not be detectable as the bacterial infection results in no increase or minimal increase in the pre-active peptide portion expression, resulting in the detectable signal being below a detection threshold (e g., not detected). In some embodiments, the active peptide portion of the defensin protein can be detected to verify the bacterial infection. In some embodiments, in addition and/or alternatively, the additional target analytes can be associated with additional proteins, such as those listed above.

[0058] In some embodiments, bacterial and viral infections can be associated with different concentration or expression levels for respective ones of the different target analytes. For example, the bacterial infection can be associated with an increase in concentration of the active peptide portion as compared to a first threshold level and without an increase or with a minimal increase in concentration of the pre-active peptide portion as compared to a second threshold level different from the first threshold level. The viral infection can be associated with an increase in concentration of the active peptide portion as compared to the first threshold level and an increase in concentration of the pre-active peptide portion as compared to the second threshold level.

[0059] The presence or expression level of the pre-active peptide portion of the defensin protein can be used to diagnosis the subject and/or select a treatment plan. For example, with a bacterial infection, an antibiotic treatment can be prescribed. With a viral infection, the subject can be advised to rest, and in some instances, prescribed an antiviral treatment. By differentiating between the viral and bacterial infections, antibiotic treatments can be quickly prescribed for bacterial infections, which can improve patient outcome. Further, antibiotic treatments can be avoided when unnecessary, such as for a subject with a viral infection and/or without either viral or bacterial infections, which can mitigate risk of antibiotic resistance and decrease healthcare costs.

[0060] The physical interaction between the plurality of first agents, the plurality of second agents, and the biological sample 102 can be performed in variety of ways and using a variety of different types of apparatuses, devices, kits, and/or assays. In various embodiments, the interaction can include physically mixing the first agents alone or prebound to the second agents (e.g., labels) and the biological sample 102, or a pre-treated form of the biological sample 102 which initiates an assay and/or other type of test. For example, physical mixing can be part of an immunochromatographic strip assay or other lateral flow test polymerase chain reaction (PCR) assay, testing for presence of the pre- active peptide portion (and, optionally the active peptide portion and/or other target analytes) using an antibody specific to the pre-active peptide portion, LC-MS/MS, including testing such as a target-specific lateral flow strip, target-specific antigen latex agglutination assays, multiplex ELISA, antibody coated latex agglutination assays, antibody coated up-converting phosphors for lateral flow assays, antibody coated bead assays, and affinity reagent coated magnetic particles.

[0061] In some embodiments, the assay used to test for the presence of the pre-active peptide portion and/or the active peptide portion can be quantitative. For example, the assay can be used to provide a true concentration of the pre-active peptide portion and the active peptide portion in the biological sample based on output detectable signals (which may be digitized) and the concentration(s) can be input into a statistical algorithm that provides a score to differentiate viral infections from bacterial infections. As a non-limiting example, the score may be on a scale from 0 to 100, with a score of “0” indicating a viral infection and a score of “100” indicating a bacterial infecting. As may be appreciated by one of ordinary skill, such statistical algorithms are commercially available and/or can be customized.

[0062] As described above, in some embodiments, the plurality of first agents can each include an antibody (or another type of affinity molecule) bound to the second agents which comprise a label. Causing the physical interaction can include binding at least one of the antibodies to the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes) and assessing for the presence of pre-active peptide portion in the biological sample 102 includes determining the physical interaction occurred by identifying the presence of the label exhibiting the detectable signal after processing. In other embodiments, causing the physical interaction includes binding respective ones of the plurality of first agents to the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes), and binding respective ones of the plurality of second agents to the first agents or an epitope or other region of the pre-active peptide portion (and, optionally the active peptide portion and/or other target analy tes). In such embodiments, assessing for the presence of the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes) in the biological sample 102 includes identifying the presence of the second agents (e.g., via the detectable signal).

[0063] In related specific embodiments, the plurality of first agents can include first antibodies and the plurality of second agents can include second antibodies bound to a label that exhibits the detectable signal (e.g., detection antibodies or labeled antiantibodies). In such embodiments, the method further includes exposing the biological sample 102 to the second antibodies and, therein, binding at least one of the second antibodies to the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes). In other embodiments, the method further includes exposing the biological sample 102 to the second antibodies and, therein, binding at least one of the second antibodies to at least one of the first antibodies. Causing the physical reaction can include applying the first antibodies to the biological sample 102 and applying the second antibodies that exhibit the detectable signal to the biological sample 102. The presence of the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes) within biological sample 102 causes binding of the first antibody to the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes) and the second antibody being specific to an epitope of the first antibody or another epitope of the pre-active peptide portion (and, optionally the active peptide portion and/or other target analytes).

[0064] As noted above, specific embodiments are directed to use of multiple target analytes. For example, causing the physical interaction can include exposing the biological sample 102 to the plurality of first agents, where subsets of plurality of first agents are specific to different ones of the set of target analytes. As a specific example, the plurality of first agents can include a plurality of antibodies. Causing the physical interaction between the biological sample 102 and the plurality of first agents can include exposing the biological sample 102 to the plurality of antibodies, the plurality of antibodies including a subset or a volume of a first antibody specific to a first target analyte of the set and a subset or a volume of a second antibody specific to a second target of the set. In some embodiments, the first target analyte can include the pre-active peptide portion and the second target analyte can include the active peptide portion. In some embodiments, in addition to the pre-active peptide portion and the active peptide portion, the set of target analytes can include PCT, TRAIL, IP 10, CRP, B2M, MxA, ISG15, UP A, and/or URP2, among other targets. For example, the set of (additional) target analytes can include CALP, S100A12, NGAL, VCAM-1, GOLM 1, CSF-1, PCT, TRAIL, IP10, CRP, B2M, MxA, ISG15, UP A, and URP2.

[0065] Although the above describes use of antibodies as the first agents (and/or second agents), embodiments are not so limited and can include a variety of different agents having an affinity to the target analytes. For example, the first agents and the second agents can include first synthetic molecules and second synthetic molecules, which respectively act like antibodies, among other variations. As used herein, designations of “first” and “second” are used to refer to one element and another of the same element, of the same type or of a different type, without reference to temporal order. For example, in different embodiments, the biological sample may be exposed to the first and second agents at the same time, the first agents followed by the second agents, or the second agents followed by the first agents.

[0066] FIG. 2 illustrates an example of a pre-active peptide portion and an active peptide portion of a defensin protein, in accordance with the present disclosure. The preactive peptide portion of the defensin protein may include the mature protein (or mature form) and/or a portion thereof, and/or may include amino acids 39-64 of the full defensin sequence. The active form of defensin protein may perform its intended function(s), and/or may include amino acids 65-94 of the full defensin sequence, with the active peptide portion including at least a portion thereof. As shown by FIG. 2, the mature form 107- A of the defensin protein includes the pre-active peptide portion 109 and the active peptide portion 111. The active form 107-B of the defensin protein which includes the active peptide portion 111 is formed by proteolysis of the mature form 107- A. A viral infected sample 103 can exhibit increased concentration of the mature form 107-A of the defensin protein and the active form 107-B of the defensin protein as compared to a non-mfected subject. A bacterial infected sample 105 can exhibit increased concentration of the active form 107-B of the defensin protein, without or with minimal increase of the mature form 107-A of the defensin protein as compared to anon-infected subject.

[0067] The (relative) expression levels of the pre-active and active-peptide portions can be used for a variety of purposes, including, but not limited to, the infection assessment. Some examples are not limited to infection assessment, and may alternatively be used for other types of assessments, such as cancer assessment and immune disease assessment. For example, in some embodiments, the various methods described herein, such as those illustrated by FIGs. 1 and 3A-3B, may further or alternatively include using the expression levels of the pro-active peptide portion and/or active peptide portion to monitor treatment of a patient associated with the biological sample for cancer and/or to monitor a cancer model system for development of a cancer therapeutic. In some embodiments, the various methods described herein may further or alternatively include using the expression levels of the pro-active peptide portion and/or active peptide portion to monitor treatment of a patient associated with the biological sample for an autoimmune disease and/or to monitor an autoimmune disease model system for development of an autoimmune disease therapeutic. In some embodiments, the various methods described herein may further or alternatively include using the expression levels of the pro-active peptide portion and/or active peptide portion to stratify a disease status of a cancer patient associated with the biological sample and/or to determine an optimal treatment for the cancer patient. In some embodiments, the various methods described herein may further or alternatively include using the expression levels of the pro-active peptide portion and/or active peptide portion to stratify a disease status of an autoimmune disease patient associated with the biological sample and/or to determine an optimal treatment for the autoimmune disease patient. In some embodiments, the various methods described herein may further or alternatively include using the expression levels of the pro-active peptide portion and/or active peptide portion to monitor the treatment of a patient with a bacterial infection or a viral infection, the patient being associated with the biological sample, and/or to monitor an infectious disease model system for development of an anti-infective therapeutic.

[0068] Example embodiments are not limited to methods and the (relative) expression levels may be used as described above, as obtained using a device, apparatus, and/or a kit as further described herein. For example, the various methods, devices, apparatuses, and kits described herein may be used to provide the expression levels of the pro-active peptide portion and/or active peptide portion which are indicative of and/or can be used for: (i) monitoring treatment of a patient associated with the biological sample for cancer and/or monitoring a cancer model system for development of a cancer therapeutic, (ii) monitoring treatment of a patient associated with the biological sample for an autoimmune disease and/or monitoring an autoimmune disease model system for development of an autoimmune disease therapeutic, (iii) stratify ing a disease status of a cancer patient associated with the biological sample and/or determining an optimal treatment for the cancer patient, (iv) stratifying a disease status of an autoimmune disease patient associated with the biological sample and/or determining an optimal treatment for the autoimmune disease patient, and/or (v) monitoring the treatment of a patient with the bacterial infection or the viral infection, the patient being associated with the biological sample, and/or monitoring an infectious disease model system for development of an anti-infective therapeutic.

[0069] FIGs. 3A-3B illustrate example methods for using a defensin protein as an indicator for differentiating between viral and bacterial infections, in accordance with the present disclosure. The methods 200, 201 of FIGs. 3A-3B can include example implementations of, and/or include at least some substantially the same features and attributes, of method 100 of FIG. 1 and/or can be implemented using any of the apparatuses, devices, and kits as illustrated further herein.

[0070] In various embodiments, both the pre-active peptide portion and the active peptide portion of a defensin protein can be assessed and used to differentiate between a viral infection and a bacterial infection. For example, and as shown by FIG. 3 A, the method 200 includes exposing a biological sample 102 to a plurality of first agents, at 104. In such embodiments, respective ones of the plurality of first agents are configured to bind to a pre-active peptide portion (e.g., of a mature form) of a defensin protein present in the biological sample and other respective ones of the plurality of first agents are configured to bind to an active peptide portion (e.g., of an active form) of the defensin protein present in the biological sample 102.

[0071] At 210, the method includes causing binding of respective ones of a plurality of second agents that exhibit a detectable signal to at least one of (i) the pre-active peptide portion or the active peptide portion or (ii) the respective ones or other respective ones of the plurality of first agents. As previously described, binding between the plurality of first agents, the pre-active peptide portion, the active peptide portion, and the plurality of second agents can generate the detectable signal which is indicative of an expression level of the pre-active peptide portion and/or an expression level of the active peptide portion in the biological sample 102. The binding may form complexes, such first complexes each containing a respective first agent, a pre-active peptide portion, and a respective second agent, as well as second complexes each containing a respective first agent, an active peptide portion, and a respective second agent.

[0072] As previously described, the pre-active peptide portion and/or the active peptide portion can be present in the biological sample at a concentration below a threshold. In such embodiments, the detectable signal(s) which accumulates responsive to the complexes forming can be below a detection threshold (e.g., the detectable signal for the pre-active peptide undetectable). This may occur, for example, with the pre-active peptide portion of the defensin protein within a bacterially-infected biological sample. In some embodiments, the active peptide portion or other target analyte can be used to confirm a bacterial infection.

[0073] At 218, the method 200 includes differentiating between a viral infection and a bacterial infection in the biological sample 102 based on the expression level of the pre- active peptide portion and the expression level of the active peptide portion. In some embodiments, differentiating between the viral infection and bacterial infection comprises comparing the expression levels of the pre-active peptide portion and the active peptide portion, which are indicative of relative quantities of the pre-active peptide portion and active peptide portion in the biological sample, to threshold levels. [0074] In some embodiments, the threshold level(s) can be associated with a noninfected (and/or healthy) biological sample or may associated with infected subjects. For example, the threshold level(s) can include an expected or anticipated expression level for infected subjects. In some embodiments, the threshold lev el (s) may be associated with expression of the mature form and/or active form of the defensm protein in a plurality of non-infected and/or heathy subjects. For example, the threshold level(s) can include an average expression level across a plurality of non-infected and/or healthy biological samples. In some embodiments, non-infected subjects and/or biological samples can include subjects, or samples obtained therefrom, which are non-virally and non-bacterially infected, such as subjects screened for infections. In some embodiments, heathy subjects and/or biological samples can include subjects, or samples obtained therefrom, which are non-virally and non-bacterially infected and are screened for additional conditions which may cause a rise in defensin biomarker expression.

[0075] In some embodiments, the threshold levels comprise a first threshold level and a second threshold level. For example, the viral infection can be associated with an increase in concentration of the active peptide portion as compared to the second threshold level and an increase in concentration of the pre-active peptide portion as compared to the first threshold level, and the bacterial infection can be associated with an increase in concentration of the active peptide portion as compared to the second threshold level and without or with a minimal increase in concentration of the pre-active peptide portion as compared to the first threshold level.

[0076] In some embodiments, multiple detection signals can be used. For example, a first detection signal can be associated with a presence of and/or indicative of an expression level of a pre-active peptide portion and a second detection signal can be associated with a presence of and/or indicative of an expression level of an active peptide portion. The first detection signal and second detection signal can include the same signal or different signals. For example, the first and second detection signals can include different colorants. As another example, the first and second detection signals can include the same colorant which are exhibited at different regions (e.g., different visible windows in a lateral flow assay, different wells). In some embodiments, different detection signals can be used for each of plurality of target analytes, such as those previously described.

[0077] FIG. 3B illustrates an example method 201, which can include an implementation of method 200 of FIG. 3 A but includes additional detail from that of method 200 of FIG. 3 A. At 104, the method 201 can include exposing a biological sample 102 to a plurality of first agents. At 212, the method 201 can include exposing the biological sample 102 to a plurality of second agents, which can occur concurrently with, before, or after the exposure to the plurality of first agents. At 214, the method 201 can include detecting the detectable signals which are associated with the presence of the pre-active peptide portion and/or the active peptide portion. As previously described, responsive to respective binding between the first agents, the second agents, and the pre- active peptide portion or the active peptide portion, the second agents including the label(s) exhibit (or are caused to exhibit) the detectable signal(s) accumulate such that the detectable signal can be detected. At 216, the method can include assessing expression levels of the pre-active peptide portion and/or the active peptide portion as compared to the threshold levels using the detectable signal(s). At 218, the method 201 can include differentiating between a viral infection and a bacterial infection in the biological sample 102, as previously described. In some embodiments, as shown at 220, the method 201 can include selecting a treatment based on the infection type, such as prescribing an antibiotic for a bacterial infection and not prescribing antibiotics for a viral infection.

[0078] Methods in accordance with the present disclosure can include a number of variations. An example method includes outputting a first detectable signal indicative of an expression level of a pre-active peptide portion of a defensin protein present in a biological sample, and outputting a second detectable signal indicative of an expression level of an active peptide portion of the defensin protein present in the biological sample. The method can further include differentiating between a viral infection and a bacterial infection in the biological sample based on the expression level of the preactive peptide portion and the expression level of the active peptide portion. As previously described, a viral infection is associated with an increase in expression level or concentration of the active peptide portion and an increase in expression level or concentration of the pre-active peptide portion as compared to a threshold level, and/or the bacterial infection is associated with an increase in expression level or concentration of the active peptide portion without an increase in expression level or concentration of the pre-active peptide portion as compared to a threshold level. In some embodiments, the pre-active peptide portion can comprise an ammo acid region including at least a portion of amino acids 39-64 of the defensin protein and/or the active peptide portion can comprise an amino acid region including at least a portion of amino acids 65-94 of the defensin protein. In some embodiments, the defensin protein is selected from neutrophil defensin 1, neutrophil defensin 2, and/or neutrophil defensin 3, such as being human DEF 1/2. In some embodiments, outputting the first detectable signal and the second detectable signal further comprises using an assay that targets the pre-active peptide portion, the active peptide portion, and a plurality of additional target analytes selected from the group consisting of: PCT, TRAIL, IP 10, CRP, B2M, MxA, ISG15, URP2, UP A, and a combinations thereof. In some embodiments, the plurality of additional targets are selected from the group consisting of: CALP, S100A12, NGAL, VCAM-1, GOLM 1, CSF-1, PCT, TRAIL, IP10, CRP, B2M, MxA, ISG15, UP A, URP2, and a combination thereof.

[0079] In some embodiments, outputting the first detectable signal and the second detectable signal further comprises exposing portions of the biological sample to a plurality of first agents disposed on a substrate or in a fluid, wherein respective ones of the plurality of first agents are configured to bind to the pre-active peptide portion and to the active peptide portion, and binding respective ones of a plurality of second agents that exhibit the first detectable signal and the second detectable signal to at least one of: the pre-active peptide portion or the active peptide portion, or the respective ones of the plurality of first agents. For example, binding between different respective ones of the plurality of first agents, the pre-active peptide portion, the active peptide portion, and the plurality of second agents generates the first detectable signal which is indicative of the expression level of the pre-active peptide portion in the biological sample and the second detectable signal which is indicative of the expression level of the active peptide portion in the biological sample. In some embodiments, differentiating between a viral infection and bacterial infection comprises comparing the expression levels of the pre- active peptide portion and the active peptide portion, which are indicative of relative quantities of the pre-active peptide portion and the active peptide portion in the biological sample, to threshold levels associated with a non-infected biological sample. [0080] FIGs. 4A-4B illustrate example apparatuses and/or kits for using a pre-active peptide portion of a defensin protein as an indicator for an infection, in accordance with the present disclosure. The apparatus 321 illustrated by FIG. 4A, in some embodiments, can form or include a kit 331. The apparatus 321 and/or kit 331 includes a support 325 and a plurality of reagents 322. In some embodiments, at least a portion of the plurality of reagents 322 can be formed on the support 325, thereby forming a device. In some embodiments, at least a portion of the plurality of reagents 322 can be separate from the support 325, and can form an apparatus 321 or a kit 331.

[0081] The plurality of reagents 322 include the plurality of first agents 326 and the plurality of second agents 328. As previously described, the plurality of first agents 326 are configured to bind to a pre-active peptide portion of a defensin protein in the biological sample. As further illustrated by FIG. 4B, in some embodiments, respective ones of the plurality of first agents 326 (e.g., 326-A) are configured to bind to the pre- active peptide portion and other respective ones of the plurality of first agents 326 (e.g., 326-B) are configured to bind to the active peptide portion. As further previously described, the plurality of second agents 328 exhibit a detectable signal and are configured to bind to at least one of the pre-active peptide portion or the respective ones of the plurality of first agents 326. As further illustrated by FIG. 4B, in some embodiments, respective ones of the plurality of second agents 328 (e.g., 328-A) are configured to bind to the pre active peptide portion or respective ones of the plurality of first agents 326 (e.g., 326-A) configured to bind to an pre-active peptide portion and other respective ones of the plurality of second agents 328 (e.g., 328-B) are configured to bind to the active peptide portion or the other respective ones of the plurality of first agents 326 (e.g., 326-B) configured to bind to the active peptide portion.

[0082] The support 325 is configured to receive a biological sample and the plurality of reagents 322. As further illustrated by FIGs. 5A-5D, in some embodiments, the biological sample can be immobilized on the support 325, the plurality of first agents 326 can be immobilized on the support 325, the plurality of second agents 328 can be immobilized on the support 325, or both the plurality of first agents 326 and the plurality of second agents 328 can be immobilized on the support 325 to form different ty pes of assays, such as directly thereon. As previously described, responsive to binding between different respective ones of the plurality of first agents 326, the pre-active peptide portion and the plurality of second agents 328 on the support 325, the detectable signal is generated which is indicative of an expression level of the pre-active peptide portion in the biological sample and which differentiates between viral and bacterial infections.

[0083] The defensin protein, the pre-active peptide portion, and the active peptide portion can include any of the above described examples. For example, the defensin protein can include defensin 1, 2, and/or 3. Further, the apparatus 321 and/or kit 331 can include any of the above described variations, including but not limited to the agents specific to different target analytes. For example, the plurality of first agents 326 can include respective agents configured to bind to additional target analytes selected from CALP, SI00A12, NGAL, VCAM-1, GOLM 1, CSF-1, PCT, TRAIL, IP10, CRP, B2M, MxA, ISG15, UP A, and URP2, among other targets and combinations thereof.

[0084] In some embodiments, as further illustrated herein, the support 325 includes: (i) a first region to receive the biological sample and configured to pass the biological sample to a second region, (ii) the second region including the plurality of second agents 328 and configured to pass at least a portion of the biological sample and the plurality of second agents 328 to a third region, and (iii) the third region including the plurality of first agents 326, wherein the presence of the pre-active peptide portion in the biological sample causes accumulation of the detectable signal in the third region. For example, such a support 325 can be used form a lateral flow assay.

[0085] In some embodiments the support 325 is configured to receive the biological sample, the plurality of first agents 326, and the plurality of second agents 328 in a region (e.g., surface, wells, etc.) of the support 325, wherein the presence of the preactive peptide portion in the biological sample causes accumulation of the detectable signal in the region. For example, such a support 325 can be used to form a direct ELISA, an indirect ELISA, and/or a sandwich assay.

[0086] As shown by FIG. 4B, in some embodiments, the plurality of the first agents 326 include a first subset of first agents 326-A configured to bind to the pre-active peptide portion and a second subset of first agents 326-B configured to bind to an active peptide portion of the defensin protein in the biological sample. In some embodiments, the plurality of second agents 328 include a first subset of the second agents 328-A configured to bind to at least one of the pre-active peptide portion or the first subset of the first agents 326-A and a second subset of the second agents 328-B configured to bind to at least one of active peptide portion or the second subset of the first agents 326- B. As previously described, binding between different respective ones of the plurality of first agents 326, the pre-active peptide portion, the active peptide portion, and the plurality of second agents 328 generates the detectable signal which is indicative of an expression level(s) of the pre-active peptide portion and/or the active peptide portion in the biological sample. The expression level of the pre-active peptide portion and the expression level of the active peptide portion can differentiate between a viral infection and a bacterial infection.

[0087] In some embodiments, the detectable signal comprises a first detectable signal and a second detectable signal. For example, the first subset of second agents 328-A can exhibit the first detectable signal and the second subset of second agents 328-B can exhibit the second detectable signal. In some such embodiments, binding between respective ones of the plurality of first agents 326 (e g., 326-A), the pre-active peptide 1 portion, and respective ones the plurality of second agents 328 (e.g., 328-A) can cause generation of the first detectable signal which is indicative of the expression level of the pre-active peptide portion. Binding between respective ones of the plurality of first agents 326 (e.g., 326-B), the active peptide portion, and respective other ones of the plurality of second agents 328 (e.g., 328-B) can cause generation the second detectable signal which is indicative of the expression level of the active peptide portion.

[0088] Accordingly, apparatuses and/or kits in accordance with FIG. 4B can include the plurality of first agents 326, wherein respective ones of the plurality' of first agents 326 (e g., 326- A) are configured to bind to a pre-active peptide portion of a defensin protein in a biological sample and other respective ones of the plurality of first agents 326 (e.g., 326-B) are configured to bind to an active peptide portion of the defensin protein in the biological sample. The apparatuses and/or kits can further include a plurality of second agents 328 that exhibit a detectable signal and with respective ones of the plurality of second agents 328 (e.g., 328-A, 328-B) configured to bind to at least one of: the pre- active peptide portion or the active peptide portion, or the respective ones or other respective ones of the plurality of first agents (e.g., 326-A, 326-B). In some embodiments, apparatuses and/or kits can further include the support 325. For example, the support 325 can receive the biological sample, the plurality of first agents 326, and plurality of second agents 328, and in response to binding between different respective ones of the plurality of first agents 326, the pre-active peptide portion, the active peptide portion, and the plurality of second agents 328 on the support 325, cause accumulation of the second agents 328 and generation of the detectable signal(s) which is indicative of an expression level of the pre-active peptide portion and/or an expression level of the active peptide portion in the biological sample. For example, the second agents 328 exhibiting the detectable signals can accumulate on the support 325 in response to the binding, and which indicates a presence and/or expression level of the pre-active peptide portion and/or the active peptide portion in the biological sample.

[0089] In some embodiments, the plurality of first agents 326 and/or the second agents 328 are disposed on the support 325 or disposed in a fluid configured to be placed on the support 325, as further illustrated herein. In some embodiments, the biological sample can be mixed with the second agents 328 that exhibit the label, which allows for the physical binding interaction between the pre-active peptide portion, the active peptide portion and/or any of the set of target analytes that may be present in the biological sample and the second agents 328. In some embodiments, the plurality of first agents 326 can be bound to a solid surface of the support 325, such as a well of a microtiter plate, a glass slide or a bead, allowing target analytes that are not bound to the first agents 326 to be removed from the bulk solution, separated and concentrated. In some embodiments, the presence of the target analytes is determined by exposing the support 325 (e.g., glass substrate or nanowell array) to a solution containing the second agents 328, which include or are a label exhibiting a detectable signal, such as a fluorescent, enzymatic, or radioactive label or an otherwise detectable molecular tag such as biotin or a molecule possessing a unique mass or isotope. After washing away unbound second agents 328, such as a secondary antibody (e.g., an anti-antibody or a second antibody that binds to another epitope of the antigen associated with the target analyte) that is labeled, the presence of detectable signal can be used to identify presence of the preactive peptide portion, the active peptide portion, and/or any of a set of target analytes. Although embodiments are not so limited, and can include exposing the support 325, which has immobilized antibodies thereon, to the biological sample. Further, other types of tests and assays can be performed, such as PCR-based test and lateral flow assays. [0090] The identification of the detection signal and/or the binding can depend on the ty pe of assay and/or test performed. For example, some tests, such as lateral flow assays, can result in detected binding that is visible. In other embodiments, the binding is detected using various components to produce and detect electromagnetic radiation, such as a light source producing monochromatic light, prisms, mirrors and lenses to direct and focus the light on the sample detection region, optical filters, apertures and a photo-detector to detect light being emitted from the sample detection region, producing a scan of the support 325 that is electronically digitized for communication with signal processing circuitry. The optical scan can be used to identify the locations on the glass slide or other support that reveal discrete spots or regions associated with the second agents 328 and that correspond to the presence of the pre-active peptide portion, the active peptide portion, and/or any of the set of target analytes. As a specific example, if a blood cell exhibits at least one of the set of target analytes, at least one agent (e g., antibodies) binds to the at least one target analyte on the glass slide. A set of second agents, such as labeled anti-antibodies, can be applied to the solid support. For example, the anti-antibody, which is fluorescently, enzymatically, or radioactively labeled and washed over the glass slide, binds to the antibody and results in a signal, such as fluorescent emission, when scanned by the optical circuitry.

[0091] Other specific embodiments are directed to an apparatus which includes optical components (e.g., fiber optic scanner), a support 325, and processing circuitry. Example optical components include a fiber optic bundle array, a laser, and imaging circuitry (e g., camera). In specific embodiments, the optical components are used to scan the biological sample, as immobilized and exposed to second agents 328 to identify first agents 326 bound to the target analyte(s). The apparatus can include various additional circuitry, such as processing circuitry for controlling the various instruments, memory circuit for storing data sets, and various computer-readable instructions for controlling the optical components and computer-executable instructions (e.g., software) for analyzing data obtained therefrom.

[0092] In various embodiments, exposing the biological sample to the first agents 326 can include forming an immuno-assay. For example, a glass substrate or other type of support 325 is coated with a biological sample from a subj ect suspected of having a viral and/or bacterial infection and used to form an immuno-complex by exposing the immobilized biological sample to the first agents 326 (e.g., detection antibodies and optionally, secondary labeled anti-antibodies). The first agents 326 can include detection antibodies in such embodiments and are referred as the “detection antibodies” below for ease of reference. An immuno-sandwich can be used to detect antibodies bound to the at least one target analyte. The glass substrate, after washing away unbound detection antibodies, can be treated with the second agents 328 that includes labeled secondary antibodies or an anti-antibodies, and an optical scan of the glass substrate is used to identify a signal (e.g., fluorescence) indicative of at least one of the labeled antiantibodies. If at least one of the set of target analytes is present, a respective detection antibody binds to a specific target analyte present on the glass substrate and the complementary anti-antibody binds to the detection antibody. For example, the anti- antibody can bind to the fragment crystallizable (FC) segment of the detection antibody. Subsequently, the label of the second agents 328 (associated with the labeled antidetection antibody) indicates presence of the particular target analytes. The antiantibodies can include various organism-specific antibodies, such as an anti-human antibodies, anti-horse antibodies, anti-dog antibodies, anti-cat antibodies, anti-fish antibodies, anti-cattle antibodies, anti-bird antibodies, among other organisms that have white blood cells and produce antibodies. As may be appreciated, the anti-antibodies used can be specific to the organism, such as an anti-horse detection antibody or an antirabbit detection antibody. Similarly, embodiments are not limited to first immobilizing the biological sample and can instead use immobilization of the antibodies or other agents, as further illustrated in FIGs. 5A-5D.

[0093] FIGs. 5A-5D illustrate example supports for determining a presence of a preactive peptide portion and/or an active peptide portion of a defensin protein, in accordance with the present disclosure. In some embodiments, the exposure of the biological sample to the plurality first agents can be used to form an immuno-assay, such as an immuno-sandwich ELISA. In FIGs. 5A and 5C-5D, the set of first agents are illustrated and described as being specific to the pre-active peptide portion 324- A. However, embodiments are not so limited and the first agents can include agents that specifically bind to more than one of the set of target analytes, such as to the pre-active peptide portion 324-A and the active peptide portion 324-B as illustrated by FIG. 5B. Similarly, embodiments are not limited to first agents and/or second agents that include antibodies. For example, agents can alternatively include synthetic molecules, among other variations.

[0094] As illustrated by FIGs. 5A-5C, the biological sample can be immobilized to a support 325. The immobilized biological sample is exposed to the plurality of first agents 326- A (and/or 326-B), such as a volume of an antibody. FIG. 5 A illustrates an embodiment in which the antibody 327 -A is labeled such that the antibody 327 -A is detectable. For example, the antibody 327 -A is bound to the second agent 328-A which comprises label 329- A. After incubation of the support 325 with a solution containing the antibody 327-A with the label 329-A, the support 325 can be washed to remove unbound antibody and is scanned to detect the presence of antibodies 327-A bound to the pre-active peptide portion 324-A. [0095] FIG. 5B is an assay configured that is substantially the same as FIG. 5A, but designed to detect the pre-active peptide portion 324-A and the active peptide portion 324-B. The plurality of first agents 326- A, 326-B include antibodies 327-A, 327-B labeled such that both are detectable. For example, the antibodies 327-A, 327-B are respectively bound to the second agents 328-A, 328-B which comprise labels 329- A, 329-B. Antibody 327-A specifically binds to pre-active peptide portion 324-A and antibody 327-B specifically binds to active peptide portion 324-B. After incubation of the support 325 with a solution containing the antibody 327-A with the label 329- A and the antibody 327-B with the label 329-B, the support 325 can be washed to remove unbound antibody and is scanned to detect the presence of antibodies 327-A bound to the pre-active peptide portion 324-A and antibodies 327-B bound to the active peptide portion 324-B.

[0096] Other embodiments, as illustrated by FIG. 5C, include an assay configuration in which a labeled secondary /anti-antibody 330 that binds to the primary antibody 327-A and is used to provide the detectable signal and indicate the presence of one or more of the set of target analytes, as illustrated by the pre-active peptide portion 324-A. In such embodiments, the second agent 328-A includes the secondary/anti-antibody 330 bound to the label 329- A.

[0097] In other embodiments, as illustrated by FIG. 5D, the antibody 327-A is immobilized to the support 325 to capture the pre-active peptide portion 324-A (and/or other target analyte(s)) when a biological sample is incubated with the support 325. The solid support 325 is washed to remove unbound biological sample (which can be further analyzed before or after to determine, e.g., a total target cell population in the biological sample and to assess the full population). The second agent 328-A, which can include a volume of a detection agent, such as a labeled secondary antibody 330, is subsequently applied to the support 325. The labeled secondary antibody 330 can be specific to a different epitope of the antigen associated with the pre-active peptide portion 324-A. After washing the support 325 to remove unbound labeled secondary antibody 330, the support 325 is scanned to detect at least one of the labeled secondary antibody 330 bound to the pre-active peptide portion 324-A via the antibody 327-A. [0098] Although the embodiments of FIGs. 5A-5D illustrate a flat solid support 325, such as a glass substrate, embodiments are not so limited and can include a variety of different supports and assays, such as beads, nano-particles, tubes, arrays, microfluidic channels, etc. Further embodiments are directed to a first agents and/or second agents designed to detect or specifically bind to the pre-active peptide portion 324-A and the active peptide portion 324-B, as well as at least one additional target analytes, such as a range of two to ten (or more) target analytes.

[0099] FIGs. 6A-6B illustrate example assays for determining a presence of a pre-active peptide portion and/or an active peptide portion of a defensin protein, in accordance with the present disclosure. More specifically, FIG. 6A illustrates a lateral flow assay and/or test that can be used to test for the presence of the pre-active peptide portion and/or active peptide portion of a defensin protein. As may be appreciated, a lateral flow test, which is also referred to as a lateral flow immunochroatographic assay, is an assay used to test for the presence or absence of a target analyte in a biological sample and is ty pically designed as a rapid test that may not require specialized equipment, having a visible readout. An example of such a test is a home pregnancy -test.

[00100] As illustrated by FIG. 6A, the assay includes a support 425 having a number of different regions 440, 442, 444, 446. The support 425 can have at least a first region 440, a second region 442, and a third region 444. At 441, the first region 440 can receive a biological sample 451 obtained from a subject. The biological sample 451 can contain at least one of a set of target analytes 452, as illustrated by the pre-active peptide portion 452-A and the active peptide portion 452-B. In some embodiments, at 443, the first region 440 can pass a portion of the biological sample 451 to the second region 442. For example, the first region 440 can act as a sponge, which can have the effect of retaining some interfering molecules while allowing the target analytes unfettered travel along the strip. Once the first region 440 is soaked by the biological sample 451, a portion of the biological sample 451 can migrate to the second region 442 and the remaining portion can be held by the first region 440.

[00101] The second region 442 can include the plurality of second agents 453 (e.g., a set of detection agents, such as a set of antibodies having a label) that can be stored within a solid matrix dried onto the support 425. In some embodiments, the second agents 453 are respectively specific to the one of the set of target analytes, such as the pre-active peptide portion 452-A and the active peptide portion 452-B, although embodiments are not so limited. For example, the second region 442 can store a conjugate that includes the plurality second agents 453 specific to the at least one target analyte (e.g., pre-active peptide portion 452-A and the active peptide portion 452-B) dried into a salt-sugar matrix that allows the second agents 453 to be stored within the second region 442. As the portion of the biological sample 451 passes into and through the second region 442, the salt-sugar matrix dissolves, releasing the second agents 453 into solution where the second agents 453 interact by diffusion with molecules present in the biological sample 451 and release to further regions of the support 425. However, embodiments are not so limited, and the second agents 453 can be temporarily connected to the second region 442 using other techniques, such as other dissolvable binding agents or otherwise being placed directly thereon. The second agents 453 can be conjugated to a label (e.g., colored dye), as shown by the particular second agent 453-A bound to a label 454-A and bound to the pre-active peptide portion 452-A, and the particular second agent 453-B bound to a label 454-B and bound to the active peptide portion 452-B. The binding of second agents 453 to the target analytes forms detectable complexes that can be revealed when reaching the third region 444 and/or the fourth region 446, as further illustrated at 445. The second region 442 passes at least some portion of the biological sample 451 to the third region 444 and, optionally the fourth region 446.

[00102] The third region 444 and, optional fourth region 446 (or more) can contain the plurality of first agents 448 (e.g., second antibodies or capture agents) immobilized to the support 425, which can be specific to a second epitope of the pre-active peptide portion 452-A and the active peptide portion 452-B. As shown, the third region 444 can include the first subset of the first agents 448, as illustrated by particular first agent 455 that are specific to the pre-active peptide portion 452-A of the defensm protein. The fourth region 446 can include the second subset of the first agents 448, as illustrated by particular first agent 457 that are specific to the active peptide portion 452-B of the defensin protein. In response to binding of the particular second agent 453-A (e.g., first antibody) to the pre-active peptide portion 452-A, a target-conjugate complex can be formed and can be passed to the third region 444. The target-conjugate complex binds to the particular first agent 4 5 that is formed in a strip or test line in the third region 444. After sufficient fluid has passed the strip, captured target-conjugate complexes can accumulate to the level to cause the strip area to become colored which is indicative of a presence and/or expression level of pre-active peptide portion 452-A. In some embodiments, a portion of the biological sample 451 is then further passed into and through the third region 444 to the fourth region 446. For example, in response to binding of the particular second agent 453-B (e.g., first antibody) to the active peptide portion 452-B, a target-conjugate complex can be formed and is passed to and through the third region 444 and to the fourth region 446. The target-conjugate complex binds to the particular first agent 457 that is formed in a strip or test line in the fourth region 446. After sufficient fluid has passed the strip, captured target-conjugate complexes can accumulate to the level to cause the strip area to become colored which is indicative of a presence and/or expression level of the active peptide portion 452-B. Although the subsets of first agents 448 are illustrated as being on different regions of the support 425, which are lateral from one other, embodiments are not so limited. In some embodiments, the third region 444 can include sub-portions which contain the subsets of first agents 448 that are otherwise geographically separated from one another.

[00103] In some embodiments, the support 425 can include at least one additional region that contains immobilized control agents that bind to any particle. The control agents can indicate the functional validity of the assay. For example, a particular second agent containing the label can binds to the control agent in the additional control region. [00104] FIG. 6B illustrates another example lateral flow assay and/or test that can be used to test for the presence of the pre-active peptide portion and/or active peptide portion of a defensin protein. The lateral flow assay of FIG. 6B can include substantially the same attributes and features of the lateral flow assay of FIG. 6A, as illustrated by the common numbering, but with the biological sample 451 being mixed with the second agents 453 in solution, at 439, and prior to placing the biological sample 451 on the first region 440 of the support 425. In some such embodiments, the assay may not include the second region 442. The common attributes and features not being repeated for ease of reference. [00105] The above described methods, apparatuses and/or kits can be used for detecting the presence of and/or expression levels of the pre-active peptide portion and/or active peptide portion of a defensin protein, and which can be used to differentiate between viral infections and bacterial infections.

[00106] A biological sample can include an aqueous solution or fluid containing a sample, in solid or fluid form, and/or reagents. A biological sample, as used herein, includes or refers to any material, collected from a subj ect, such as biologic material and carried in a fluid. Examples of biological samples include, but are not limited to, whole blood, blood plasma, and other body fluids, as well as tissue cell cultures obtained from humans, plants, or animals. Such biological samples may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. The biological material can comprise all ty pes of mammalian and non-mammalian animal cell. Non-limiting sample examples include whole blood and blood-derived products such as plasma, serum and buffy coat, urine, feces, cerebrospinal, synovial and interstitial fluid or other body fluids, tissues, cell cultures, cell suspensions, etc. The term “fluid” refers to any substance that flows under applied forces. Reagents include or refer to a substance, compound, or mixture for use in a reaction, which may be added to cause the reaction to occur.

[00107] The various ranges provided herein include the stated range and any value or sub-range within the stated range. Furthermore, when “about” is utilized to describe a value, this includes, refers to, and/or encompasses variations (up to +/- 10%) from the stated value.

[00108] Reference throughout the specification to “examples”, “an example”, “some examples”, “embodiments”, and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in the example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any embodiment can be combined in any suitable manner in the various embodiments unless the context clearly dictates otherwise. [00109] In describing and claiming embodiments disclosed herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[00110] Additionally, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list. Conjunctive language such as the phrase “at least one of X, Y, or Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, Z, X and Y, X and Z, Y and Z, or X, Y, and Z. Thus, such conjunctive language is not generally intended to imply that certain embodiments require at least one of X, at least one of Y, and at least one of Z to each be present, unless otherwise stated. For example, in some embodiments, the plurality of second agents include second agents that bind to the pre-active peptide portion and second agents that bind to the active peptide portion, or second agents that bind to the respective ones of the plurality of first agents that bind to pre-active peptide portion and second agents that bind to respective ones of the plurality of first agents that bind to the active peptide portion, or combinations thereof. Similarly, the use of the term “and/or” is used in its inclusive sense to connect a list of elements, such that the term “and/or” means one, some, or all of the elements in the list. For example, in some embodiments, the detectable signals are indicative of the expression level of the pre-active peptide portion, the expression level of the active peptide portion, or are indicative of both expression levels of the pre-active peptide portion and the active peptide portion. Thus, such conjunctive language of “X and/or Y” can imply that certain embodiments may include at least one of X, include at least one of Y, or include least one of X and at least one of Y.

[00111] Although specific embodiments have been illustrated and described herein, a variety of alternate and/or equivalent implementations can be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific embodiments discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof. EXPERIMENTAL EMBODIMENTS

[00112] A number of experimental embodiments were conducted to assess target analytes for differentiating between viral and bacterial infections and to characterize properties for differentiating there between. In some experiments, new assays were developed that target the differences between the pre-active and active forms of defensin protein by itself and in combination with other proteins. Such assays have application in the management of antibiotic treatment of infections, such as patients with lower respiratory tract infections or septics. Example biomarkers which were used to assess for infections include amino acid sequences set forth in SEQ ID NOs: 1-7, such as shown in Table 1.

Assessing Target Analytes

[00113] In some experiments, a proteomics method was used to identify host-response biomarkers in blood that can be utilized for rapid characterization and discrimination between viral and bacterial infections. Defensin was identified as a target. The mass spectrometry analysis coupled with reverse-phase liquid chromatographic separation demonstrated the use of two separate regions from the defensin protein, the pre-active peptide portion and active peptide portion, that were used to distinguish viral infection from bacterial infection. The data showed that in PBMCs and the contained low-density neutrophils for patients with influenza infection, peptides form both pre-active peptide portion (amino acids 39-64) and active peptide portion (amino acids 65-94) were greatly elevated compared to healthy controls. For patients with bacterial infection, only the active form was elevated. Assays that target the differences between the mature and active-forms of the defensin protein by itself, or in combination with other proteins, can allow for rapid characterization of bacterial and viral infections, contributing to early diagnosis and management of antibiotic treatment. In addition, the white blood cells known as neutrophils are involved in both the early and prolonged cell response to immune system challenges by, among their myriad known and continually being elucidated functions, migrating into tissues and producing a number of antimicrobial molecules. Assignment of different molecular markers to distinct subpopulations of neutrophils may have diagnostic, prognostic and disease stratification utility for not only infectious diseases but also various cancers and autoimmune conditions. Assays with markers that elaborate a subpopulation of neutrophils can also be used to monitor disease modification during the treatment of patients with drugs or cell-based therapies. [00114] In some embodiments, a plurality of different target analytes were observed in infected patient blood samples. Example target analytes analyzed using LC-MS/MS methods in lysed white blood cell and plasma samples collected from patients, and combined with those including PCT, MxA, TRAIL, IP10, CRP, B2M, ISG15, DEF1/2, and UP A. Table 1 below summarizies examples regions/portions of a defensin protein:

Table 1: Defensin Protein Regions

Human Samples

[00115] For the samples, human blood samples were collected at a single time point with informed consent under an appropriate Institutional Review Board (IRB)-approved protocol. Paired blood plasma and cell samples from patients infected with various ty pes of bacterial and viral infections were either obtained at Stanford University School of Medicine (Palo Alto, CA) or procured from BioIVT Westbury, NY. Samples from healthy donors were obtained at Stanford Blood Center (Palo Alto, CA). Table 2 below summarizes the sample information. [00116] For the plasma sample, an 8.5 milliliter (mL) blood sample was collected from each subject in a P100 tube coated with K2EDTA anticoagulant and protease inhibitor cocktail for the protein stabilization (BD Biosciences). This tube was centrifuged at 1600 gravitation force (G) for 30 minutes in room temperature and the plasma was collected, aliquoted, and stored at -80 degrees Celsius (C) before analysis.

[00117] For the paired cell sample, an additional tube of blood sample was drawn into an 8 mL Cell Preparation Tube (CPT) with sodium heparin (BD, BD Biosciences). This tube was centrifuged at 1600 G for 30 minutes in room temperature into three fractions, where the upper fraction containing plasma and a whitish cloudy band of cells, e g., PBMCs, just under plasma. After centrifugation, one half of the plasma in the upper fraction was removed and discarded carefully without disturbing the cell layer. The remaining plasma and cells in the upper fraction were collected and transferred to a 15 mL conical centrifuge tube. Calcium and magnesium free phosphate-buffered saline (PBS) at pH = 7.4 (Gibco, Life Technologies) was added to bring the volume up to 15 mL and centrifuged at 300 x G for 15 minutes. The supernatant was aspirated without disturbing pellet. To wash the cells, the pelleted cells were resuspended in 10 mL of PBS and centrifuged at 300 x G for 10 minutes. The supernatant was removed and the cell pellet after the wash was lysed in 1 mL of completed Lysis-M buffer (Roche, Cat# 04719956001). The cell lysate was aliquoted and stored at -80 degrees C until downstream processing or analysis.

Table 2: Human subjects whose samples were collected

EXAMPLE 1: DISCOVERY BY PROTEOMICS PROFILING

[00118] To discover the form of proteins for discriminating different types of infections, the PBMC fraction of a representative subset of blood samples taken from patients with viral infections and patients with bacterial infections (Table 2) was compared using a liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) proteomics method.

[00119] Approximately 0.3 rnL of cell lysate from each sample was added into 0.6 mL of a denaturing buffer containing Tris-buffered (pH 8) 6M guanidinium chloride (Sigma-Aldrich). These denatured samples were reduced with 10 mM dithiothreitol (Sigma- Aldrich) at 60 degrees C for 1 hour and alkylated with 25 mM iodoacetic acid (Sigma- Aldrich) in the dark at room temperature for 30 minutes. A buffer exchange against 50 mM ammonium bicarbonate at pH 8.3 (Fisher Scientific) was performed to remove denaturant and reduction-alkylation reagents in the sample using an Amicon Ultra-4 centrifugal filter unit (Millipore) with a molecular weight cutoff of 3000 Da. Sequencing grade trypsin (Promega) at a concentration of 4 micrograms (pg)/mL was added to each buffer exchanged sample for digestion at 37 degrees C overnight. Digested samples were acidified using 10% formic acid to give a final formic concentration of 0.1% for analysis.

[00120] Digested samples were analyzed with a nanoflow LC-MS/MS method by injecting and separating on a 75 micrometer (pm) x 10 centimeter (cm) nanocolumn (New Objectives) at a flow rate of 200 normal L per minute (nL/min) using a fluidics scheme, which was conducted as described by Bondarenko, et al., “Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry”, Anal Chem, 2002, 74(18): p. 4741-9, which is incorporated herein in its entirety for its teachings. The chromatography system consisted of an Agilent 1100 capillary binary solvent delivery pump and solvent degasser, together with an HTC-Pal autosampler (Leap Technologies). An increasing gradient of acetonitrile in water (0% to 55% over 2 hours), was used for on-line separation with both solvents containing 0.1% formic acid. The effluent of the nano column was fed directly to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Scientific) utilizing a nanospray source. During a run, high- resolution MSI scans performed at nominal mass resolution setting of 60,000 (m/z 400), were acquired in positive-ion mode and saved as profile data. Tandem mass spectra (MS2) were performed in the LTQ linear ion-trap on each of the top 5 or 7 non- dynamically excluded ions from MS 1 scan.

[00121] Peptide sequence and protein identification were determined using a commercial database searching software (ByOnic/ComByne, Protein Metrics, CA) against a human protein database concatenated with reverse sequences. A cutoff <5% for false discovery rate was used in peptide identifications.

[00122] Using an established mass spectrometry' (MS)-based proteomics method described, differentially processed forms of the defensin protein were identified as a new biomarker for distinguishing bacterial and viral infections by analyzing hundreds of host proteins in the lysed cell samples. The MS-based proteomics method was used as described by Shaler, et al., “Particle radiation-induced dysregulation of protein homeostasis in primary human and mouse neuronal cells”, Life Sci Space Res (Amst), 2020. 25: p. 9-17; and Balog, et al., “Development of a point-of-care radiation biodosimeter: studies using novel protein biomarker panels in non-human primates”, Int J Radiat Biol, 2020. 96(1): p. 35-46, each of which are incorporated herein in their entireties for their teachings.

[00123] The results indicated bacterial infections exclusively result in abundant production of the active form (amino acid 65-94), whereas in viral infections the preactive peptide is retained and both the mature and active forms are detected (amino acid 39-94). FIGs. 7A-7C show an example of the ability to distinguish viral from bacterial infections by measuring the amount of a peptide unique to the pre-active peptide of the defensin protein in a biological sample from patients with an active viral infection. A further demonstration of the pre-active peptide, showing the sequence that bridges the site between the pre-active peptide and active form of the defensin protein is provided in FIGs. 8A-8C. An additional demonstration showing the detection of the active form of defensin protein in biological samples of patients with either viral or bacterial infections is shown in FIGs. 9A-9C. By analyzing the levels of both the pre-active peptide and the active form in collected patient samples, experiments demonstrated the power of our quantitative “label-free” shotgun proteomics platform for host-biomarker analysis at a level of specificity that has not been previously performed. An example pre-active peptide portion is set forth in SEQ ID NO: 1 (e g., defensin amino acids 39-64) and an example active peptide portion is set forth in SEQ ID NO: 2 (e.g.. defensin amino acids 65-94).

[00124] FIGs. 7A-7C illustrate example levels of expression of a pre-active peptide portion of a defensin protein in a viral infected sample and a bacterial infected sample, in accordance with the present disclosure. In particular, FIG. 7A shows an example of a defensin pre-active peptide portion used to differentiate bacterial from viral infections as shown by the exact-mass chromatograms for the quadruply-charged peptide corresponding to amino acids 39-56 of pre-active peptide portion of the defensin protein and as set forth in SEQ ID NO: 3. FIG. 7B shows an example of a defensin pre-active peptide portion used to differentiate bacterial from viral infections as shown by the zoomed-in high-resolution mass spectra averaged over the region of the chromatographic peak. FIG. 7C shows an example of a defensin pre-active peptide portion used to differentiate bacterial from viral infections as shown by the tandem mass spectrum of the peptide from the strong signal in the virus-infected patient sample. [00125] FIGs. 8A-8C illustrate further example levels of expression of a pre-active peptide portion of a defensin protein in a viral infected sample and a bacterial infected sample, in accordance with the present disclosure. The data demonstrates the preponderance of the pre-active peptide portion of the defensin protein in a virus- infected patient compared to a patient with a bacterial infection as shown by the FIG. 8A of the exact-mass chromatograms for the doubly-charged peptide corresponding to amino acids 63-69 of mature form/chain of the defensin protein and as set forth in SEQ ID NO: 4; FIG. 8B showing the zoomed-in high-resolution mass spectra averaged over the region of the chromatographic peak and; FIG. 8C showing the tandem mass spectrum of the peptide from the strong signal in the virus-infected patient sample.

[00126] FIGs. 9A-9C illustrate example levels of expression of an active peptide portion of a defensin protein in a viral infected sample and a bacterial infected sample, in accordance with the present disclosure. More particularly, FIGs. 9A-9C shows an example of an active peptide portion of a defensin protein detected in biological samples from patients with either viral or bacterial infections as shown by: FIG. 9A showing extracted mass chromatograms from the LC-MS proteomic analysis of biological samples from patients; FIG. 9B showing the zoomed-in high-resolution mass spectra averaged over the region of the chromatographic peak and; FIG. 9C showing the tandem mass spectrum of the peptide from the strong signal in the virus-infected patient sample. The chromatograms are produced by extracting the exact mass of the doubly -charged peptide corresponding to the N-terminal tryptic peptide of the active form of defensin having the sequence corresponding to defensin amino-acids 65-69, ACYCR, and as set forth in SEQ ID NO: 5.

EXAMPLE 2: VERIFICATION OF DEFENSIN AS BIOMARKER BY ELISA [00127] A commercially available human alpha-defensin 1 (DEFA) DuoSet ELISA Kit was used (R&D Systems) to confirm the potential clinical utility of alpha defensin- 1 (DEFA) in discriminating bacterial from viral infections. The antibody pair included in the kit binds to a purified E. coli-e resssA recombinant human DEFA with epitopes in the active form of DEFA (amino acids 65-94).

[00128] An assay was conducted at room temperature, following the manufacturer protocol, on individual cell lysate samples collected from 33 human subjects as shown in Table 2. The samples comprised 12 healthy controls, 11 patients with diagnostically- confirmed bacterial infections, and 10 patients with confirmed viral infections. To prepare seven-point standards ranging from 0.5 to 32 nanograms (ng)/mL, 2-fold serial dilutions of DEFA calibrator stock in a reagent diluent buffer (1% BSA in PBS at pH 7.4 (R&D Systems) was performed. The human subject cell lysate samples were then thawed and diluted before being analyzed in the reagent diluent at an optimized dilution factor of 1000 to 5000-fold.

[00129] Positive and negative quality controls (QCs) were generated by spiking samples with or without the calibrator, respectively. To ensure the quality and reproducibility of the analysis, duplicate analyses of standards, QCs, and samples was performed, and block randomized the samples to minimize analysis order or position bias.

[00130] To perform the ELISA assay, a mouse antihuman alpha-Defensin 1 antibody was immobilized onto microtiter wells of a 96-well MaxiSorp plate (Nunc) at 1 mg/mL in coating buffer (PBS at pH 7.4) (Life Technologies) overnight. The wells were washed 3 cycles with the wash buffer (0.05% Tween 20 in PBS at pH 7.4) (Teknova) using a plate washer (BioTek, 405LS) and then blocked with the reagent diluent buffer for 1 hour. After blocking, the wash processes were repeated. Calibration standards, QCs and diluted cell lysate samples were added into wells and incubated for 2 hours. After incubation and wash, the captured antigen was detected with a sheep antihuman DEFA biotinylated polyclonal antibody at 75 ng/mL and a solution of streptavidin conjugated horseradishperoxidase. Wells were washed again with the wash buffer. A substrate solution, Tetramethylbenzidine (TMB) (Sigma) was added to the wells and incubated in dark for 20 minutes. The color development was terminated by adding a stop solution (2 N H2SO4) (BDH). The plate was scanned utilizing a microplate reader (Biotek, PowerWave HT340). The optical density (OD) value of each well was obtained from the absorbance at 450 nanometers (nm) with the wavelength correction set to 570 nm. The concentration of each sample was calculated using an averaged value of duplicated measurements based on a 4-parameter logistic fit of the standard curve (BioTek, KC4 Kineticalc software).

[00131] Statistical analysis of the concentration data was performed using GraphPad Prism 9.5.1 (GraphPad Software, CA, USA). Results as displayed in FIG. 10 show that DEFA effectively distinguishes viral from bacterial infections, with high levels of DEFA observed in patients with influenza and respiratory syncytial virus infections compared to those with bacterial infections (p=0.0015) or healthy controls (p<0.0001). An area under the ROC curve value of 0.89 is obtained as shown in FIG. 11 for stratifying between viral and bacterial infection patients.

[00132] FIG. 10 is a graph showing ELISA measurements for DEFA (amino acids 65- 94), in accordance with the present disclosure. Box and whisker plots of ELISA measurements for DEFA in lysed blood cell samples showed statistically significant elevations in patients with viral infections (n=10) compared to bacterial infections (n=l 1) (p=0.0015) or healthy controls with no infections (n=12) (p<0.0001). The box represents the interquartile range with median as a bisecting line. The whiskers represent the minimum and maximum values. The p-values are calculated using Mann Whitney U test (nonparametric) for unpaired two group comparisons, ** P<0.01, **** P<0.0001. [00133] FIG. 11 is a graph showing receiver operator characteristic (ROC) curve of DEFA, in accordance with the present disclosure. As shown, the ROC curve of DEFA discriminates between bacterial and viral infections among patient samples. An area under the curve (AUC) of 0.89 (95% confidence interval 0.76 to 1.0, p=0.0025) demonstrates a good discrimination using this marker alone. Use of additional targets can further discriminate between bacterial and viral infections.

EXAMPLE 3: DEFA PERFORMANCE COMPARED TO OTHER BIOMARKERS [00134] Myxovirus resistance protein 1 (MxA) is a host-response protein for bacterial and viral discrimination. Concentrations of MxA protein were measured on the same patient samples used to obtain DEFA results. Aliquots of frozen lysed blood cell samples were thawed and diluted 5 to 25-fold for assaying using the MxA protein human ELISA kit (BioVendor R&D) according to manufacturer instructions.

[00135] C-reactive protein (CRP) and IFN-gamma-inducible protein 10 (IP-10;

CXCL10) in blood plasma samples are other two biomarkers for distinguishing between acute bacterial and viral infection. To compare the performance of these two proteins with DEFA, frozen aliquots of paired plasma samples were thawed and analyzed using the Quantikine® colorimetric sandwich ELISA kits for CRP and IP-10 respectively (R&D Systems). Assays were conducted following manufacturer’s suggested protocols. For the CRP ELISA measurements, plasma samples were diluted 2500-fold while as for the IP-10, a 5-fold dilution was used.

[00136] The result summarized in Table 3 show good performance for stratifying infection types using DEFA with an AUC value of 0.89 which is significantly better than those obtained with other biomarkers, AUC of 0.56 for MxA, 0.70 for CRP, and 0.57 for IP10.

Table 3. Summary of classification performance characterization EXAMPLE 4: DEFA PERFORMANCE IN A TWO-BIOMARKER PANEL [00137] To test if the performance of DEFA is further improved by combining with another biomarker, additional proteins were measured by commercially available ELISA kits. These include neutrophil gelatinase-associated lipocalin (NGAL) (Bioporto) and calprotectin (CALP) (DRG) assayed using plasma samples, as well as an ubiquitin-like protein ISG15 in lysed cell samples (Creative diagnostics). A multiple logistic regression model was used in selecting a biomarker panel to evaluate the two- biomarker panel performance. Among all proteins analyzed, DEFA in combination with ISG15 yields the best AUC value of 0.93 shown in FIG. 12. This further illustrates the potential of additional improvement to include DEFA in a panel of protein biomarkers in distinguishing viral from bacterial infections.

[00138] FIG. 12 is a graph showing the ROC of DEFA in two-biomarker pannel, in accordance with the present disclosure. In particular, FIG. 12 shows the ROC of DEFA in the panel with ISG15 discriminating between bacterial and viral infections among patient samples. An AUC of 0.93 (95% confidence interval 0.81 to 1.0, p=0.0009) shows an improved discrimination compared to each biomarker measured alone.