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WO2025208075A1 - Methods of detecting persistent and latent viral infections using media enriched by newly synthesized antibodies (mensa) - Google Patents

Methods of detecting persistent and latent viral infections using media enriched by newly synthesized antibodies (mensa)

Info

Publication number
WO2025208075A1
WO2025208075A1 PCT/US2025/022104 US2025022104W WO2025208075A1 WO 2025208075 A1 WO2025208075 A1 WO 2025208075A1 US 2025022104 W US2025022104 W US 2025022104W WO 2025208075 A1 WO2025208075 A1 WO 2025208075A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
cov
coronavirus
mensa
human
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/022104
Other languages
French (fr)
Inventor
Frances Eun-Hyung Lee
Natalie Haddad
Ignacio Sanz
John DAISS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Emory University
Original Assignee
Emory University
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Filing date
Publication date
Application filed by Emory University filed Critical Emory University
Publication of WO2025208075A1 publication Critical patent/WO2025208075A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035—Herpes simplex virus I or II
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus

Definitions

  • kits of any preceding aspect further comprising one or more reagents to obtain whole blood or PBMC, one or more reagents to separate newly proliferated ASC from plasma (including, but not limited to one or more antibodies specific one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and/or Ki67), media to incubate the newly proliferated ASC, one or more antigens, and/or one or more reagents for detecting the presence of antibodies secreted by the newly proliferated ASC.
  • a persistent or latent viral infection such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS (-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (SARS-CoV-2) (SARS-Co
  • the MENSA is obtained by obtaining whole blood or PBMC from a subject; separating plasma from the whole blood or PBMC to produce separated cells; isolating newly proliferated ASC from the separated cells (such as, for example, using magnetic bead separation, ficoll gradient separation, elutriation, or FACS including, but not limited to the use of antibodies specific for one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and Ki67); washing the newly proliferated ASC; and incubating the washed newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion to thereby producing MENSA; wherein the MENSA comprises at least a 10 6 or 10 7 -fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
  • disclosed herein are methods of detecting a persistent or latent viral infection of any preceding aspect, wherein the method further comprises assessing any symptoms of infection that the subject is experiencing.
  • Also disclosed herein are methods of detecting a subsequent acute viral infection such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)- 229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Sever
  • a subsequent acute viral infection of any preceding aspect wherein the MENSA is detected by immunoassay (such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD).
  • immunoassay such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2,
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • reduce or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • terapéuticaally effective refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Biocompatible generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
  • compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • control is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or “negative.”
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the term When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect.
  • Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • antibody secreting cell or “plasma cell” refers to any B lineage cell capable of secreting antibody including but not limited to plasmablasts, short-lived antibody secreting cells, long-lived plasma cell.
  • MENSA Media enriched by newly synthesized antibodies
  • MENSA differs from plasma in that the antibodies present in plasma are from long-lived plasma cells in the bone marrow and not newly synthesized antibodies.
  • MENSA also differs from media containing PBMC in that there are no memory B cells present and the ASC therein are secreting new synthesized antibody specific for an ongoing antigenic insult.
  • media substantially free of preexisting antibody refers to media where the amount of contaminating pre-existing plasma antibodies in the media is reduced at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 10 3 , 2xl0 3 , 3xl0 3 , 4xl0 3 , 5xl0 3 , 6xl0 3 , 7xl0 3 , 8xl0 3 , 9xl0 3 , 10 4 , 10 s , 10 6 , 10 7 -fold relative to whole blood, plasma, or PBMC of the biological sample from which the newly proliferating ASC were obtained.
  • the “media substantially free of preexisting antibody,” can refer to media where any contaminating pre-existing plasma antibody has been reduced at least 0.76-3.90 mg/dL when the contaminating pre-existing plasma antibody is IgA; at least 6.50-15.00 mg/dL when the contaminating pre-existing plasma antibody is IgG; or at least 0.40-3.45 mg/dL when the contaminating pre-existing plasma antibody is IgM.
  • the specific IgG subclass is IgGl
  • the contaminating pre- existing plasma antibody can be reduced at least 3.41-8.94 mg/dL.
  • the specific IgG subclass is IgG2
  • the contaminating pre-existing plasma antibody can be reduced at least 1.71-6.32 mg/dL.
  • the contaminating pre-existing plasma antibody can be reduced at least 0.184-1.060 mg/dL.
  • the specific IgG subclass is IgG4 the contaminating pre-existing plasma antibody can be reduced at least 0.024-1.210 mg/dL.
  • MENSA can function as a new serologic surrogate with similarly high specificities of antibodies, but with several major advantages.
  • the pathogen-specific ASCs require only a single time point and can identify patients during the acute illness. Since the “historic or old” contaminating antibodies found in plasma are removed and only the newly synthesized antibodies measured from newly proliferated ASC, only antibodies or immune reactions to the current illness are measured.
  • Another advantage of measuring MENSA over insensitive low affinity IgM measurements is reliability.
  • MENSA can detect all antibody isotypes such as high affinity IgG or IgA as well as low affinity IgM antibodies which can increase its reliability.
  • MENSA is free from substances that interfere with clinical assays including but not limited to pharmaceutical agents and nonpharmaceutical drugs, lipemia, icterus, bile salts, hemoglobin, heterophilic antibodies, autoimmune antibodies, vitamins, antioxidants, and nutritional supplements.
  • MENSA can be cell free comprising antibodies and media which provides for compatibility with a large number of immune-analytical readouts that do not work with non-cell free samples.
  • MENSA as disclosed herein provides a snapshot of a moment in time and because it is not hampered by elevated levels of pre-existing antibodies (indeed the processing to generate MENSA removes said antibodies), MENSA can be used to detect persistent and latent viral infections.
  • the ASC Elispots used herein measure microbe- specific antibodies and not cytokines as known with commercially available IFNy cytokine Elispot assays. While ASC Elispots are well established as a research tool, herein is the first to demonstrate the striking detection potential of MENSA from circulating ASC for persistent and latent viral infections. Accordingly, these methods are the first reliable rapid immune-based assay that yields high pathogen- specific diagnostic sensitivity and specificity at initial presentation thereby providing real-time information for treatment and quarantine decisions.
  • MENSA final elaboration fluid
  • MENSA is separated from contaminating preexisting antibody (typically present in the plasma) and other circulating plasma cell and B- cell populations.
  • the separation of MENSA from the contaminating pre-existing antibody (typically present in the plasma) and other circulating plasma cell and B-cell populations can comprise any means known in the art including but not limited to magnetic bead cell sorting, FACS, and ficoll gradient separation.
  • the methodology used to separate the plasma from the newly proliferated ASC can actively separate either component so long as the end result is the removal of contaminating pre-existing antibody (and other circulating plasma cell and B-cell populations from the newly proliferated ASC.
  • a major candidate to improve the separation step is to capture the newly proliferated ASCs using magnetic beads bearing one, two, three, four, five, six, or more antibodies specific for cell surface markers unique to ASCs, specifically CD19, CD138, CD27, IgD, Ki67, and/or CD38.
  • CD19, CD138, CD27, IgD, Ki67, and/or CD38 can also be used in FACS sorting of the newly proliferated ASC.
  • isolation will result in a 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 10 3 , 2xl0 3 , 3xl0 3 , 4xl0 3 , 5xl0 3 , 6xl0 3 , 7xl0 3 , 8xl0 3 , 9xl0 3 , 10 4 , 10 5 , 10 6 , 10 7 -fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
  • the purity of the sample can comprise at least a 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 10 3 , 2xl0 3 , 3xl0 3 , 4xl0 3 , 5xl0 3 , 6xl0 3 , 7xl0 3 , 8xl0 3 , 9xl0 3 , 10 4 , 10 s , 10 6 , 10 7 -fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC. It is understood and herein contemplated that there are many mechanisms that can be used to separate the newly proliferated ASC from plasma.
  • cell surface markers for separating newly proliferated ASC include but are not limited to CD38, CD27, CD19, CD138, IgD, and Ki67.
  • methods of isolating MENSA comprising obtaining whole blood or PBMC from a subject, separating the plasma from the newly proliferated ASC using magnetic bead separation comprising one, two, three, four, five, or six or more of anti- CD38, anti- CD27, anti- CD19, anti- CD138, anti- IgD, or anti- Ki67 beads, and incubating the newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion.
  • the media should comprise sugars and amino acids needed for protein synthesis, newly activated ASC, and at least a two, three, four, five, six, seven, eight, nine, or ten log reduction in plasma or serum from the subject.
  • MENSA may contain survival factors such as IL-2, IL-6, IL-15, IL-21, and IFN-a, APRIL, enhancers of antibody secretion such as IL-21, other nonantibody secreting cells such as T cells or macrophage, but not red blood cells.
  • the MENSA may also contain magnetic beads or compounds needed for rapid separation of ASC from whole blood, PBMC, or plasma.
  • the MENSA is obtained by obtaining whole blood or PBMC from a subject; separating plasma from the whole blood or PBMC to produce separated cells; isolating newly proliferated ASC from the separated cells (such as, for example, using magnetic bead separation, ficoll gradient separation, elutriation, or FACS including, but not limited to the use of antibodies specific for one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and Ki67); washing the newly proliferated ASC; and incubating the washed newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion to thereby producing MENSA; wherein the MENSA comprises at least a 10 6 or 10 7 -fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
  • the persistent and/or latent viral antigen specific MENSA can be detected by any immunoassay known in the art including, but not limited to ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD.
  • immunoassays are enzyme linked immunosorbent assays (ELISAs), enzyme linked immunospot assay (ELISPOT), radioimmunoassays (RIA), radioimmune precipitation assays (RIP A), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/ FLAP).
  • ELISAs enzyme linked immunosorbent assays
  • ELISPOT enzyme linked immunospot assay
  • RIA radioimmunoassays
  • RIP A radioimmune precipitation assays
  • immunobead capture assays Western blotting, dot blotting, gel-shift assays
  • Flow cytometry protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, flu
  • immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed biomarkers) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed biomarkers) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • a molecule of interest such as the disclosed biomarkers
  • an antibody to a molecule of interest such as antibodies to the disclosed biomarkers
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed biomarkers or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process.
  • a molecule of interest such as the disclosed biomarkers or their antibodies
  • the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S.
  • a label can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence.
  • a fluorescent dye also known herein as fluorochromes and fluorophores
  • enzymes that react with colorimetric substrates (e.g., horseradish peroxidase).
  • colorimetric substrates e.g., horseradish peroxidase
  • each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.
  • Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1 ,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4- methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- 1 methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine (ACMA); AB
  • APTRA-BTC APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAGTM CBQCA; ATTO-TAGTM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBOTM -1; BOBOTM-3; Bodipy492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bo
  • Hydroxystilbamidine (FluoroGold); Hydroxy tryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;
  • mBBr-GSH Monobromobimane
  • MPS Metal Green Pyronine Stilbene
  • NBD NBD Amine
  • Nile Red Nitrobenzoxedidole
  • Nuclear Fast Red i Nuclear Yellow
  • a modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation.
  • radionuclides useful in this embodiment include, but are not limited to, tritium, iodine- 125, iodine- 131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18.
  • the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker.
  • radionuclides useful in the aspect include, but are not limited to, Tc-99m, Re- 186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry.
  • immunoassays that utilize electrophoresis to separate and detect the specific proteins of interest allow for evaluation of protein size, they are not very sensitive for evaluating protein concentration.
  • immunoassays wherein the protein or antibody specific for the protein is bound to a solid support (e.g., tube, well, bead, or cell) to capture the antibody or protein of interest, respectively, from a sample, combined with a method of detecting the protein or antibody specific for the protein on the support.
  • a solid support e.g., tube, well, bead, or cell
  • examples of such immunoassays include Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexed bead assay, and magnetic capture.
  • Radioimmunoassay is a classic quantitative assay for detection of antigenantibody reactions using a radioactively labeled substance (radioligand), either directly or indirectly, to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Radioimmunoassay is used, for example, to test hormone levels in the blood without the need to use a bioassay. Non-immunogenic substances (e.g., haptens) can also be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation.
  • carrier proteins e.g., bovine gamma-globulin or human serum albumin
  • Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha. -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In this assay a nitrocellulose microtiter plate is coated with antigen.
  • Enzyme-Linked Immunosorbent Assay is an immunoassay that can detect an antibody specific for a protein.
  • a detectable label bound to either an antibody-binding or antigenbinding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha. -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • ELISA procedures see Voller, A. et al., J.
  • ELISA techniques are know to those of skill in the art.
  • antibodies that can bind to proteins can be immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a marker antigen can be added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen can be detected. Detection can be achieved by the addition of a second antibody specific for the target protein, which is linked to a detectable label.
  • ELISA is a simple “sandwich ELISA.” Detection also can be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • competition ELISA Another variation is a competition ELISA.
  • test samples compete for binding with known amounts of labeled antigens or antibodies.
  • the amount of reactive species in the sample can be determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
  • ELIS As have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunecomplexes.
  • Antigen or antibodies can be linked to a solid support, such as in the form of plate, beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody.
  • a solid support such as in the form of plate, beads, dipstick, membrane or column matrix
  • any remaining available surfaces of the wells can then be “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera.
  • a nonspecific protein that is antigenically neutral with regard to the test antisera.
  • these include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure can also be used.
  • the immobilizing surface is contacted with the control clinical or biological sample to be tested under conditions effective to allow immunecomplex (antigen/antibody) formation.
  • Detection of the immunecomplex then requires a labeled secondary binding agent or a secondary binding agent in conjunction with a labeled third binding agent.
  • Under conditions effective to allow immunecomplex (antigen/antibody) formation means that the conditions include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween so as to reduce non-specific binding and to promote a reasonable signal to noise ratio.
  • solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween so as to reduce non-specific binding and to promote a reasonable signal to noise ratio.
  • the suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be from about 1 minute to twelve hours, at temperatures of about 20° to 30° C, or can be incubated overnight at about 0° C to about 10° C.
  • the contacted surface can be washed so as to remove non-complexed material.
  • a washing procedure can include washing with a solution such as PBS/Tween or borate buffer. Following the formation of specific immunecomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunecomplexes can be determined.
  • Protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles.
  • the assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems.
  • Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc.
  • the capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.
  • Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDotsTM, Quantum Dot, Hayward, CA), and barcoding for beads (UltraPlexTM, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., NanobarcodesTM particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).
  • Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to.
  • a good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems.
  • the immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity.
  • Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein.
  • Both covalent and noncovalent methods of protein immobilization are used and have various pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable.
  • Covalent coupling methods provide a stable linkage, can be applied to a range of proteins and have good reproducibility; however, orientation may be variable, chemical derivatization may alter the function of the protein and requires a stable interactive surface.
  • Biological capture methods utilizing a tag on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the biological reagent must first be immobilized adequately and the array may require special handling and have variable stability.
  • Fluorescence labeling and detection methods are widely used.
  • the same instrumentation as used for reading DNA microarrays is applicable to protein arrays.
  • capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance.
  • Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences).
  • TSA tyramide signal amplification
  • Planar waveguide technology Zeptosens
  • High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot).
  • Luminex phycoerythrin as label
  • Quantum Dot semiconductor nanocrystals
  • HTS Biosystems Intrinsic Bioprobes, Tempe, AZ
  • rolling circle DNA amplification Molecular Staging, New Haven CT
  • mass spectrometry Intrinsic Bioprobes; Ciphergen, Fremont, CA
  • resonance light scattering Gene Sciences, San Diego, CA
  • BioForce Laboratories atomic force microscopy
  • Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.
  • high affinity capture reagents such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.
  • Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli. after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids or engineered human equivalents (Domantis, Waltham, MA) may also be useful in arrays.
  • ligand-binding domains of proteins which are engineered into multiple variants capable of binding diverse target molecules with antibodylike properties of specificity and affinity.
  • the variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display.
  • ligand-binding scaffolds or frameworks include ‘Affibodies’ based on Staph, aureus protein A (Affibody, Bromma, Sweden), ‘Trinectins’ based on fibronectins (Phylos, Lexington, MA) and ‘Anticalins’ based on the lipocalin structure (Pieris Proteolab, Freising - Weihenstephan, Germany). These can be used on capture arrays in a similar fashion to antibodies and may have advantages of robustness and ease of production.
  • Nonprotein capture molecules notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO).
  • Aptamers are selected from libraries of oligonucleotides by the SelexTM procedure and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the cross reactivity of aptamers due to the specific steric requirements.
  • Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains can be used to detect binding. Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labelling is used for comparison of different samples with different colors. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label- free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand.
  • An alternative to an array of capture molecules is one made through ‘molecular imprinting’ technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrintTM, Aspira Biosystems, Burlingame, CA).
  • ProteinChip® array (Ciphergen, Fremont, CA), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins.
  • protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulfide bridges.
  • High- throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilised on a microarray.
  • Large-scale ‘proteome chips’ promise to be very useful in identification of functional interactions, drug screening, etc. (Proteometrix, Branford, CT).
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1987)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the antibodies are generated in other species and “humanized” for administration in humans.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity.
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993) and Chothia et al., J. Mol. Biol., 196:901 (1987)).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences.
  • Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679, published 3 March 1994).
  • hybridoma cells that produces the monoclonal antibody.
  • monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988).
  • a hybridoma method a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the generation of monoclonal antibodies has depended on the availability of purified protein or peptides for use as the immunogen.
  • DNA-based immunization can be used, wherein DNA encoding a portion of an antigen expressed as a fusion protein with human IgGl is injected into the host animal according to methods known in the art (e.g., Kilpatrick KE, et al. Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Fit- 3 receptor. Hybridoma. 1998 Dec;17(6):569-76; Kilpatrick KE et al. High-affinity monoclonal antibodies to PED/PEA-15 generated using 5 microg of DNA. Hybridoma. 2000 Aug;19(4):297-302, which are incorporated herein by referenced in full for the methods of antibody production) and as described in the examples.
  • immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment.
  • ELISPOT and solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • the binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • Serology is useful when virus isolation is negative or inadequate and is the only available test using the host immune response.
  • IgM serology offers low diagnostic yields with frequent false positives, and a single IgG level is not helpful in diagnosing secondary respiratory infections in adults because they require longitudinal »4-fold rises to determine a new infection.
  • serology has limited clinical utility and is only used for retrospective diagnosis since both an acute and convalescent sample is necessary. This limitation is particularly relevant to adults with history of multiple influenza infections in whom increases of strain specific antibody titers must be interpreted with caution. So, serology is helpful for epidemiological studies but is rarely used in clinical management.
  • the subject has prior antigen exposure, but is not presently experiencing latent reactivation can be useful.
  • the method further comprises measuring serum antibody levels specific for the same antigens as assayed for MENSA.
  • a therapeutic goal can be the treatment of the subject to alleviate, reduce, and or eliminate the effects of the persistent or latent viral infection including, but not limited to Long-COVID or PASC including, but not limited to a reduction, decrease, and or elimination of a persistent or latent virus.
  • methods of detecting a persistent or latent viral infection wherein when a persistent or latent viral infection is detected, the method further comprises the administration of a therapeutically effective amount of an antiviral therapy to the subject.
  • a subsequent acute viral infection such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-
  • a virus such as, for example, Herpes Simplex virus- 1 (HSV-l)(such as, for example, glycoprotein gG-1 and glycoprotein D), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein gG-2 and glycoprotein D), Varicella-Zoster virus (glycoprotein E, glycoprotein B, glycoprotein H, and glycoprotein L), Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl 8,
  • a subsequent acute viral infection of any preceding aspect wherein the MENSA is detected by immunoassay (such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD).
  • immunoassay such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2,
  • kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • the kits can include magnetic beads or antibodies for use in separating newly proliferated ASC from plasma such as anti-CD38, anti-CD19, or anti-Cd27 antibodies or magnetic beads as well as the necessary labware to perform the isolation. Kits can also include media and enhancers to stimulate antibody production in MENSA.
  • kits can include antigens to coat the wells of microtiter plates for diagnosis, efficacy, or biodetection assays embodied in some of the methods, as well as the primary antibody, and reagents required to detect the antibody as intended. It is further understood that the kit can further comprise secondary antibodies and assay support structures such as, for example, microtiter plates.
  • kits for detecting the presence of a persistent or latent viral infection such as, for example, an infection with Herpes Simplex virus- 1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea vims (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS- CoV), Severe Acute Respiratory Syndrome (SARS) -Coronavirus (CoV)-2 (SARS
  • the disclosed kits can further comprise instructions directing the practitioner to wash the newly proliferated and separated ASC sufficiently to obtain at least a two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 10 3 , 2xl0 3 , 3xl0 3 , 4xl0 3 , 5xl0 3 , 6xl0 3 , 7xl0 3 , 8xl0 3 , 9xl0 3 , 10 4 , 10 5 , 10 6 , 10 7 -fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC. Additionally, the disclosed kits can comprise a washing solution as disclosed herein to perform the washing.
  • an antibody reagent kit comprising containers of the monoclonal antibody or fragment thereof and one or more reagents for detecting binding of the antibody or fragment thereof to an antigen.
  • the reagents can include, for example, fluorescent tags, enzymatic tags, or other tags.
  • the reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that can be visualized.
  • the reagents can further include a microtiter plate with nitrocellulose wells. Also disclosed herein are kits, wherein the kit further comprises a Luminex microsphere.
  • kits may detect the presence of an antigen from a latent of persistent viral infection and thereby the presence of a latent or viral infection
  • the kit can further comprise one or more reagents to obtain whole blood or PBMC, one or more reagents to separate newly proliferated ASC from plasma (including, but not limited to one or more antibodies specific one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and/or Ki67), media to incubate the newly proliferated ASC, one or more antigens, and/or one or more reagents for detecting the presence of antibodies secreted by the newly proliferated ASC.
  • kits disclosed herein can comprise any array of panels to which for the detection of a persistent or viral infection or detecting a latent or persistent viral infection that is involved in Long-COVID or post-acute sequelae of SARS-CoV-2 infection (PASC).
  • the kit can specifically include a panel of antigens from SARS-CoV-2 and one or more of EBV, CMV, and/or HSV-2.
  • Example 1 MENSA to Identify SARS-CoV-2 Persistence and Latent Viral Reactivation in Long-COVID a) Results
  • MENSA reactivity was greater overall in the PASC group than in the CR group (Fig. 7A).
  • EBV more PASC patients had positive MENSA samples 22/60 (37%) compared to CR subjects 4/23 (17%).
  • a MENSA test was scored positive if any one of the 3 antigens (EBNA1, VCA, and gB350) was positive. Nearly all individuals in the general population have been exposed to EBV and our results were consistent with this finding.
  • EBV serologies are positive for 59/60 (98%) in the PASC group and all 23 CR samples.
  • MENSA assays are positive in 47% (28/60) of the PASC patients for EBV, CMV, and/or HSV2 whereas only 17% (4/23) are positive in the CR cohort.
  • a positive MENSA for SARS2 in PASC patients demonstrates ongoing new immune responses consistent with a reservoir for the persistence of SARS2 virus.
  • a positive MENSA for any of the 3 herpes viruses also demonstrates reactivation of latent EBV, CMV, and HSV2 identifying underlying viral triggers in this condition.
  • MENSA Since MENSA measures antibodies only from these newly-minted ASC and not from old LLPC, MENSA antibodies provide a unique antibody signature to reveal the cause of the present-day illness. We show that, during convalescence, the MENSA responses become negative because memory B cells are no longer differentiating into ASC and released into the blood. Thus, MENSA offers an immune snapshot to uncover the sources of the patient’ s ailment.
  • SARS2 and latent herpes viruses such as EBV, CMV, and HSV2 due to strong T cell responses that mediate rapid viral clearance.
  • SARS2 antigen assays have been shown to identify the spike protein, but quantities are extremely low and require ultrasensitive assays which carry a high risk of false positives.
  • Autopsies up to 230 days after acute SARS2 infection detected SARS2 RNA in multiple tissues such as the gut, central nervous system (CNS), muscle, myocardium, and the respiratory tract demonstrating viral reservoirs.
  • measuring the MENSA has advantages over pathogen detection by PCR amplification or protein since MENSA is in the blood, agnostic to viral reservoir locations, and would not require invasive tissue sampling. Since MENSA culminates from the total newly-minted ASC traveling in the blood during acute illness, knowledge of the viral reservoir location is not necessary since the MENSA reveals infections in deep-seated sites similar to a liquid biopsy.
  • Breakthrough or repeat infections can be diagnosed with serum assays, but they typically require serial blood samples during acute infection and convalescence for comparison. Another advantage of the MENSA over serum is that only a single blood sample during illness is needed. The decline of MENSA to negative levels after infection or vaccination demonstrates its clinical utility in measuring secondary or breakthrough infections.
  • MENSA antibodies are also exact in that they can distinguish infection from vaccination based on spike and the nucleocapsid proteins in some patients. This specificity and sensitivity along with the kinetics make MENSA an ideal diagnostic platform to reveal viral triggers that were previously difficult to measure with just serum or PCR tests. The MENSA diagnostic would be the first of its kind to understand the main viral drivers of this chronic disease.
  • MENSA antibodies are expected to peak within days after exposure, and then quickly decline back to baseline within a month after the infection has resolved. Interestingly, CR MENSA does not revert to pre-pandemic baseline levels and these mechanisms are not clear. Perhaps non-specific plasma antibody binding to monocytes in the MENSA cultures may be the reason and will require more studies. Another possibility is low-level bystander responses which have been suggested to explain the difference between pre- and post-pandemic samples. Interestingly, even when using the post-pandemic MENSA samples as controls, 60% of the PASC patients have higher SARS2, EBV, CMV, and HSV2 responses in the MENSA.
  • the real-time immune snapshots provided by MENSA may be leveraged to inform therapeutic strategies and successful treatment of chronically ill PASC patients. Whether MENSA can also be useful to identify persistence of viral reservoirs in other chronic illnesses such as multiple sclerosis, HIV, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and other infections with post-sequelae are yet to be determined.
  • EBV was recently implicated in multiple sclerosis.
  • infection with SARS2 is associated with increased susceptibility and severity of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and dementia but interpreting these correlations has been difficult. If MENSA can lead to early diagnosis of these chronic neurodegenerative disorders or help identify the cause of these disease flares, perhaps treatments may prove more effective in preventing progression and severity of these pathological conditions.
  • PASC can include symptoms, such as dyspnea, fatigue, and depression, and serious clinical indications, such as cardiovascular disease or diabetes. Recent studies also suggest increased risk for autoimmune inflammatory rheumatic diseases in PASC and CR patients. Future clinical trials are necessary to perform proof-of-concept studies where MENSA data could be used to inform treatment modalities for mitigating symptoms associated with SARS2 viral persistence, reactivation of viruses, reactivation of viruses in other chronic illnesses, or early activation of other chronic illnesses.
  • MENSA MEN-derived mono-nuclear cells
  • ASC antibody- secreting cells
  • PBMC peripheral blood mono-nuclear cells
  • Peripheral blood samples were collected in sodium heparin tubes and PBMC were isolated by centrifugation (1,000 xg; 10 min) using Lymphocyte Separation Media (Coming) and Leucosep tubes (Greiner Bio-One).
  • RPMI-1640 Five washes with RPMI-1640 (Coming) were performed to remove serum immunoglobulins (800 x g; 5 min) with erythrocyte lysis (3 mL; 3 min), and harvested PBMCs were cultured at 10 6 cells/mL in R10 Medium (RPMI-1640, 10% Sigma FBS, 1% Gibco Antibiotic/ Anti-mycotic) for 24 h at 37° C and 5% CO2. After incubation, the cell suspension was centrifuged (800 xg; 5 min), and the supernatant (MENSA) was separated from the PBMC pellet, aliquoted, and stored at -80°C for testing.
  • MENSA supernatant
  • Antigens of interest were selected from literature, coupled to Luminex MagPlex Microspheres of spectrally distinct regions via carbodiimide coupling, and tested for antigen specific IgG reactivity against patient samples as previously described.
  • SARS-CoV-2 Spike S I Receptor Binding Domain (RBD; catalog no. Z03483; expressed in HEK293 cells) and Nucleocapsid protein (N; catalog no. Z03480; expressed in Escherichia coli), were purchased from GenScript.
  • SI catalog no. S1N-C52H3; HEK293
  • S2 catalog no. S2N-C52H5; HEK293
  • SI N-terminal domain (NTD; catalog no. S1D- C52H6; HEK293) were purchased from ACROBiosystems.
  • ORF3a The C-terminus sequence of ORF3a (Accession: QHD43417.1, amino acids 134-275 plus N-terminal His6-Tag) was sent to Genscript for custom protein expression in E. coli. Each protein was expressed with an N-terminal His6-Tag to facilitate purification, at least 90% pure, and appeared as a predominant single band on SDS-PAGE analysis.
  • EBV, CMV, and HSV2 antigens were carefully selected for antigenicity based on previous reports.
  • the following proteins were used: EBV EBNA1 protein from Abeam (produced in E. coli, N-Terminus His Tag, CAT#abl38345); EBV VCA pl 8 from RayBiotech (produced in E.
  • EBV gp350 protein from AcroBiosystems (produced in HEK293 cells, His Tag, MALS verified, CAT#GP0-E52H6); CMV glycoprotein B from AcroBiosystems (strain AD169, expressed from HEK293 cells, His Tag, MALS verified, CAT#CMB-V52H4); CMV gH pentamer complex, consisting of gH, gL, UL128, UL130 and UL131A proteins, produced in mammalian HEK293 cells from The Native Antigen Company (CAT#CMV-PENT); HSV2 envelope glycoprotein D from AcroBiosystems (gD, expressed HEK293 cells, His Tag, MALS verified, CAT#GLD- V52H4).
  • Serum samples were tested at 1:500 dilution in assay buffer (1XPBS, 1% BSA) while MENSA samples were tested neat with no dilution. Results were analyzed on a Luminex FLEXMAP 3D instrument. Median fluorescent intensity (MFI) using phycoerythrin-conjugated detection antibodies (Goat Anti-Human IgG-PE, Southern Biotech cat. #2040-09) was measured for each sample using the Luminex xPONENT software on Enhanced PMT setting. The background value of assay buffer or R10 media was subtracted from the serum or MENSA results, respectively, to obtain MFI minus background (Net MFI). All samples were tested in duplicate and the average of the two results were used for analysis.
  • MFI Median fluorescent intensity
  • R10 media was subtracted from the serum or MENSA results, respectively, to obtain MFI minus background (Net MFI). All samples were tested in duplicate and the average of the two results were used for analysis.
  • SARS2 Serum Co was calculated as the average plus three standard deviations of the 16 Healthy Donor samples collected prior to SARS2 exposure. Since the majority of the population is expected to be positive for EBV, CMV, and/or HSV2 antibodies in their serum, we obtained de-identified clinically confirmed negative sera from the Emory clinical laboratory. For each virus, three confirmed negative serum samples were used.
  • T7 bacteriophage library consisting of 149,259 peptides tiling all protein-coding sequences from viruses with human hosts. Viral sequences were downloaded from Uniprot, collapsed on 90% identity, and bioinformatically parsed into 90 amino-acid peptide tiles with 45 amino-acid overlaps between adjacent tiles. Healthy and Covid patients’ plasma or serum and matched MENSA reactivities were profiled using phage immunoprecipitation and sequencing (PhlP-seq). MENSA samples were profiled in duplicate and plasma/serum samples in triplicate. PhlP-seq was performed as previously described with some modifications.
  • T7 bacteriophage libraries were aliquoted into 96-well plates and incubated with 20pl each of protein A and G Dynabeads on a rotator for 4 h at room temperature. Next, plates were placed on a magnet and supernatants were transferred to a fresh 96-well plate, to which we added patient plasma containing 2 pg of total IgG, and continued with the immunoprecipitation and washing steps, as previously described. Following the washes, protein A and protein G Dynabeads were resuspended in PCR master mix, amplified with 16 rounds of PCR, SPRI cleaned to remove primers, and indexed for sequencing with 8 rounds of PCR with primers containing Illumina p5 and p7 barcodes. NGS libraries were quantified on a Tapestation4200 and normalized for sequencing on Illumina Nextseq 2000 or Novaseq 6000 instruments. Each sequencing library received a minimum of 3M reads.
  • PhlP-seq single-end DNA sequences were aligned to a library of reference DNA sequences (149,259 75bp for viral) with the bowtie2 aligner (v2.0) using end-to-end matching.
  • Read counts were summarized using samtools (vl.14) and collated into a counts matrix. The raw counts were converted to counts per million (CPM) using the ‘cpm‘ function from the R package edgeR (v3.36.0).
  • CPM values for healthy controls were summarized by computing the peptide-wise mean and standard deviation across all healthy control samples.
  • CPM values for each patient sample were collapsed by computing the peptide-wise minimum across technical replicates.
  • Peptide-wise z-scores were then computed as: where Zi,j is the z-score for patient i, peptide j; Ci,j is the minimum CPM for patient i, peptide j; is the mean of peptide j in the healthy control samples, and 07 is the standard deviation of peptide j in the healthy control samples. For each patient, hits were identified as those peptides with cij > 10 AND ZIJ > 10.
  • Circulating antibody-secreting cells are a biomarker for early diagnosis in patients with Lyme disease.
  • Nakagawa, S. & Takahashi, M.U. gEVE a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes. Database (Oxford) 2016(2016).

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Abstract

Disclosed are methods and kits for the detection of persistent and latent viral infections using media elaborated with newly synthesized antibodies.

Description

METHODS OF DETECTING PERSISTENT AND LATENT VIRAL INFECTIONS USING MEDIA ENRICHED BY NEWLY SYNTHESIZED ANTIBODIES (MENSA)
I. CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 63/571,271, filed on March 28, 2024, which is incorporated herein by reference in its entirety.
IL STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under AI125180 awarded by the National Institutes of Health. The Government has certain rights in the invention.
III. BACKGROUND
In December 2019, the world was changed when the SARS-CoV-2 (SARS2) virus was identified in Wuhan, China and rapidly spread throughout the world. The first U.S. case was identified in January 2020, and by March 2020 the World Health Organization (WHO) had officially declared COVID-19 to be a global pandemic. By the end of 2020, there had been a total of 79 million reported cases and over 1.7 million deaths globally. Vaccines and antiviral therapies allowed us to combat this new global threat and emerge from this devastating pandemic. However, after many in the US received the primary mRNA vaccines, a new Delta virus surged, followed by the Omicron (B.1.1.529) variant by the end of 2021, which had increased transmissibility.
In addition to new viral variant infections, some patients suffered from sequelae after the initial acute infection. Long-COVID or post-acute sequelae of SARS-CoV-2 infection (PASC) is a condition described as ongoing, relapsing, or new symptoms present after the acute phase of the infection. Incidence of PASC was notable in approximately 10% of patients after acute infection. Definitions by the CDC used symptoms > 30 days after acute infection whereas the WHO described continuation or development of new symptoms 3 months after the initial SARS2 infection, with symptoms lasting for at least 2 months with no other explanation. The diversity of symptoms and differences in plasma proteomics between inflammatory and quiescent PASC subsets attest to the heterogeneity of long- COVID. Additionally, many multiomic studies have identified metabolic and inflammatory derangements such as decreased cortisol or serotonin levels, complement dysregulation, and alternations of cytotoxic T cell, atypical B cell, or neutrophil signatures in PASC patients compared to adults who recovered from SARS2 without sequelae. Some studies suggest that these inflammatory changes may be triggered by viral persistence of SARS2 and/or reactivation of latent EBV. Interestingly, these studies used viral PCR testing which is known to be less sensitive in the blood or requires ultrasensitive spike antigen tests. Another study showed higher serum levels of EBV antibodies in PASC patients (47%) compared to healthy adults (28%) but there was significant overlap between the two groups due to serum antibodies in response to a previous infection, confounding the observation of new or ongoing immune responses. Given the deficiencies of current detection methods, what are needed are new tests and testing methods for the detection of latent and persistent viral infection.
IV. SUMMARY
Disclosed herein are kits and compositions related to the detection of persistent and/or latent viral infections.
In one aspect, disclosed herein are kits for detecting the presence of a persistent or latent viral infection (such as, for example, an infection with Herpes Simplex virus- 1 (HSV- 1), Herpes Simplex virus-2 (HS V-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the Bl.351 variant, B.1.1.7 variant, and P.l variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2) in a subject comprising one or more viral antigens from a persistent or latent virus; wherein the latent or persistent virus is selected from the group consisting of Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein D), Varicella-Zoster virus, Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl8, and/or gp350), Cytomegalovirus (CMV)(such as, for example, glycoprotein B, and/or gH pentamer (such as, for example, gH, gL, UL128, UL130 and UL131A proteins), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2 (including, but not limited to the Bl.351 variant, B.1.1.7 variant, and P.l variant)(antigens such as, for example, SI Receptor Binding Domain (RBD), Nucleocapsid protein, SI, S2, SI N-terminal domain (NTD), and/or the C-terminus of 0RF3a), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.
Also disclosed herein are kits of any preceding aspect, wherein the kit further comprises a Luminex microsphere.
In one aspect disclosed herein are kits of any preceding aspect further comprising one or more reagents to obtain whole blood or PBMC, one or more reagents to separate newly proliferated ASC from plasma (including, but not limited to one or more antibodies specific one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and/or Ki67), media to incubate the newly proliferated ASC, one or more antigens, and/or one or more reagents for detecting the presence of antibodies secreted by the newly proliferated ASC.
In one aspect, disclosed herein are methods of detecting a persistent or latent viral infection (such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS (-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the B 1.351 variant, B.l.1.7 variant, and P.l variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2) in a subject comprising: a) obtaining media enriched by newly synthesized antibodies (MENSA) from the subject; b) contacting an antigen from a persistent or latent virus (such as, for example, Herpes Simplex virus- 1 (HSV-l)(such as, for example, glycoprotein gG-1 and glycoprotein D), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein gG-2 and glycoprotein D), Varicella-Zoster virus (glycoprotein E, glycoprotein B, glycoprotein H, and glycoprotein L), Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl8, and/or gp350), Cytomegalovirus (CMV)(such as, for example, glycoprotein B, and/or gH pentamer (such as, for example, gH, gL, UL128, UL130 and UL131A proteins), Human Herpes virus-6 (such as, for example Ul i, U24, U94, glycoprotein H, glycoprotein Q), Variola virus, Hepatitis B virus (such as, for example Hepatitis B surface antigen (HbsAg)), Hepatitis C virus (such as, for example, Hepatitis C virus core antigen (HCVcAg)), Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15)(such as, for example SI), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)- 229E (such as, for example SI), HCoV-OC43 (such as, for example SI), HCoV-HKUl (such as, for example SI), HCoV-NL63 (such as, for example SI), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV) )(antigens such as, for example, SI Receptor Binding Domain (RBD), SI, and S2), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2 (including, but not limited to the Bl.351 variant, B.1.1.7 variant, and P.l variant)(antigens such as, for example, S I Receptor Binding Domain (RBD), Nucleocapsid protein, SI, S2, SI N-terminal domain (NTD), and/or the C-terminus of 0RF3a), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)(such as, for example the nucleocapsid protein (NP)), Measles virus (such as, for example the nucleocapsid protein (NP)), Polyomavirus (such as, for example the SV40 large T antigen), Human Papillomavirus (such asl for example LI, E6, and E7), Adenovirus (such as, for example the hexon protein), Human T-cell Leukemia virus type-1 (such as, for example, gp21, p24, pl9, and gp46), Rubella virus (such as, for example spike glycoprotein El and capsid nucleoprotein), Simian Immunodeficiency virus (such as, for example p27), Human Immunodeficiency virus type-1 (such as, for example p24), and Human Immunodeficiency virus type-2 (such as, for example p24)) with the MENSA; and c) detecting the presence of MENSA bound to the antigen; wherein the presence of MENSA bound to an antigen indicates the presence of a persistent or latent viral infection.
Also disclosed herein are methods of detecting a persistent or latent viral infection of any preceding aspect, wherein the MENSA is obtained by obtaining whole blood or PBMC from a subject; separating plasma from the whole blood or PBMC to produce separated cells; isolating newly proliferated ASC from the separated cells (such as, for example, using magnetic bead separation, ficoll gradient separation, elutriation, or FACS including, but not limited to the use of antibodies specific for one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and Ki67); washing the newly proliferated ASC; and incubating the washed newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion to thereby producing MENSA; wherein the MENSA comprises at least a 106 or 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
In one aspect, disclosed herein are methods of detecting a persistent or latent viral infection of any preceding aspect, wherein the MENSA is detected by immunoassay (such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD).
Also disclosed herein are methods of detecting a persistent or latent viral infection of any preceding aspect, wherein the method further comprises measuring serum antibody levels specific for the same antigens as assayed for MENSA.
In one aspect, disclosed herein are methods of detecting a persistent or latent viral infection of any preceding aspect, wherein the method further comprises assessing any symptoms of infection that the subject is experiencing.
In one aspect, disclosed herein are methods of detecting a persistent or latent viral infection of any preceding aspect, wherein when a persistent or latent viral infection is detected, the method further comprises the administration of a therapeutically effective amount of an antiviral therapy to the subject.
Also disclosed herein are methods of detecting a subsequent acute viral infection (such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)- 229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)- Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the Bl.351 variant, B.1.1.7 variant, and P.l variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Hantavirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2) comprising a) obtaining media enriched by newly synthesized antibodies (MENSA) from the subject; b) contacting an antigen from a virus (such as, for example, Herpes Simplex virus- 1 (HSV-l)(such as, for example, glycoprotein gG-1 and glycoprotein D), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein gG-2 and glycoprotein D), Varicella-Zoster virus (glycoprotein E, glycoprotein B, glycoprotein H, and glycoprotein L), Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl 8, and/or gp350), Cytomegalovirus (CMV)(such as, for example, glycoprotein B, and/or gH pentamer (such as, for example, gH, gL, UL128, UL130 and ULI 31 A proteins), Human Herpes virus-6 (such as, for example U11, U24, U94, glycoprotein H, glycoprotein Q), Variola virus, Hepatitis B virus (such as, for example Hepatitis B surface antigen (HbsAg)), Hepatitis C virus (such as, for example, Hepatitis C virus core antigen (HCVcAg)), Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15)(such as, for example SI), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E (such as, for example SI), HCoV-OC43 (such as, for example SI), HCoV-HKUl (such as, for example SI), HCoV-NL63 (such as, for example SI), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV) )(antigens such as, for example, SI Receptor Binding Domain (RBD), SI, and S2), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2 (including, but not limited to the B1.351 variant, B.1.1.7 variant, and P.l variant)(antigens such as, for example, SI Receptor Binding Domain (RBD), Nucleocapsid protein, SI, S2, SI N- terminal domain (NTD), and/or the C-terminus of 0RF3a), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-C V)(such as, for example the nucleocapsid protein (NP)), Measles virus (such as, for example the nucleocapsid protein (NP)), Polyomavirus (such as, for example the SV40 large T antigen), Human Papillomavirus (such asl for example LI, E6, and E7), Adenovirus (such as, for example the hexon protein), Human T-cell Leukemia virus type-1 (such as, for example, gp21, p24, pl9, and gp46), Rubella virus (such as, for example spike glycoprotein El and capsid nucleoprotein), Simian Immunodeficiency virus (such as, for example p27), Vesicular stomatitis virus, Hepatitis A virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Influenza virus A, Influenza virus B, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Hantavirus, Human Immunodeficiency virus type-1 (such as, for example p24), and Human Immunodeficiency virus type-2 (such as, for example p24)) with the MENSA; and c) detecting the presence of MENSA bound to the antigen; wherein the presence of MENSA bound to an antigen indicates the presence of an acute viral infection.
In one aspect, disclosed herein are methods of detecting a subsequent acute viral infection of any preceding aspect, wherein the MENSA is detected by immunoassay (such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD).
Also disclosed herein are methods of detecting a subsequent acute viral infection of any preceding aspect, wherein when a persistent or latent viral infection is detected, the method further comprises the administration of a therapeutically effective amount of an antiviral therapy to the subject.
V. BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.
Figures 1 A, IB, 1C, and I D show primary wild type COVID- 19 infection responses in MENSA and serum. Dot plots 596 show IgG antibody reactivity against Sl-RBD in the MENSA (1A) and serum (IB) of patients 597 experiencing Wild Type SAR-CoV-2 infections in 2020. Similar results are also shown for anti598 Nucleocapsid IgG in the MENSA (1C) and serum (ID). Samples from patients with acute 599 Mild/Moderate (59 samples from 54 patients; blue dots) and Severe/Critical (60 samples from 56 600 patients; red dots) infections were collected less than 30 DPSO (acute) and/or after 60 DPSO 601 (convalescence). Early pandemic healthy controls, with no prior exposure to SARS2 (n=60), are 602 shown as black dots on the left of each panel. All units are represented as Median Fluorescent 603 Intensity minus background (Net MFI). Dashed lines indicate the CO threshold of positivity for 604 each sample type and antigen. Serum COs were calculated as the average Net MFI plus five 605 standard deviations of the 60 healthy controls (RBD: 1724; N: 682). For MENSA COs, a subset 606 of the convalescent patients was identified as a confirmed COVID Recovered (CR) population 607 (no sequelae; N=19) and was used as a contemporary control group to calculate the average 608 Net MFI plus 3 standard deviations (RBD: 570; N: 441). Pair-wise comparisons were performed 609 using the Kruskal-Wallis test in GraphPad Prism (unpaired, nonparametric test; ns p > 0.05, * p 610 < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Figures 2A, 2B, 2C, and 2D show primary vaccination responses in MENSA and serum. Dot plots show IgG 613 antibody reactivity against Sl-RBD in the MENSA (2A) and serum (2B) of subjects receiving their 614 primary COVID- 19 mRNA vaccination, with no prior infection. Similar results are also shown for 615 anti-nucleocapsid IgG in the MENSA (2C) and serum (2D). Samples were collected from 11 616 healthy adults prior to vaccination (Baseline; n=7), after Dose 1 Peak (9-20 DPV; n=8), Dose 2 617 Peak (6-12 DPV; n=8), Dose 3 Baseline (>80 DPV dose 2 through 0 DPV dose 3; n=6), and 618 Dose 3 Peak (4-12 DPV dose 3; n=7). All values are reported as average Net MFI (Median 619 Fluorescent Intensity - Background). Dashed lines indicate the Co threshold of positivity for
620 each sample type and antigen as determined in Figure 1. Pair- wise comparisons were
621 performed using the Kruskal- Wallis test in GraphPad Prism (unpaired, nonparametric test; ns p 622 > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Figures 3 A, 3B, 3C, and 3D show the kinetics of MENSA and serum after SARS2 infection and multiple vaccine 625 doses. Line graphs show MENSA (dark blue) and serum (light blue) IgG antibody responses to 626 Sl-RBD (3A and 3B) and Nucleocapsid (3C and 3D) over time for a single patient starting with an initial 627 primary SARS2 infection in 2020, through three doses of Pfizer COVID- 19 mRNA vaccination in 628 2021 , and a breakthrough Omicron infection in 2022. All values are reported as average Net 629 MFI (Median Fluorescent Intensity - Background). Red vertical dashed lines represent a new 630 exposure event. The primary and breakthrough infection events are symbolized as virions. Each 631 vaccination dose event is symbolized as a syringe. Horizontal dashed black lines represent the 632 Co threshold of positivity for each sample and antigen combination as determined from Figure 1.
Figure 4 shows the percentage of PASC patients with symptoms. Sixty PASC patients recruited in the Emory Long-COVID clinic with self-reported symptom questionnaires at enrollment. Percentages of each symptom is shown to the right of each bar. The twelve- symptom PASC scores are shown in black with point scores in parentheses after each symptom.
Figures 5A, 5B, 5C, and 5D show prolonged, elevated MENSA for SARS2 in a subset of PASC patients. Dot plots show MENSA and serum IgG antibody responses to Sl- RBD (5A and 5B) and 642 Nucleocapsid (5C and 5D) in samples collected between 60-279 DPSO since initial CO VID- 19 Wild 643 Type infection from patients who completely recovered from their acute illness (CR; n=19) and 644 patients who suffer PASC (n=39). Blue dots represent a Mild/Moderate acute disease severity. 645 Red dots represent a Severe/Critical acute disease severity. All values are reported as average 646 Net MFI (Median Fluorescent Intensity - Background). Dashed lines indicate the CO threshold of 647 positivity for each sample type and antigen combination as determined from Figure 1. Pair- wise 648 comparisons were performed using the Mann- Whitney test in GraphPad Prism (unpaired, 649 nonparametric test; ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Figure 6 shows Phage immunoprecipitation sequencing (PhlP-Seq) analysis of MENSA of PASC and CR patients for discovery. PhlP-seq analysis determines the level of binding of antibodies to 149,259 peptides tiling all protein-coding sequences from viruses with human hosts in MENSA samples from three CR (left column) and 10 PASC patients (middle column). Data are presented as z-scores of the anti-viral antibodies detected. Each row represents a linear peptide of viruses that were differentially bound in PASC and CR groups. Color code identifies virus species (right column).
Figures 7A and 7B show MENSA and serum in PASC, CR, and healthy adults for SARS2, EBV, CMV, and HSV2. MENSA (7 A) and serum (7B) samples were collected from healthy donors prior to SARS2 exposure (top), CO VID recovered (middle), and PASC (bottom) patients, tested for IgG reactivity against SARS2, EBV, CMV, and HSV2 antigens, and presented in a heat map. Green cells represent Net MFI values > the Co thresholds calculated for each sample type and antigen combination, while white or grey cells represent values below the Co. In MENSA, a Co was calculated as the average Net MFI of 22/23 CR samples plus three standard deviations for each antigen. Each SARS2 Serum Co was calculated as the average plus three standard deviations of the 16 Healthy Donor samples collected prior to SARS2 exposure. For each of the remaining viruses, three clinically confirmed negative serum samples were obtained as virus specific negative controls and the average Net MFI plus three standard deviations were used to calculate Co for each antigen.
Figures 8A and 8B show anti-SARS-CoV-2 IgG levels are higher in the MENSA and serum of infected patients than in unexposed healthy controls. MENSA (8A) and serum (8B) samples were collected from healthy adults during the early pandemic period, prior to any SARS-CoV-2 exposure, and from patients within 30 days post symptom onset (DPSO) of their primary wild type infections in 2020. All samples were tested for IgG reactivity against four spike-associated proteins (RBD, SI, S2, NTD) and two non-spike proteins (Nucleocapsid, ORF3a). All values are reported as average Net MFI (Median Fluorescent Intensity - Background). Pair-wise comparisons were performed using the Mann- Whitney test in GraphPad Prism (unpaired, nonparametric test; ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
Figures 9A and 9B show that MENSA anti-RBD IgG offers superior diagnostic value while MENSA anti-Nucleocapsid IgG offers a non-spike option for natural infection. Receiver operating characteristic (ROC) curves were performed for antiSARS-CoV-2 IgG levels in MENSA (9 A) and serum (9B) samples collected from healthy adults with no SARS-CoV-2 exposure and acute infected primary wild type patients. The Area Under the Curve (AUC) measurement provides an index of diagnostic potential. An AUC near 0.5 suggests no diagnostic value while an AUC near 1.0 indicates strong diagnostic potential.
Figures 10A, 10B, 10C, and 10D show the kinetics of MENSA and serum after three vaccine doses and a breakthrough infection. Line graphs show MENSA (dark blue) and serum (light blue) IgG antibody responses to Sl-RBD (10A and 10B) and Nucleocapsid (10C and 10D) over time for a single patient through two primary Modema COVID-19 mRNA vaccine doses in February-March 2021, a breakthrough Omicron infection in December 2021, and a third booster dose of mRNA COVID-19 vaccine in February 2022. All values are reported as average Net MFI (Median Fluorescent Intensity - Background). Red vertical dashed lines represent a new exposure event. Each vaccination dose event is symbolized as a syringe. The breakthrough infection event is symbolized as a virion. Horizontal dashed black lines represent the CO threshold of positivity for each sample and antigen combination as determined from Figure 1.
Figures 11 A, 1 IB, 11C, and 1 ID show the kinetics of MENSA and serum after four vaccine doses and a breakthrough infection. Line graphs show MENSA (dark blue) and serum (light blue) IgG antibody responses to Sl-RBD (11A and 1 IB) and Nucleocapsid (11C and HD) over time for a single patient through two primary Modema C0V1D-19 mRNA vaccine doses in January 2021, a third booster dose of mRNA COVID-19 vaccine in October 2021, a breakthrough Omicron infection in February 2022, and a fourth mRNA CO VID- 19 vaccine dose in April 2022. All values are reported as average Net MFI (Median Fluorescent Intensity - Background). Red vertical dashed lines represent a new exposure event. Each vaccination dose event is symbolized as a syringe. The breakthrough infection event is symbolized as a virion. Horizontal dashed black lines represent the CO threshold of positivity for each sample and antigen combination as determined from Figure 1.
VI. DETAILED DESCRIPTION
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Definitions
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.
The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
"Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.
"Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of” when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."
“Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
"Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
“Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
“Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
Media enriched by newly synthesized antibodies (MENSA)
Following antigen exposure from vaccination or infection, naive and/or memory B cells proliferate and differentiate into ASCs in lymph nodes. These ASCs are thought to be generated in activated lymph nodes, a compartment that is difficult to sample. These recently blasted ASCs leave the lymph node and burst into the circulation as they migrate to other tissue sites such as the bone marrow, spleen, or sites of inflammation presumably reflecting the migration of these effector cells to survival niches in the bone marrow and spleen. These highly informative cells are readily detectable from as little as 1-5 cc of blood and in the absence of measurable bystander effect on unrelated antigenic specificities. As opposed to historic plasma antibodies produced by resident bone marrow plasma cells, newly synthesized antibodies reflect an ongoing immune response as they are secreted from newly proliferated antibody secreting cells (ASC) that burst dramatically in the peripheral blood just a few days after acute infections and can persist in the circulation for several weeks. It is understood and herein contemplated that “antibody secreting cell” or “plasma cell” refers to any B lineage cell capable of secreting antibody including but not limited to plasmablasts, short-lived antibody secreting cells, long-lived plasma cell.
Media enriched by newly synthesized antibodies (MENSA) comprises “newly synthesized antibodies” directly elaborated from specialized cells during acute illness or following vaccination. MENSA differs from plasma in that the antibodies present in plasma are from long-lived plasma cells in the bone marrow and not newly synthesized antibodies. MENSA also differs from media containing PBMC in that there are no memory B cells present and the ASC therein are secreting new synthesized antibody specific for an ongoing antigenic insult. Thus, put another way, disclosed herein is media substantially free of preexisting antibody, but comprising newly synthesized antibodies from recently proliferating ASC in the blood. Accordingly, MENSA further differs from plasma antibody in that the antibody to MENSA, being essentially free of pre-existing antibody, is directed to a single antigen or foreign substance or organism; whereas, plasma antibody has antibodies specific to every prior antigenic experience. Moreover, MENSA is not merely isolated natural antibody or ASC but a mixture of media and at a minimum newly synthesized antibodies grown in culture in the absence of contaminating plasma antibodies and in that through the creation of the analytical matrix a properties and functions are gained such as the absence of pre-existing antibodies, the ability to detect recent antigenic or ongoing antigenic exposure; non of which can be accomplished with antibodies directly removed from a subject.
It is understood and herein contemplated that “media substantially free of preexisting antibody,” refers to media where the amount of contaminating pre-existing plasma antibodies in the media is reduced at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 103, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, 104, 10s, 106, 107-fold relative to whole blood, plasma, or PBMC of the biological sample from which the newly proliferating ASC were obtained. Alternatively, the “media substantially free of preexisting antibody,” can refer to media where any contaminating pre-existing plasma antibody has been reduced at least 0.76-3.90 mg/dL when the contaminating pre-existing plasma antibody is IgA; at least 6.50-15.00 mg/dL when the contaminating pre-existing plasma antibody is IgG; or at least 0.40-3.45 mg/dL when the contaminating pre-existing plasma antibody is IgM. Where the specific IgG subclass is IgGl the contaminating pre- existing plasma antibody can be reduced at least 3.41-8.94 mg/dL. Where the specific IgG subclass is IgG2 the contaminating pre-existing plasma antibody can be reduced at least 1.71-6.32 mg/dL. Where the specific IgG subclass is IgG3 the contaminating pre-existing plasma antibody can be reduced at least 0.184-1.060 mg/dL. Where the specific IgG subclass is IgG4 the contaminating pre-existing plasma antibody can be reduced at least 0.024-1.210 mg/dL. Put another way, in one aspect, disclosed herein are analytical matrixes comprising media elaborated with newly synthesized antibodies (MENSA) from recently proliferated antibody secreting cells (ASC) circulating in the blood; wherein the analytical matrix comprises at least a 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 103, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, 104, 10s, 106, 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or peripheral blood mononuclear cells (PBMC). It is understood and herein contemplated that the reduction of contaminating plasma antibodies, plasma cells, and in some cases neutrophils and red blood cells is an important aspect of the disclosed methods and failure to sufficiently remove the contaminants will affect the ability to perform the disclosed methods and obtain a reliable result. In one aspect, where reduction of contaminating preexisting plasma antibodies is claimed or discussed it is further contemplated a contemporaneous reduction in other contaminants such as red blood cells, plasma cells, neutrophils, and/or B cells.
MENSA can function as a new serologic surrogate with similarly high specificities of antibodies, but with several major advantages. First, the pathogen-specific ASCs require only a single time point and can identify patients during the acute illness. Since the “historic or old” contaminating antibodies found in plasma are removed and only the newly synthesized antibodies measured from newly proliferated ASC, only antibodies or immune reactions to the current illness are measured. Another advantage of measuring MENSA over insensitive low affinity IgM measurements is reliability. MENSA can detect all antibody isotypes such as high affinity IgG or IgA as well as low affinity IgM antibodies which can increase its reliability. Herein is shown that the following characteristics of MENSA as measured by ASC Elispots: (1) high pathogen-specificity with no bystander effect (2) specificity only during acute illness and not during asymptomatic periods 1 (3) detection at the time of initial clinical presentation. Third, MENSA is free from substances that interfere with clinical assays including but not limited to pharmaceutical agents and nonpharmaceutical drugs, lipemia, icterus, bile salts, hemoglobin, heterophilic antibodies, autoimmune antibodies, vitamins, antioxidants, and nutritional supplements. Fourth, in some aspects, it is contemplated that MENSA can be cell free comprising antibodies and media which provides for compatibility with a large number of immune-analytical readouts that do not work with non-cell free samples.
In this study, we use a novel diagnostic platform whereby we capture antibodies secreted from 116 plasmablasts or newly-minted antibody secreted cells (ASC). This method can identify new or 117 repeat infections despite elevated serum antibodies since these ASC appear in circulation shortly after infection or vaccination, then rapidly disappear from the bloodl6-20 21,22 118 . By capturing 119 antibodies from these special ASC in a new matrix called Media Enriched with Newly 120 Synthesized Antibodies (MENSA), we provide a signature response from only the new illness. 121 MENSA antibodies often appear prior to seroconversion and differ from serum antibodies, which 122 confound results with the patient’s entire historical microbial record. In prior studies, we have 123 successfully used MENSA to diagnose acute Lyme disease, Clostridioides difficile, Streptococcus pneumoniae, and deep bone/tissue infections with Staphylococcus aureus 19,23- 124 27 125 . In all, the novel MENSA assay can successfully diagnose repeat bacterial and viral infections 126 from a single blood sample even when serum antibody titers are extremely high, showing the 127 assay’s exceptional ability to resolve complexity of antibody signals. 128 129 Here, we provide proof-of-concept testing that the MENSA technology can capture the new host 130 immune response to accurately diagnose acute primary and breakthrough infections when 131 known SARS2 virus or proteins are present. It is also positive after vaccination when spike 132 proteins elicit an acute immune response. Applying the same principles for long-COVID 133 patients, we use MENSA to identify SARS2, EBV, CMV, and/or HSV2 as the underlying viral 134 drivers in 60% of PASC patients. With a single blood sample, this novel detection assay shows 135 persistence of SARS2 and/or reactivation of latent herpes viruses in long-COVID patients.
Methods of detecting persistent and/or latent viral infections
The presently described methods are the first in a category of reliable immunoassays at a single time point and thus methods disclosed herein measure MENSA found in the blood only during the acute illness. The problem combating persistent and latent viral infection in particular is the serum antibodies do not work as there is no way to distinguish preexisting antibodies that were generated from prior antigen exposure from antibodies that were generated in response to viral persistence or reactivation of a latent infection. Once generated, serum antibody levels remain high. By contrast, MENSA while antibody based has the surprising benefit of being present only in active infections. That is, it does not suffer the drawbacks of serum antibodies. Once an infection is controlled, MENSA levels fall below detection levels. Thus, MENSA as disclosed herein provides a snapshot of a moment in time and because it is not hampered by elevated levels of pre-existing antibodies (indeed the processing to generate MENSA removes said antibodies), MENSA can be used to detect persistent and latent viral infections. The ASC Elispots used herein measure microbe- specific antibodies and not cytokines as known with commercially available IFNy cytokine Elispot assays. While ASC Elispots are well established as a research tool, herein is the first to demonstrate the striking detection potential of MENSA from circulating ASC for persistent and latent viral infections. Accordingly, these methods are the first reliable rapid immune-based assay that yields high pathogen- specific diagnostic sensitivity and specificity at initial presentation thereby providing real-time information for treatment and quarantine decisions.
ASCs are circulating antibody-making cells that have been recently stimulated by ongoing infection. During an acute illness the total ASC increases to 2-20% of all B cells even though they constitute only <0.5% of B cell population in steady state. More importantly, 20 to 90% of ASCs are secreting antibodies specific for the infecting pathogen, resulting in a massive expansion of pathogen specific antibodies during illness. By isolating ASCs and measuring the secreted antibody products, two critical parameters are gained that make this technology superb for detection of latent and persistent viral infections. First, sensitivity is enhanced because the newly synthesized antibodies are relatively abundant and most are specific for the infecting pathogen. Second, specificity is improved by removing all of the contaminating pre-existing plasma antibodies among which are potentially confounding antibodies elicited by prior infections (from long-lived bone marrow resident plasma cells) and well-known interfering substances such as rheumatoid factor and heterophilic antibodies. This final elaboration fluid (MENSA) is a relatively “clean” which contains ONLY newly synthesized antibodies from the specialized ASC isolated from the blood.
In one aspect, disclosed herein are methods of detecting a persistent or latent viral infection (such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the Bl.351 variant, B.l.1.7 variant, and P.l variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type- 2) in a subject comprising: a) obtaining media enriched by newly synthesized antibodies (MENSA) from the subject; b) contacting an antigen from a persistent or latent virus (such as, for example, Herpes Simplex virus- 1 (HSV-l)(such as, for example, glycoprotein gG-1 and glycoprotein D), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein gG-2 and glycoprotein D), Varicella-Zoster virus (glycoprotein E, glycoprotein B, glycoprotein H, and glycoprotein L), Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl8, and/or gp350), Cytomegalovirus (CMV)(such as, for example, glycoprotein B, and/or gH pentamer (such as, for example, gH, gL, UL128, UL130 and UL131A proteins), Human Herpes virus-6 (such as, for example Ull, U24, U94, glycoprotein H, glycoprotein Q), Variola virus, Hepatitis B virus (such as, for example Hepatitis B surface antigen (HbsAg)), Hepatitis C virus (such as, for example, Hepatitis C virus core antigen (HCVcAg)), Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15)(such as, for example SI), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)- 229E (such as, for example SI), HCoV-OC43 (such as, for example SI), HCoV-HKUl (such as, for example SI), HCoV-NL63 (such as, for example SI), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV) )(antigens such as, for example, SI Receptor Binding Domain (RBD), SI, and S2), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2 (including, but not limited to the Bl.351 variant, B.l.1.7 variant, and P.l variant)(antigens such as, for example, SI Receptor Binding Domain (RBD), Nucleocapsid protein, SI, S2, SI N-terminal domain (NTD), and/or the C-terminus of 0RF3a), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)(such as, for example the nucleocapsid protein (NP)), Measles virus (such as, for example the nucleocapsid protein (NP)), Polyomavirus (such as, for example the SV40 large T antigen), Human Papillomavirus (such asl for example LI, E6, and E7), Adenovirus (such as, for example the hexon protein), Human T-cell Leukemia virus type-1 (such as, for example, gp21, p24, pl9, and gp46), Rubella virus (such as, for example spike glycoprotein El and capsid nucleoprotein), Simian Immunodeficiency virus (such as, for example p27), Human Immunodeficiency virus type-1 (such as, for example p24), and Human Immunodeficiency virus type-2 (such as, for example p24)) with the MENSA; and c) detecting the presence of MENSA bound to the antigen; wherein the presence of MENSA bound to an antigen indicates the presence of a persistent or latent viral infection.
To isolate the MENSA analyte, MENSA is separated from contaminating preexisting antibody (typically present in the plasma) and other circulating plasma cell and B- cell populations. The separation of MENSA from the contaminating pre-existing antibody (typically present in the plasma) and other circulating plasma cell and B-cell populations can comprise any means known in the art including but not limited to magnetic bead cell sorting, FACS, and ficoll gradient separation. Moreover, the methodology used to separate the plasma from the newly proliferated ASC can actively separate either component so long as the end result is the removal of contaminating pre-existing antibody (and other circulating plasma cell and B-cell populations from the newly proliferated ASC. For example, the methodology could seek to remove plasma from the ASC such as a ficoll gradient and washing. Alternatively, the methodology could use markers on ASC for use on a column, filter, or sorting mechanism. Thus, it is understood and herein contemplated that whether any method disclosed herein recites that plasma is separated from the ASC or the ASC are separated from the plasma, there is no implied target of separation. Merely, what is meant is that resulting ASC will be free of plasma or B cell contaminant. When performed, the resulting analyte can comprise 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 103, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, 104, 10s, 106, 107- fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
A major candidate to improve the separation step is to capture the newly proliferated ASCs using magnetic beads bearing one, two, three, four, five, six, or more antibodies specific for cell surface markers unique to ASCs, specifically CD19, CD138, CD27, IgD, Ki67, and/or CD38. One or more of CD19, CD138, CD27, IgD, Ki67, and/or CD38 can also be used in FACS sorting of the newly proliferated ASC.
Whole blood and PBMC CD 19+ isolation was less efficient than density purified CD38+ enriched selections for both total IgG and influenza- specific ASC frequencies per mL of blood. This result is not surprising since CD19 expression can be slightly lower on ASC than naive and memory B cells even though circulating ASCs have both CD19 and CD38 cell surface expression. Therefore, a custom blended panel of bead marker sets can be used which includes CD19 and CD38 and others including but not limited to CD138, CD27, IgD, and Ki67 to optimally isolate the circulating ASC fraction from whole blood. These commercially available bead isolation steps typically require magnetic bead-laden ASCs that are retained by a magnet while the remainder of the blood cells and plasma are washed away. Thus, in one aspect disclosed herein are methods of detecting persistent and/or latent viral infections that further comprise isolating MENSA comprising obtaining whole blood, or PBMC from a subject, separating the newly proliferated ASC from the plasma, washing the newly proliferated ASC, and incubating the newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion. It is understood and herein contemplated that the disclosed isolation will result in a 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 103, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, 104, 105, 106, 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
As used throughout this application, the term “washing” refers to a process of removing contaminants such as plasma, PBMC, whole blood, and pre-existing antibodies from population of cells of interest, such as, for example newly proliferated ASC. It is understood and herein contemplated that washing comprises the administration of an excess volume washing solution to dilute any contaminants. It is understood that washing can comprise a means for separating the newly proliferated ASC from the excess washing solution such as, for example, centrifugation (also referred to as spinning). The washing solution can then be discarded and washed cells re- suspended in a suitable media. In one aspect the wash solution can comprise any media suitable for said purpose including but not limited saline, buffered saline, and tissue culture media such as, for example MEM, DMEM, RPMI, Media 199, Opti-MEM, F10, Ham’s F12, IMDM, each with or without serum, such as, Fetal Calf serum or Fetal Bovine serum. It is further understood that a rinse and spin wash cycle can be performed more than one time each time decreasing the contaminants and increasing the purity of the newly proliferated ASC. For example, the rinse and centrifugation cycle can be performed one, two, three, four, five, six, seven, eight, nine, ten, or more times. It is further understood that through washing, the purity of the sample can comprise at least a 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 103, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, 104, 10s, 106, 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC. It is understood and herein contemplated that there are many mechanisms that can be used to separate the newly proliferated ASC from plasma. For example, ASC can be separated using ficoll gradients, elutriation, cell sorting methods such as magnetic bead sorting or fluorescence acquired cell sorting (FACS). While careful multicolor flow cytometry can identify several distinct subsets of circulating ASC populations, the majority of these cells can be captured within the CD19+, CD27hi, CD38hi population containing > 90% of recently proliferated cells as indicated by the almost universal expression of the nuclear proliferation antigen Ki67. Accordingly, where ASC are sorted based using magnetic beads or fluorescence, the sort is based on the presence and/or absence of one or more surface markers to which a tagged antibody can be bound. Examples of cell surface markers for separating newly proliferated ASC include but are not limited to CD38, CD27, CD19, CD138, IgD, and Ki67. Thus, for example, in one aspect disclosed herein are methods of isolating MENSA comprising obtaining whole blood or PBMC from a subject, separating the plasma from the newly proliferated ASC using magnetic bead separation comprising one, two, three, four, five, or six or more of anti- CD38, anti- CD27, anti- CD19, anti- CD138, anti- IgD, or anti- Ki67 beads, and incubating the newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion.
In order for MENSA to be useful for detection of antigenic exposure or for diagnosis, secreted antibodies that are newly synthesized in response to activation of ASC must be present and pre-existing antibodies should be absent. Thus, the media should comprise sugars and amino acids needed for protein synthesis, newly activated ASC, and at least a two, three, four, five, six, seven, eight, nine, or ten log reduction in plasma or serum from the subject. Additionally, MENSA may contain survival factors such as IL-2, IL-6, IL-15, IL-21, and IFN-a, APRIL, enhancers of antibody secretion such as IL-21, other nonantibody secreting cells such as T cells or macrophage, but not red blood cells. Also, depending on the ASC separation method employed, the MENSA may also contain magnetic beads or compounds needed for rapid separation of ASC from whole blood, PBMC, or plasma.
Thus, in one aspect, disclosed herein are methods of detecting a persistent or latent viral infection, wherein the MENSA is obtained by obtaining whole blood or PBMC from a subject; separating plasma from the whole blood or PBMC to produce separated cells; isolating newly proliferated ASC from the separated cells (such as, for example, using magnetic bead separation, ficoll gradient separation, elutriation, or FACS including, but not limited to the use of antibodies specific for one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and Ki67); washing the newly proliferated ASC; and incubating the washed newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion to thereby producing MENSA; wherein the MENSA comprises at least a 106 or 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
It is understood and herein contemplated that the persistent and/or latent viral antigen specific MENSA can be detected by any immunoassay known in the art including, but not limited to ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD.
1. Immunoassays
The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), enzyme linked immunospot assay (ELISPOT), radioimmunoassays (RIA), radioimmune precipitation assays (RIP A), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAP/ FLAP).
In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed biomarkers) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed biomarkers) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes. Contacting a sample with the antibody to the molecule of interest or with the molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply bringing into contact the molecule or antibody and the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any molecules (e.g., antigens) present to which the antibodies can bind. In many forms of immunoassay, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed biomarkers or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process. In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, for example, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety and specifically for teachings regarding immunodetection methods and labels.
As used herein, a label can include a fluorescent dye, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence. Substances suitable for delectably labeling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts. Furthermore, in the case where multiple antigens are reacted with a single array, each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.
Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1 ,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4- methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- 1 methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITS A; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7;
APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ - 3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A;
Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (nonratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin;
Hydroxystilbamidine (FluoroGold); Hydroxy tryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;
Monobromobimane (mBBr-GSH); Monochlorobimane ; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B | PE]; Phycoerythrin R |PE|; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL- 1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO- l;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.
A modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine- 125, iodine- 131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18. In another aspect, the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker. Examples of radionuclides useful in the aspect include, but are not limited to, Tc-99m, Re- 186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry.
The radiolabeled compounds are useful as imaging agents to diagnose neurological disease (e.g., a neurodegenerative disease) or a mental condition or to follow the progression or treatment of such a disease or condition in a mammal (e.g., a human). The radiolabeled compounds described herein can be conveniently used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT).
Labeling can be either direct or indirect. In direct labeling, the detecting antibody (the antibody for the molecule of interest) or detecting molecule (the molecule that can be bound by an antibody to the molecule of interest) include a label. Detection of the label indicates the presence of the detecting antibody or detecting molecule, which in turn indicates the presence of the molecule of interest or of an antibody to the molecule of interest, respectively. In indirect labeling, an additional molecule or moiety is brought into contact with, or generated at the site of, the immunocomplex. For example, a signalgenerating molecule or moiety such as an enzyme can be attached to or associated with the detecting antibody or detecting molecule. The signal-generating molecule can then generate a detectable signal at the site of the immunocomplex. For example, an enzyme, when supplied with suitable substrate, can produce a visible or detectable product at the site of the immunocomplex. ELIS As use this type of indirect labeling.
As another example of indirect labeling, an additional molecule (which can be referred to as a binding agent) that can bind to either the molecule of interest or to the antibody (primary antibody) to the molecule of interest, such as a second antibody to the primary antibody, can be contacted with the immunocomplex. The additional molecule can have a label or signal-generating molecule or moiety. The additional molecule can be an antibody, which can thus be termed a secondary antibody. Binding of a secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complexes can be contacted with the labeled, secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes can then be generally washed to remove any non-specifically bound labeled secondary antibodies, and the remaining label in the secondary immune complexes can then be detected. The additional molecule can also be or include one of a pair of molecules or moieties that can bind to each other, such as the biotin/avadin pair. In this mode, the detecting antibody or detecting molecule should include the other member of the pair.
Other modes of indirect labeling include the detection of primary immune complexes by a two step approach. For example, a molecule (which can be referred to as a first binding agent), such as an antibody, that has binding affinity for the molecule of interest or corresponding antibody can be used to form secondary immune complexes, as described above. After washing, the secondary immune complexes can be contacted with another molecule (which can be referred to as a second binding agent) that has binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (thus forming tertiary immune complexes). The second binding agent can be linked to a detectable label or signalgenerating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide for signal amplification.
Immunoassays that involve the detection of as substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample. Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample. Finally, in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.
Provided that the concentrations are sufficient, the molecular complexes ([Ab-Ag]n) generated by antibody-antigen interaction are visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light. The formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody is used to measure specific antigen ([Ab-Ag]n), and reagent antigens are used to detect specific antibody ([Ab-Ag]/r). If the reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay), “clumping” of the coated particles is visible at much lower concentrations. A variety of assays based on these elementary principles are in common use, including Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidometric and nephelometric assays. The main limitations of such assays are restricted sensitivity (lower detection limits) in comparison to assays employing labels and, in some cases, the fact that very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards that make the procedures more complex. Some of these Group 1 assays date right back to the discovery of antibodies and none of them have an actual “label” (e.g. Ag-enz). Other kinds of immunoassays that are label free depend on immunosensors, and a variety of instruments that can directly detect antibody-antigen interactions are now commercially available. Most depend on generating an evanescent wave on a sensor surface with immobilized ligand, which allows continuous monitoring of binding to the ligand. Immunosensors allow the easy investigation of kinetic interactions and, with the advent of lower-cost specialized instruments, may in the future find wide application in immunoanalysis.
The use of immunoassays to detect a specific protein can involve the separation of the proteins by electrophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.
Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e., the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.
Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by “wrapping around” the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4: 1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length. Further, it is usually necessary to reduce disulfide bridges in proteins (denature) before they adopt the random-coil configuration necessary for separation by size; this is done with 2-mercaptoethanol or dithiothreitol (DTT). In denaturing SDS- PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.
Determination of molecular weight is done by SDS-PAGE of proteins of known molecular weight along with the protein to be characterized. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. loglOMW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel.
In two-dimensional electrophoresis, proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another. For example, isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel, and SDS electrophoresis in a slab gel can be used for the second dimension. One example of a procedure is that of O’Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), herein incorporated by reference in its entirety for its teaching regarding two-dimensional electrophoresis methods. Other examples include but are not limited to, those found in Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421 -5425 (1977), Ornstein, L., Disc electrophoresis, L. Ann. N.Y. Acad. Sci. 121:321349 (1964), each of which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods.
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods, discloses a discontinuous system for resolving proteins denatured with SDS. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Accordingly, the resolving gel and the stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.
One example of an immunoassay that uses electrophoresis that is contemplated in the current methods is Western blot analysis. Western blotting or immunoblotting allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each of which is herein incorporated by reference in their entirety for teachings regarding Western blot methods. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel. The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein.
The attachment of specific antibodies to specific immobilized antigens can be readily visualized by indirect enzyme immunoassay techniques, usually using a chromogenic substrate (e.g. alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possibilities for probing include the use of fluorescent or radioisotope labels (e.g., fluorescein, 125I). Probes for the detection of antibody binding can be conjugated anti-immunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin/ streptavidin).
The power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass. Proteins are first separated by mass in the SDS-PAGE, then specifically detected in the immunoassay step. Thus, protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample.
The gel shift assay or electrophoretic mobility shift assay (EMSA) can be used to detect the interactions between DNA binding proteins and their cognate DNA recognition sequences, in both a qualitative and quantitative manner. Exemplary techniques are described in Omstein L., Disc electrophoresis - 1: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which is herein incorporated by reference in its entirety for teachings regarding gel-shift assays.
In a general gel-shift assay, purified proteins or crude cell extracts can be incubated with a labeled (e.g., 32P-radiolabeled) DNA or RNA probe, followed by separation of the complexes from the free probe through a nondenaturing polyacrylamide gel. The complexes migrate more slowly through the gel than unbound probe. Depending on the activity of the binding protein, a labeled probe can be either double-stranded or single-stranded. For the detection of DNA binding proteins such as transcription factors, either purified or partially purified proteins, or nuclear cell extracts can be used. For detection of RNA binding proteins, either purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used. The specificity of the DNA or RNA binding protein for the putative binding site is established by competition experiments using DNA or RNA fragments or oligonucleotides containing a binding site for the protein of interest, or other unrelated sequence. The differences in the nature and intensity of the complex formed in the presence of specific and nonspecific competitor allows identification of specific interactions.
Gel shift methods can include using, for example, colloidal forms of COOMASSIE (Imperial Chemicals Industries, Ltd) blue stain to detect proteins in gels such as polyacrylamide electrophoresis gels. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985), and Neuhoff et al., Electrophoresis 9:255-262 (1988), each of which is herein incorporated by reference in its entirety for teachings regarding gel shift methods. In addition to the conventional protein assay methods referenced above, a combination cleaning and protein staining composition is described in U.S. Patent 5,424,000, herein incorporated by reference in its entirety for its teaching regarding gel shift methods. The solutions can include phosphoric, sulfuric, and nitric acids, and Acid Violet dye. Radioimmune Precipitation Assay (RIPA) is a sensitive assay using radiolabeled antigens to detect specific antibodies in serum/plasma. The antigens are allowed to react with the serum and then precipitated using a special reagent such as, for example, protein A sepharose beads. The bound radiolabeled immunoprecipitate is then commonly analyzed by gel electrophoresis. Radioimmunoprecipitation assay (RIPA) is often used as a confirmatory test for diagnosing the presence of HIV antibodies. RIPA is also referred to in the art as Farr Assay, Precipitin Assay, Radioimmune Precipitin Assay; Radioimmunoprecipitation Analysis; Radioimmunoprecipitation Analysis, and Radioimmunoprecipitation Analysis.
While the above immunoassays that utilize electrophoresis to separate and detect the specific proteins of interest allow for evaluation of protein size, they are not very sensitive for evaluating protein concentration. However, also contemplated are immunoassays wherein the protein or antibody specific for the protein is bound to a solid support (e.g., tube, well, bead, or cell) to capture the antibody or protein of interest, respectively, from a sample, combined with a method of detecting the protein or antibody specific for the protein on the support. Examples of such immunoassays include Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexed bead assay, and magnetic capture.
Radioimmunoassay (RIA) is a classic quantitative assay for detection of antigenantibody reactions using a radioactively labeled substance (radioligand), either directly or indirectly, to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Radioimmunoassay is used, for example, to test hormone levels in the blood without the need to use a bioassay. Non-immunogenic substances (e.g., haptens) can also be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. RIA involves mixing a radioactive antigen (because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes 125I or l3lI are often used) with antibody to that antigen. The antibody is generally linked to a solid support, such as a tube or beads. Unlabeled or “cold” antigen is then adding in known quantities and measuring the amount of labeled antigen displaced. Initially, the radioactive antigen is bound to the antibodies. When cold antigen is added, the two compete for antibody binding sites - and at higher concentrations of cold antigen, more binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones in solution and the radioactivity of each used to plot a binding curve. The technique is both extremely sensitive, and specific. Enzyme-Linked Immunospot Assay (ELISPOT is an immunoassay that can detect an antibody specific for a protein or antigen. In such an assay, a detectable label bound to either an antibody -binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha. -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In this assay a nitrocellulose microtiter plate is coated with antigen. The test sample is exposed to the antigen and then reacted similarly to an ELISA assay. Detection differs from a traditional ELISA in that detection is determined by the enumeration of spots on the nitrocellulose plate. The presence of a spot indicates that the sample reacted to the antigen. The spots can be counted and the number of cells in the sample specific for the antigen determined.
Enzyme-Linked Immunosorbent Assay (ELISA), or more generically termed EIA (Enzyme ImmunoAssay), is an immunoassay that can detect an antibody specific for a protein. In such an assay, a detectable label bound to either an antibody-binding or antigenbinding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha. -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. For descriptions of ELISA procedures, see Voller, A. et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980; Butler, J. E., In: Structure of Antigens, Vol. 1 (Van Regenmortel, M., CRC Press, Boca Raton, 1992, pp. 209-259; Butler, J. E., In: van Oss, C. J. et al., (eds), Immunochemistry, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed.), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991 ); Crowther, “ELISA: Theory and Practice,” In: Methods in Molecule Biology, Vol. 42, Humana Press; New Jersey, 1995;U.S. Patent 4,376,110, each of which is incorporated herein by reference in its entirety and specifically for teachings regarding ELISA methods.
Variations of ELISA techniques are know to those of skill in the art. In one variation, antibodies that can bind to proteins can be immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a marker antigen can be added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen can be detected. Detection can be achieved by the addition of a second antibody specific for the target protein, which is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection also can be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
Another variation is a competition ELISA. In competition ELISA’ s, test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the sample can be determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.
Regardless of the format employed, ELIS As have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunecomplexes. Antigen or antibodies can be linked to a solid support, such as in the form of plate, beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate can then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells can then be “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
In ELIS As, a secondary or tertiary detection means rather than a direct procedure can also be used. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control clinical or biological sample to be tested under conditions effective to allow immunecomplex (antigen/antibody) formation.
Detection of the immunecomplex then requires a labeled secondary binding agent or a secondary binding agent in conjunction with a labeled third binding agent.
“Under conditions effective to allow immunecomplex (antigen/antibody) formation” means that the conditions include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween so as to reduce non-specific binding and to promote a reasonable signal to noise ratio.
The suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be from about 1 minute to twelve hours, at temperatures of about 20° to 30° C, or can be incubated overnight at about 0° C to about 10° C.
Following all incubation steps in an ELISA, the contacted surface can be washed so as to remove non-complexed material. A washing procedure can include washing with a solution such as PBS/Tween or borate buffer. Following the formation of specific immunecomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunecomplexes can be determined.
To provide a detecting means, the second or third antibody can have an associated label to allow detection, as described above. This can be an enzyme that can generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one can contact and incubate the first or second immunecomplex with a labeled antibody for a period of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for 2 hours at room temperature in a PBS- containing solution such as PBS -Tween).
After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label can be quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2’-azido-di-(3-ethyl- benzthiazoline-6-sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantitation can then be achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
Protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems.
One of the chief formats is the capture array, in which ligand-binding reagents, which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays can be used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and testing many serum samples simultaneously. In proteomics, capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling. Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.
For construction of arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high throughput production. For capture arrays and protein function analysis, it is important that proteins should be correctly folded and functional; this is not always the case, e.g. where recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying autoantibodies and selecting ligand binding proteins.
Protein arrays have been designed as a miniaturization of familiar immunoassay methods such as ELISA and dot blotting, often utilizing fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdrops of protein delivered onto planar surfaces are the most familiar format, alternative architectures include CD centrifugation devices based on developments in microfluidics (Gyros, Monmouth Junction, NJ) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, Biotrove, Woburn, MA) and tiny 3D posts on a silicon surface (Zyomyx, Hayward CA). Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, CA), and barcoding for beads (UltraPlex™, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).
Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to. A good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems. The immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity. Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein.
Both covalent and noncovalent methods of protein immobilization are used and have various pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control; it may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide a stable linkage, can be applied to a range of proteins and have good reproducibility; however, orientation may be variable, chemical derivatization may alter the function of the protein and requires a stable interactive surface. Biological capture methods utilizing a tag on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the biological reagent must first be immobilized adequately and the array may require special handling and have variable stability.
Several immobilization chemistries and tags have been described for fabrication of protein arrays. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the Versalinx™ system (Prolinx, Bothell, WA) reversible covalent coupling is achieved by interaction between the protein derivatized with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence and allows the immobilized proteins to retain function. Noncovalent binding of unmodified protein occurs within porous structures such as HydroGel™ (PerkinElmer, Wellesley, MA), based on a 3-dimensional polyacrylamide gel; this substrate is reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function. Widely used biological coupling methods are through biotin/streptavidin or hexahistidine/Ni interactions, having modified the protein appropriately. Biotin may be conjugated to a poly-lysine backbone immobilised on a surface such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).
Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography. A number of commercial arrayers are available [e.g. Packard Biosciences] as well as manual equipment [V & P Scientific]. Bacterial colonies can be robotically gridded onto PVDF membranes for induction of protein expression in situ.
At the limit of spot size and density are nanoarrays, with spots on the nanometer spatial scale, enabling thousands of reactions to be performed on a single chip less than 1mm square. BioForce Laboratories have developed nanoarrays with 1521 protein spots in 85sq microns, equivalent to 25 million spots per sq cm, at the limit for optical detection; their readout methods are fluorescence and atomic force microscopy (AFM).
Fluorescence labeling and detection methods are widely used. The same instrumentation as used for reading DNA microarrays is applicable to protein arrays. For differential display, capture (e.g., antibody) arrays can be probed with fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no intervening washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot). A number of novel alternative readouts have been developed, especially in the commercial biotech arena. These include adaptations of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonance light scattering (Genicon Sciences, San Diego, CA) and atomic force microscopy [BioForce Laboratories].
Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.
Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli. after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids or engineered human equivalents (Domantis, Waltham, MA) may also be useful in arrays.
The term “scaffold” refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibodylike properties of specificity and affinity. The variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display. Such ligand-binding scaffolds or frameworks include ‘Affibodies’ based on Staph, aureus protein A (Affibody, Bromma, Sweden), ‘Trinectins’ based on fibronectins (Phylos, Lexington, MA) and ‘Anticalins’ based on the lipocalin structure (Pieris Proteolab, Freising - Weihenstephan, Germany). These can be used on capture arrays in a similar fashion to antibodies and may have advantages of robustness and ease of production.
Nonprotein capture molecules, notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the cross reactivity of aptamers due to the specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains can be used to detect binding. Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labelling is used for comparison of different samples with different colors. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label- free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand. What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Since analyte concentrations cover a wide range, sensitivity has to be tailored appropriately; serial dilution of the sample or use of antibodies of different affinities are solutions to this problem. Proteins of interest are frequently those in low concentration in body fluids and extracts, requiring detection in the pg range or lower, such as cytokines or the low expression products in cells.
An alternative to an array of capture molecules is one made through ‘molecular imprinting’ technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, CA).
Another methodology which can be used diagnostically and in expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins.
Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc. Generally they require an expression library, cloned into E. coli. yeast or similar from which the expressed proteins are then purified, e.g. via a His tag, and immobilized. Cell free protein transcription/translation is a viable alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems.
For detecting protein-protein interactions, protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulfide bridges. High- throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilised on a microarray. Large-scale ‘proteome chips’ promise to be very useful in identification of functional interactions, drug screening, etc. (Proteometrix, Branford, CT).
As a two-dimensional display of individual elements, a protein array can be used to screen phage or ribosome display libraries, in order to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, ‘library against library’ screening can be carried out. Screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects is another application of the approach.
A multiplexed bead assay, such as, for example, the BD™ Cytometric Bead Array, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assay generates data that is comparable to ELISA based assays, but in a “multiplexed” or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve. Further, multiplexed bead assay allows quantification of soluble analytes in samples never previously considered due to sample volume limitations. In addition to the quantitative data, powerful visual images can be generated revealing unique profiles or signatures that provide the user with additional information at a glance.
2. Antibodies
As used herein, the term “antibody” encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
The term “variable” is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1987)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
As used herein, the term “antibody or fragments thereof’ encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab’)2, Fab’, Fab and the like, including hybrid fragments. Thus, fragments of the antibodies that retain the ability to bind their specific antigens are provided. For example, fragments of antibodies which maintain antigen binding activity are included within the meaning of the term “antibody or fragment thereof.” Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
Also included within the meaning of “antibody or fragments thereof’ are conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference.
Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab’)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the “best- fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993) and Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).
It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding (see, WO 94/04679, published 3 March 1994).
Disclosed are hybridoma cells that produces the monoclonal antibody. The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988). In a hybridoma method, a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. Traditionally, the generation of monoclonal antibodies has depended on the availability of purified protein or peptides for use as the immunogen. More recently DNA based immunizations have shown promise as a way to elicit strong immune responses and generate monoclonal antibodies. In this approach, DNA-based immunization can be used, wherein DNA encoding a portion of an antigen expressed as a fusion protein with human IgGl is injected into the host animal according to methods known in the art (e.g., Kilpatrick KE, et al. Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Fit- 3 receptor. Hybridoma. 1998 Dec;17(6):569-76; Kilpatrick KE et al. High-affinity monoclonal antibodies to PED/PEA-15 generated using 5 microg of DNA. Hybridoma. 2000 Aug;19(4):297-302, which are incorporated herein by referenced in full for the methods of antibody production) and as described in the examples.
An alternate approach to immunizations with either purified protein or DNA is to use antigen expressed in baculovirus. The advantages to this system include ease of generation, high levels of expression, and post-translational modifications that are highly similar to those seen in mammalian systems. Use of this system involves expressing domains of antigen-specific antibody as fusion proteins. The antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the antigen-specific antibody nucleotide sequence. This results in the display of the foreign proteins on the surface of the virion. This method allows immunization with whole virus, eliminating the need for purification of target antigens.
Generally, either peripheral blood lymphocytes (“PBLs”) also referred to as peripheral blood mononuclear cells (PBMC) are used in methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, “Monoclonal Antibodies: Principles and Practice” Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., “Monoclonal Antibody Production Techniques and Applications” Marcel Dekker, Inc., New York, (1987) pp. 51-63). The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against an antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art, and are described further in the Examples below or in Harlow and Lane Antibodies, A Laboratory Manul Cold Spring Harbor Publications, New York, (1988).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco’s Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994, U.S. Pat. No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual , Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen.
The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
An isolated immunogenically specific paratope or fragment of the antibody is also provided. A specific immunogenic epitope of the antibody can be isolated from the whole antibody by chemical or mechanical disruption of the molecule. The purified fragments thus obtained are tested to determine their immunogenicity and specificity by the methods taught herein. Immunoreactive paratopes of the antibody, optionally, are synthesized directly. An immunoreactive fragment is defined as an amino acid sequence of at least about two to five consecutive amino acids derived from the antibody amino acid sequence.
One method of producing proteins comprising the antibodies is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert - butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to the antibody, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer- Verlag Inc., NY. Alternatively, the peptide or polypeptide is independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.
For example, enzymatic ligation of cloned or synthetic peptide segments allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic pep tide -alpha- thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J.Biol.Chem., 269: 16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
Alternatively, unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
Also disclosed are fragments of antibodies which have bioactivity. The polypeptide fragments can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system. For example, amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity. For example, in various embodiments, amino or carboxy-terminal amino acids are sequentially removed from either the native or the modified non-immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays. In another example, a fragment of an antibody comprises a modified antibody wherein at least one amino acid has been substituted for the naturally occurring amino acid at a specific position, and a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the antibody, has been replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified antibody. For example, a modified antibody can be fused to a maltose binding protein, through either peptide chemistry or cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide. The hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified antibody receptor can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, pg. 164.). Similar purification procedures are available for isolating hybrid proteins from eukaryotic cells as well.
The fragments, whether attached to other sequences or not, include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antigen. (Zoller MJ et al. Nucl. Acids Res. 10:6487-500 (1982).
A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment. For example, ELISPOT and solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding. The binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
As needed, prior exposure to a virus or the presence of an ongoing infection can be determined by serology, immunofluorescence, RT-PCR, or viral culture. Viral culture had been used as the gold standard in the past; however, now the reference standard for diagnosis of influenza virus infection is RT-PCR or virus culture. In several small studies in patients with influenza-like illness (ILI), higher numbers of positives are detected by RT- PCR tests compared to culture. Since viral culture requires 3-14 days and is clinically impractical, it is no longer routinely used. In contrast, direct antigen detection can yield results in approximately 15 minutes; however, sensitivity is usually much lower ranging 50- 70% with a specificity of 90%. More than 10 rapid influenza tests have been approved by the FDA, but during some influenza seasons, sensitivity can be much lower, as low as 27%. As recent as the 2009 HlNlpandemic, sensitivity of the rapid antigen tests reached 45-51%, with results slightly better in infants under 2 years of agel07 because primary infection often leads to higher viral shedding.
RT-PCR is considered the most sensitive and specific of the diagnostic influenza assays since it does not require isolation of intact virus but can detect viral components. Primers that amplify the RNA encoding the relatively conserved influenza proteins (matrix or nucleoprotein) have been successful in detecting all viral strains to date. HA-specific RT- PCR can identify different influenza A virus subtypes but is not always performed. Sensitivity of RT-PCR is described as 100% compared to culture and may even exceed culture results by 5-15% since viral RNA may be detected days after live virus isolation. However, without a true gold standard, it is not known what the true sensitivity and specificity of PCR. Of note, one ICU study during the 2009 pandemic showed that PCR missed over 30% of the acute infections when compared to serology. This is indirect evidence that the sensitivity the technology disclosed herein is equal to or surpasses that of PCR for ICU patients.
Serology is useful when virus isolation is negative or inadequate and is the only available test using the host immune response. However, currently available immune assays preclude their routine use. IgM serology offers low diagnostic yields with frequent false positives, and a single IgG level is not helpful in diagnosing secondary respiratory infections in adults because they require longitudinal »4-fold rises to determine a new infection. Unfortunately, serology has limited clinical utility and is only used for retrospective diagnosis since both an acute and convalescent sample is necessary. This limitation is particularly relevant to adults with history of multiple influenza infections in whom increases of strain specific antibody titers must be interpreted with caution. So, serology is helpful for epidemiological studies but is rarely used in clinical management.
With the exception of serology, all the above-mentioned tests involve viral detection; and therefore, sensitivity can be dependent on timing of specimen collection during the clinical phase. With regards to detection of latent or persistent viral infections, in some aspects, the methods can involve assessing if the subject has symptoms of infection. If a subject maintains symptoms of an acute infection, such as, in the case of long-COVID, where a subject may continue to feel ill 60 days or more following the start of SARs-CoV-2 infection, the continued elevated levels of MENSA would indicate a persistent infection. If a subject is asymptomatic or had an asymptomatic period but is showing symptoms of an illness, the detection of MENSA indicates a latent viral reactivation. Accordingly, in one aspect, disclosed herein are methods of detecting a persistent or latent viral infection, wherein the method further comprises assessing if the subject has continued symptoms of an infection, presently exhibits symptoms of an illness, or had an asymptomatic period.
In some aspects, it is understood and herein contemplated that showing the subject has prior antigen exposure, but is not presently experiencing latent reactivation can be useful. Thus, in one aspect disclosed herein are methods of detecting a persistent or latent viral infection, wherein the method further comprises measuring serum antibody levels specific for the same antigens as assayed for MENSA.
It is further understood and herein contemplated that the detection of a latent viral infection and/or persistent viral infection while important to understanding sequelae a subject may be experiencing or why a subject has long-COVID, or addressing scientific questions relating to persistent or latent viral infections; the therapeutic/clinical goal would to be treatment of the subject. A therapeutic goal can be the treatment of the subject to alleviate, reduce, and or eliminate the effects of the persistent or latent viral infection including, but not limited to Long-COVID or PASC including, but not limited to a reduction, decrease, and or elimination of a persistent or latent virus. Thus, in one aspect, disclosed herein are methods of detecting a persistent or latent viral infection, wherein when a persistent or latent viral infection is detected, the method further comprises the administration of a therapeutically effective amount of an antiviral therapy to the subject.
A further advantage of MENSA over serum antibodies extends beyond the ability to detect persistent or latent viral infections. As serum antibody levels increase and remain high following antigenic exposure, there is no way to distinguish via serum antibodies if a subsequent illness is a new infection with a virus to which the subject has been previously exposed (or received a vaccination) or a first exposure to a new virus as the serum antibodies for a previously encountered antibody would already be elevated. By contrast, because MENSA levels fall below detectable levels, if a subject has elevated MENSA to a particular antigen, then they are experiencing a new primary infection or reactivation. If the virus is not a virus that undergoes latency, then the infection would be a new primary infection. Accordingly, in one aspect, disclosed herein are methods of detecting a subsequent acute viral infection (such as, for example, an infection with Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the Bl.351 variant, B.1.1.7 variant, and P.l variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Hantavirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2) comprising a) obtaining media enriched by newly synthesized antibodies (MENSA) from the subject; b) contacting an antigen from a virus (such as, for example, Herpes Simplex virus- 1 (HSV-l)(such as, for example, glycoprotein gG-1 and glycoprotein D), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein gG-2 and glycoprotein D), Varicella-Zoster virus (glycoprotein E, glycoprotein B, glycoprotein H, and glycoprotein L), Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl 8, and/or gp350), Cytomegalovirus (CMV)(such as, for example, glycoprotein B, and/or gH pentamer (such as, for example, gH, gL, UL128, UL130 and UL131A proteins), Human Herpes virus-6 (such as, for example Ul i, U24, U94, glycoprotein H, glycoprotein Q), Variola virus, Hepatitis B virus (such as, for example Hepatitis B surface antigen (HbsAg)), Hepatitis C virus (such as, for example, Hepatitis C virus core antigen (HCVcAg)), Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15)(such as, for example SI), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E (such as, for example SI), HCoV-OC43 (such as, for example SI), HCoV-HKUl (such as, for example SI), HCoV-NL63 (such as, for example SI), Severe Acute Respiratory Syndrome (SARS)- Coronavirus (CoV)(SARS-CoV) )(antigens such as, for example, SI Receptor Binding Domain (RBD), SI , and S2), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2 (including, but not limited to the B1.351 variant, B.1.1.7 variant, and P.l variant)(antigens such as, for example, SI Receptor Binding Domain (RBD), Nucleocapsid protein, S I, S2, SI N-terminal domain (NTD), and/or the C-terminus of ORF3a), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS- CoV)(such as, for example the nucleocapsid protein (NP)), Measles virus (such as, for example the nucleocapsid protein (NP)), Polyomavirus (such as, for example the SV40 large T antigen), Human Papillomavirus (such asl for example LI, E6, and E7), Adenovirus (such as, for example the hexon protein), Human T-cell Leukemia virus type-1 (such as, for example, gp21, p24, pl9, and gp46), Rubella virus (such as, for example spike glycoprotein El and capsid nucleoprotein), Simian Immunodeficiency virus (such as, for example p27), Vesicular stomatitis virus, Hepatitis A virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Influenza virus A, Influenza virus B, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Hantavirus, Human Immunodeficiency virus type-1 (such as, for example p24), and Human Immunodeficiency virus type-2 (such as, for example p24)) with the MENSA; and c) detecting the presence of MENSA bound to the antigen; wherein the presence of MENSA bound to an antigen indicates the presence of an acute viral infection.
In one aspect, disclosed herein are methods of detecting a subsequent acute viral infection of any preceding aspect, wherein the MENSA is detected by immunoassay (such as, for example, ELISA, immunohistochemistry, chemiluminescence, ELIspot (including, but not limited to plasma cell ELIspot), surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, Multi- Array, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD).
Also disclosed herein are methods of detecting a subsequent acute viral infection of any preceding aspect, wherein when a persistent or latent viral infection is detected, the method further comprises the administration of a therapeutically effective amount of an antiviral therapy to the subject.
Kits
Disclosed herein are kits that are drawn to reagents that can be used in practicing the methods disclosed herein. The kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods. For example, the kits can include magnetic beads or antibodies for use in separating newly proliferated ASC from plasma such as anti-CD38, anti-CD19, or anti-Cd27 antibodies or magnetic beads as well as the necessary labware to perform the isolation. Kits can also include media and enhancers to stimulate antibody production in MENSA. The kits can include antigens to coat the wells of microtiter plates for diagnosis, efficacy, or biodetection assays embodied in some of the methods, as well as the primary antibody, and reagents required to detect the antibody as intended. It is further understood that the kit can further comprise secondary antibodies and assay support structures such as, for example, microtiter plates. For example, disclosed herein are kits for detecting the presence of a persistent or latent viral infection (such as, for example, an infection with Herpes Simplex virus- 1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus(including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea vims (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS- CoV), Severe Acute Respiratory Syndrome (SARS) -Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the Bl.351 variant, B.1.1.7 variant, and P.l variant), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2) in a subject comprising one or more viral antigens from a persistent or latent virus; wherein the latent or persistent virus is selected from the group consisting of Herpes Simplex virus-1 (HSV-l)(such as, for example, glycoprotein gG-1 and glycoprotein D), Herpes Simplex virus-2 (HSV-2)(such as, for example, glycoprotein gG-2 and glycoprotein D), Varicella-Zoster virus (glycoprotein E, glycoprotein B, glycoprotein H, and glycoprotein L), Epstein-Barr virus (EBV)(such as, for example, EBNA1, VCA pl 8, and/or gp350), Cytomegalovirus (CMV)(such as, for example, glycoprotein B, and/or gH pentamer (such as, for example, gH, gL, UL128, UL130 and UL131A proteins), Human Herpes virus-6 (such as, for example Ul i, U24, U94, glycoprotein H, glycoprotein Q), Variola virus, Hepatitis B virus (such as, for example Hepatitis B surface antigen (HbsAg)), Hepatitis C virus (such as, for example, Hepatitis C virus core antigen (HCVcAg)), Coronavirus (including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15)(such as, for example SI), Porcine epidemic diarrhea vims (PEDV), human coronavirus (HCoV)-229E (such as, for example SI), HCoV-OC43 (such as, for example SI), HCoV-HKUl (such as, for example SI), HCoV-NL63 (such as, for example SI), Severe Acute Respiratory Syndrome (SARS)- Coronavirus (CoV)(SARS-CoV) )(antigens such as, for example, SI Receptor Binding Domain (RBD), S I, and S2), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2 (including, but not limited to the Bl.351 variant, B.l.1.7 variant, and P.l variant)(antigens such as, for example, SI Receptor Binding Domain (RBD), Nucleocapsid protein, SI, S2, SI N-terminal domain (NTD), and/or the C-terminus of ORF3a), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS- CoV)(such as, for example the nucleocapsid protein (NP)), Measles virus (such as, for example the nucleocapsid protein (NP)), Polyomavirus (such as, for example the SV40 large T antigen), Human Papillomavirus (such asl for example LI, E6, and E7), Adenovirus (such as, for example the hexon protein), Human T-cell Leukemia virus type-1 (such as, for example, gp21, p24, pl9, and gp46), Rubella virus (such as, for example spike glycoprotein El and capsid nucleoprotein), Simian Immunodeficiency virus (such as, for example p27), Human Immunodeficiency virus type-1 (such as, for example p24), and Human Immunodeficiency virus type-2 (such as, for example p24).
In one aspect, the disclosed kits can further comprise instructions directing the practitioner to wash the newly proliferated and separated ASC sufficiently to obtain at least a two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 103, 2xl03, 3xl03, 4xl03, 5xl03, 6xl03, 7xl03, 8xl03, 9xl03, 104, 105, 106, 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC. Additionally, the disclosed kits can comprise a washing solution as disclosed herein to perform the washing.
Also provided is an antibody reagent kit comprising containers of the monoclonal antibody or fragment thereof and one or more reagents for detecting binding of the antibody or fragment thereof to an antigen. The reagents can include, for example, fluorescent tags, enzymatic tags, or other tags. The reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that can be visualized. The reagents can further include a microtiter plate with nitrocellulose wells. Also disclosed herein are kits, wherein the kit further comprises a Luminex microsphere. It is further understood that wherein a kit may detect the presence of an antigen from a latent of persistent viral infection and thereby the presence of a latent or viral infection, the kit can further comprise one or more reagents to obtain whole blood or PBMC, one or more reagents to separate newly proliferated ASC from plasma (including, but not limited to one or more antibodies specific one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and/or Ki67), media to incubate the newly proliferated ASC, one or more antigens, and/or one or more reagents for detecting the presence of antibodies secreted by the newly proliferated ASC.
It is understood and herein contemplated that the kits disclosed herein can comprise any array of panels to which for the detection of a persistent or viral infection or detecting a latent or persistent viral infection that is involved in Long-COVID or post-acute sequelae of SARS-CoV-2 infection (PASC). For example, the kit can specifically include a panel of antigens from SARS-CoV-2 and one or more of EBV, CMV, and/or HSV-2.
Examples
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.
1. Example 1: MENSA to Identify SARS-CoV-2 Persistence and Latent Viral Reactivation in Long-COVID a) Results
(1) Patient enrollment
For the purpose of measuring serum and MENSA responses against SARS2 during infection and after vaccination, we enrolled a total of 241 adults between 2020 and 2024 at Emory University in Atlanta, GA and collected blood to generate the MENSA matrix and serum samples (Table 1). During the first year of the pandemic in 2020, we enrolled 110 adults with PCR-positive nasopharyngeal swabs (NFS) during their primary SARS2 infection. Fifty-four adults were outpatients with mild/moderate (M/M) disease as defined by the NIH criteria, and 56 adults had severe/critical (S/C) illness in the Intensive Care Units. From the 54 M/M adults, we collected 59 blood samples: 16 during acute infection (within 30 days post-symptom onset (DPSO)) and 43 during convalescence (60 DPSO). Five patients provided both acute and convalescent samples. Of the 56 S/C patients, we had 60 samples: 40 provided acute illness time points and 20 at convalescence. Four patients provided samples at both time points. These patients will be referred to as the primary SARS2 infection population. We also enrolled 60 healthy adults who had no known exposure to SARS2 and provided blood samples during the initial lockdown period, between March and June 2020. This group is referred to as the healthy controls without prior SARS2 exposure.
Serum and MENSA samples were collected from 11 vaccinated subjects who had no SARS2 infection prior to their primary two-dose mRNA vaccine series (Pfizer n=5, Moderna n=6). We also enrolled 3 adults with Omicron breakthrough infections from December 2021 -June 2022. Finally, we enrolled 61 PASC patients from the Emory Long- COVID clinic from January 2021 to February 2024 and compared against 25 CO VID recovered patients without any sequelae (CR). Of note, some of the same subjects were enrolled in different groups. For example, some of the healthy controls were later vaccinated and/or tested positive for breakthrough infections in the 4 years of the study; some patients after acute primary and/or breakthrough COVID infections without any sequelae were also included in the CR groups.
(2) SARS2 RBD and N antigen selection
SARS2 antigens were selected for measuring responses to SARS2 infection and vaccination. Fluorescent bead assays were used as previously described for the SARS2 antigens with spike SI, SI receptor binding domain (RBD), S I N-terminal domain (NTD), S2, nucleocapsid (N), and ORF-3a. We measured the acute IgG antibody responses against all six antigens in 56 adults with primary acute SARS2 infection between 6-28 DPSO and compared them against 60 healthy control adults with no known SARS2 exposure. MENSA and serum antibody reactivity was significantly higher in the infected groups than in the healthy control group for each of the six antigens (Fig. 8). Receiver operating characteristic (ROC) curves yielded Area Under the Curve (AUC) values of >0.89 for the four spike- associated proteins, with MENSA anti-RBD demonstrating the highest value, AUC=1.0 (Fig. 9). To distinguish natural infection responses from vaccination responses, we examined the diagnostic potential of two non-spike proteins, N and ORF3a. N yielded much higher signals in the infected groups and a slightly higher AUC value than ORF3a. Therefore, we focus on anti-RBD and anti-N IgG for all subsequent analyses. (3) Primary SARS2 infection responses in MENSA and serum
Primary infected patients were further divided based on the severity of their acute infections. During the acute stage of primary SARS2 infection, we observe a rise in both MENSA and serum anti-RBD IgG for M/M (69%, 88%) and S/C (95%, 95%) patients (Fig. 1A,B). We see a similar rise in MENSA and serum anti-N IgG for M/M (69%, 88%) and S/C (80%, 95%) patients as well (Fig. 1C,D). During convalescence (60-360 DPSO, 2-12 months after), MENSA levels drop significantly while serum levels rise quickly and remain elevated for months to years especially for anti-RBD IgG (Fig. 1). The MENSA levels from S/C were higher than in M/M patients during the acute infection because of higher frequencies of circulating early-minted ASC as previously shown by flow cytometry. Additionally, for S/C compared to MM patients, serum levels rise higher during the acute illness and remain elevated during convalescence, suggesting more ASC survived in other tissues such as the spleen, bone marrow, or mucosal sites. During convalescence, 87% of MENS As become negative, but are overall slightly higher than pre-pandemic levels. In all, MENSA rises during acute infection and then rapidly falls to negative whereas serum titers rise and remain elevated.
(4) Determining Co thresholds for positivity
Since the convalescent baseline MENSA negative values could be slightly higher than in pre-pandemic controls, we identify a subset of the convalescent patients from Fig. 1 as COVID Recovered (CR) who had fully recovered with no sequelae (N=19). CR MENSA samples were used as controls to calculate the MENSA Co using the average Net MFI plus 3 standard deviations. In contrast to MENSA, serum levels rise and remain high indefinitely in both CR and PASC patients; therefore, the serum Co values are calculated using the average Net MFI plus 5 standard deviations of the 60 healthy controls prior to SARS2 exposure. These calculated Net MFI Co values for MENSA (RBD: 570; N: 441) and serum (RBD: 1724; N: 682) are used to distinguish positive and negative samples in Figures 1-3, 5, 10, and 11.
(5) MENSA and serum from primary and booster mRNA vaccination
Similar to observations in primary acute SARS2 infections, adults receiving the COVID- 19 vaccination, with no known prior exposure, have positive MENSA IgG for SARS2 but only to RBD and not specific to N since only spike proteins are engineered in the mRNA vaccines (Fig. 2). This rise in MENSA IgG to RBD 1-2 weeks after the first dose further increases to a higher peak after the second dose. MENSA anti-RBD antibodies decline to negative levels prior to the third vaccine dose and then increase again within a week after the third booster (Fig. 2A). Serum anti-RBD antibody levels increase after dose one and remain elevated throughout months to years (Fig. 2B).
Similar to serum titers, MENSA antibody levels to N are also negative providing accurate responses to only the known proteins in the vaccines (Fig. 2C, D).
(6) Longitudinal time-course of MENSA and serum after SARS2 infection and vaccination
Patients were recruited during the primary SARS2 infection or prior to the primary mRNA vaccine series. Serial blood samples were collected for MENSA and serum at the first and each subsequent SARS2 exposure events (infection and vaccination) to characterize the kinetics of the immune response during repeated exposure. In Figure 3, we follow a 30-year-old Caucasian male subject from his initial mild SARS2 infection in 2020, before and after three doses of the Pfizer mRNA COVID-19 vaccine in 2021, and finally during his breakthrough Omicron infection and recovery in 2022. This subject’s first draw was collected at 9 DPSO from his primary SARS2 infection in early 2020. MENSA is positive for anti-RBD and anti-N IgG during the acute infection (Fig. 3A,C). The serum antibody titers are also weakly positive for anti-RBD and anti-N (Fig. 3B,D), as expected in most primary M/M infections. By 80 DPSO, the serum levels have increased further and remained elevated for several months while the MENSA levels rapidly decrease back to baseline. Upon three vaccine doses, the MENSA anti-RBD antibody levels rise and fall as expected whereas the serum antibodies to RBD remain positive from their previous infection and demonstrate a modest increase. Again, MENSA to the N protein is negative since it is not a component of the mRNA vaccine (Fig. 3C). However, serum anti-N levels remain weakly positive for several months to years after initial infection and before declining (Fig. 3D). In 2022, two years after the primary SARS2 infection and 8 months since his last vaccine booster vaccine, this subject had a PCR-confirmed Omicron breakthrough infection. Once again, the MENSA for anti-RBD and anti-N increases together. As expected, serum anti-N titers rise rapidly while the serum anti-RBD antibody levels, which were already high, remain elevated (Fig. 3). Unlike serum, the kinetics of MENSA demonstrate a rapid rise after each SARS2 exposure, whether it is due to vaccination or infection, but then decline to baseline negative values. MENSA is also highly discriminatory for spike (RBD) only after vaccination but shows a combination of spike (RBD) and N antibody levels during acute infections. We present additional kinetics spanning several years from a 24-year-old Caucasian female through three doses of the Moderna mRNA COVID- 19 vaccine and an Omicron breakthrough infection (Fig. 10) and from a 35-year-old Asian male through four doses of the Moderna mRNA COVID- 19 vaccine and an Omicron breakthrough infection (Fig. 11). In all, during SARS2 exposures from infection or vaccination, the MENSA antibody levels rise and then fall while serum titers rise after the primary exposure and stay elevated.
(7) MENSA and serum in PASC vs COVID Recovered (CR)
In addition to the 25 COVID recovered patients with no subsequent sequelae, we enrolled 61 PASC patients from the Emory Long-CO VID clinic from 2021 to 2024 with self-reported symptom questionnaires at enrollment (Fig. 4). We reconciled symptoms collected at enrollment and with a physician chart review and follow-up. The most common self-reported symptom in this PASC cohort was enduring fatigue in 97% of patients, followed by persistent shortness of breath (SOB) in 75% with some who received new diagnoses of asthma or lung disease following their initial infection. Other common symptoms included brain fog (67%), dizziness (52%), post-exertional malaise (47%), headache (42%), chest and muscle pain (38% respectively), chronic cough (37%), depression/anxiety (37%), palpitations (32%), sleep disturbance (32%) joint pain/arthralgia (32%), and persistent loss of taste or smell (30%) (Fig. 4). In addition to follow-up in the Long-COVID clinic, 35% were referred for advanced neurology/neurocognitive evaluation and 58% for cardiac issues with new diagnoses of tachycardia and/or postural orthostatic tachycardia syndrome (POTS) (31%), arrhythmia/atrial fibrillation (19%), heart failure (11%), ongoing chest pain (11%), venous reflux/vein compression by doppler ultrasound (11%), dysautonomia (8%) and pericarditis/myocarditis (8%). Attempting to reconcile the 12 PASC symptom scores to better predict this long-COVID as recently reported in November 2023, we calculated the average PASC score of 11.8 at enrollment for the 60 patients. Total scores equal to or greater than 12 correlated with PASC patients at 6 months, while scores less than 12 were more likely to be PASC indeterminate.
For the initial PASC experiment, we tested for only SARS2 antigens in MENSA and serum samples prepared from the subset of 19 CR subjects mentioned above and 39 PASC patients recruited during the first year of the Long-COVID clinic at Emory University December 2020-May 2021. For the CR group, fifteen of the samples were taken directly from the Figure 1 convalescent data while four of the patients donated additional follow-up samples. All patients were enrolled from day 60-279 DPSO after their initial acute infection and prior to any COVID- 19 vaccination. Of the 39 PASC patients, 56% had initial M/M acute infections and 44% had initial S/C acute infections. Of the 19 CR patients, 95% had M/M acute infections and 5% had S/C acute infections. In the 39 PASC patients, 33% still had positive MENSA for spike RBD after 60 DPSO compared to none in the CR group (Fig. 5A). Only 10% of PASC patients and none of the CR patients had a positive MENSA for N (Fig. 5C). In serum, nearly all PASC (97%) and CR (95%) patients had positive antibodies for RBD and N (Fig. 5B,D). One patient from each group was negative for both antigens in their serum and MENSA.
(8) Human viral scan in MENSA of PASC and CR
For discovery of other human virus reactivation in the MENSA samples, we compared MENSAs collected from 10 PASC patients in 2021 and three CR patients using the human PhlP-seq single-end DNA sequences that were aligned to a library of reference DNA sequences of 149,259 peptides tiling protein-coding sequences from all viruses with human hosts. Since it was early in the pandemic, the PhlP-seq was not optimized for SARS2. After quality control, we identified 227 peptides which were positive in MENSA in greater than three PASC patients and identified three additional major viruses, EBV, CMV, and HSV2 (Fig. 6). Thus, a new PASC MENSA assay was developed using viral antigens from SARS2, EBV, CMV, and HSV2.
(9) MENSA for SARS2, EBV, CMV, and HSV2 in PASC and CR
In a cohort of 60 PASC patients (39 patients in 2021 and 21 patients from 2022- 2024), 21 CR patients (2020-2022), and 16 healthy adult controls (2020), we tested MENSA and serum for a combined SARS2, EBV, CMV, and HSV2 IgG immunoassay. We had 23 samples from 21 CR patients since two individuals suffered repeat SARS2 infections in 2020 and 2022. As expected, all healthy controls drawn prior to SARS2 exposure were negative for SARS2 IgG in both MENSA and serum (Fig. 7).
Only 2/16 (13%) were positive in the MENSA for any of the viruses tested (EBV) in the healthy control group, whereas 14/16 (88%) were positive for EBV, CMV, and/or HSV2 in the serum. In the PASC vs CR groups, we show positive MENSA for SARS2 in 24/60 (40%) of PASC patients and none in the CR (Fig. 7A). Nearly all PASC and CR patients are positive for the antibodies to SARS2 in the serum (98% and 96%, respectively) (Fig. 7B). Interestingly, the lack of correlation between MENSA and serum suggests they function as independent variables, although frequencies of plasmablasts may be linked with rise in serum titers. When examining the latent herpes viruses, MENSA reactivity was greater overall in the PASC group than in the CR group (Fig. 7A). For EBV, more PASC patients had positive MENSA samples 22/60 (37%) compared to CR subjects 4/23 (17%). A MENSA test was scored positive if any one of the 3 antigens (EBNA1, VCA, and gB350) was positive. Nearly all individuals in the general population have been exposed to EBV and our results were consistent with this finding. Here, EBV serologies are positive for 59/60 (98%) in the PASC group and all 23 CR samples.
MENSAs for CMV are positive in 14/60 (23%) of the PASC patients compared to 1/23 (4%) in the CR group. Again, a test was scored positive if one of the two CMV antigens (gB or pentamer) was positive. Similar frequencies of positive CMV serology are notable in the PASC patients 43/60 (72%) versus CR individuals 18/23 (78%). For HSV2, MENSA samples are positive in 9/60 (15%) of the PASC patients, and 1/23 (4%) in the CR samples. The serum was positive in 49/60 (82%) and 12/23 (52%) of the PASC and CR samples, respectively. Overall, MENSA assays are positive in 47% (28/60) of the PASC patients for EBV, CMV, and/or HSV2 whereas only 17% (4/23) are positive in the CR cohort. In all, we identify a positive MENSA in 36/60 (60%) PASC for any of the 4 viruses (SARS2, EBV, CMV, or HSV2) compared to 4/23 (17%) for the CR group. In conclusion, a positive MENSA for SARS2 in PASC patients demonstrates ongoing new immune responses consistent with a reservoir for the persistence of SARS2 virus. Moreover, a positive MENSA for any of the 3 herpes viruses also demonstrates reactivation of latent EBV, CMV, and HSV2 identifying underlying viral triggers in this condition. b) DISCUSSION
Understanding the main viral drivers of the inflammatory and metabolic changes in patients with long-COVID has been challenging. Multiomic studies provide a wealth of information but have not identified the underlying triggers of this chronic condition. Although suggestions of viral persistence have been raised, it has been difficult to demonstrate ongoing reservoirs or reactivation of the latent virus by PCR due to the limited sensitivity of the current tests. Thus, detecting the pathogen has been challenging in patients with normal or even heightened immune responses. In this paper, we offer MENSA as a novel approach to identify the main viral drivers of long-COVID. MENSA ascertains unique immune signatures by capturing the antibodies from the circulating plasmablasts. These antibodies in the MENSA provide an immune snapshot that reveals the underlying drivers of the current illness. As proof of concept, we show that with known exposure to SARS2 by infection or vaccination, MENSA from the blood is positive. Applying these same principles, in a cohort of 60 PASC patients of whom we did not know the underlying cause, we show that 60% have a positive MENSA response against SARS2, EBV, CMV, and/or HSV2, thereby demonstrating ongoing reservoirs of SARS2 and/or reactivation of latent herpes viruses.
With first time infections, naive B cells are activated and undergo massive expansions through extrafollicular and germinal center reactions in the lymph nodes to form memory B cells and newly-minted ASC that produce antibodies. Interestingly, the majority of these ASC die, but a few successfully migrate to the bone marrow or tissue sites where they can undergo further maturation to become long-lived plasma cells (LLPC). Nearly all ASC circulating in blood are newly generated and display markers of recent proliferation such as Ki67, unlike LLPC which stop proliferating. Memory B cells persist over a lifetime and differentiate into plasmablasts when re-encountering the same antigens. During breakthrough or repeat infections, newly-minted ASC mostly originate from memory B cells and circulate transiently in the blood. Since MENSA measures antibodies only from these newly-minted ASC and not from old LLPC, MENSA antibodies provide a unique antibody signature to reveal the cause of the present-day illness. We show that, during convalescence, the MENSA responses become negative because memory B cells are no longer differentiating into ASC and released into the blood. Thus, MENSA offers an immune snapshot to uncover the sources of the patient’ s ailment.
Despite the high sensitivity of PCR testing in the nasopharyngeal swabs (NPS), blood PCR tests have limited utility for SARS2 and latent herpes viruses, such as EBV, CMV, and HSV2 due to strong T cell responses that mediate rapid viral clearance. SARS2 antigen assays have been shown to identify the spike protein, but quantities are extremely low and require ultrasensitive assays which carry a high risk of false positives. Autopsies up to 230 days after acute SARS2 infection detected SARS2 RNA in multiple tissues such as the gut, central nervous system (CNS), muscle, myocardium, and the respiratory tract demonstrating viral reservoirs. Thus, measuring the MENSA has advantages over pathogen detection by PCR amplification or protein since MENSA is in the blood, agnostic to viral reservoir locations, and would not require invasive tissue sampling. Since MENSA culminates from the total newly-minted ASC traveling in the blood during acute illness, knowledge of the viral reservoir location is not necessary since the MENSA reveals infections in deep-seated sites similar to a liquid biopsy.
Breakthrough or repeat infections can be diagnosed with serum assays, but they typically require serial blood samples during acute infection and convalescence for comparison. Another advantage of the MENSA over serum is that only a single blood sample during illness is needed. The decline of MENSA to negative levels after infection or vaccination demonstrates its clinical utility in measuring secondary or breakthrough infections.
Specificity of MENSA antibodies are also exact in that they can distinguish infection from vaccination based on spike and the nucleocapsid proteins in some patients. This specificity and sensitivity along with the kinetics make MENSA an ideal diagnostic platform to reveal viral triggers that were previously difficult to measure with just serum or PCR tests. The MENSA diagnostic would be the first of its kind to understand the main viral drivers of this chronic disease.
MENSA antibodies are expected to peak within days after exposure, and then quickly decline back to baseline within a month after the infection has resolved. Interestingly, CR MENSA does not revert to pre-pandemic baseline levels and these mechanisms are not clear. Perhaps non-specific plasma antibody binding to monocytes in the MENSA cultures may be the reason and will require more studies. Another possibility is low-level bystander responses which have been suggested to explain the difference between pre- and post-pandemic samples. Interestingly, even when using the post-pandemic MENSA samples as controls, 60% of the PASC patients have higher SARS2, EBV, CMV, and HSV2 responses in the MENSA.
Autoantigen triggers have also been implicated in PASC patients, and so we tested MENSA from a limited number of PASC patients for autoantigens using the PhlP-seq human peptidome library which consists of 605,656 peptides tiling protein-coding sequences, splice variants, non-coding open reading frames, and endogenous retroviral sequences in the human genome. No differences were observed between the PASC and CR MENSA against the human peptidome (our unpublished results). However, a larger number of patients using the 3-D conformational epitopes of the human proteome may be needed to definitively rule out MENSA responses to autoantigens. Since the original PhlP-seq assays used linear viral peptides, we may also consider a panel of 3-D conformational epitopes to identify important unique immune signatures for viruses that infect humans to ensure comprehensive testing for other viruses.
There are several limitations of this study. One is that we do not have longitudinal samples from the PASC patients and thus, it is unclear how consistent the MENSA responses are in the patients with chronic illness over time. Second, large clinical trials are needed to evaluate responses to anti-viral therapies in MENSA positive patients identified with SARS2 persistence and reactivation of EBV, CMV, or HSV2 infections. Finally, the utility of MENSA may be challenging in immunocompromised patients since the MENSA requires B cell activation to form new ASC.
The real-time immune snapshots provided by MENSA may be leveraged to inform therapeutic strategies and successful treatment of chronically ill PASC patients. Whether MENSA can also be useful to identify persistence of viral reservoirs in other chronic illnesses such as multiple sclerosis, HIV, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and other infections with post-sequelae are yet to be determined. For example, EBV was recently implicated in multiple sclerosis. Interestingly, infection with SARS2 is associated with increased susceptibility and severity of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and dementia but interpreting these correlations has been difficult. If MENSA can lead to early diagnosis of these chronic neurodegenerative disorders or help identify the cause of these disease flares, perhaps treatments may prove more effective in preventing progression and severity of these pathological conditions.
PASC can include symptoms, such as dyspnea, fatigue, and depression, and serious clinical indications, such as cardiovascular disease or diabetes. Recent studies also suggest increased risk for autoimmune inflammatory rheumatic diseases in PASC and CR patients. Future clinical trials are necessary to perform proof-of-concept studies where MENSA data could be used to inform treatment modalities for mitigating symptoms associated with SARS2 viral persistence, reactivation of viruses, reactivation of viruses in other chronic illnesses, or early activation of other chronic illnesses.
In summary, MENSA is a novel immune diagnostic which captures unique signatures of the early-minted ASC in the blood to reveal the cause of illness. In chronic conditions such as PASC where serum antibody titers are high, MENSA is an independent matrix that identifies persistence of SARS2 viruses or antigens and can also recognize the reactivation of latent herpes viruses, such as EBV, CMV, and HSV2 in 60% of patients. This host immune snapshot reveals the fundamental drivers of viral persistence and reactivation in this chronic disease. c) METHODS
(1) Patient enrollment
We enrolled 241 adults between 2020 and 2024 at Emory University in Atlanta, GA and collected blood to generate the MENSA matrix and serum samples (Table 1). Tn 2020, we enrolled 110 adults with PCR-positive NPS during their primary SAR2 infection. Fifty - four adults were outpatients with mild/moderate (M/M) disease as defined by the NIH criteria, and 56 adults had severe/critical (S/C) illness in the Intensive Care Units. We also enrolled 60 healthy adults with no known exposure to SARS2 early during the pandemic with blood samples collected between March and June 2020 during the initial lockdown as the healthy adult controls without prior SARS2 exposure. Eleven vaccinated subjects who had no prior SARS2 infection were enrolled before and during their primary two-dose mRNA vaccine series (Pfizer n=5, Moderna n=6) and drawn again before and after their third booster dose. We also enrolled 3 adults experiencing Omicron breakthrough infections between December 2021 and June 2022.
Finally, we enrolled 61 long-COVID or PASC patients from the Emory Long- COVID Clinic which was started in January 2021 until February 2024 (Fig. 4). Initially, 40 patients were enrolled in 2021 (Figs. 5, 6, 7) and an additional 21 patients were enrolled in 2022-2024 (Fig. 7). All patients filled patient-reported symptom questionnaires. Samples from nine of the initial 39 PASC patients from Fig. 5 and one additional PASC patient recruited in 2021 were sent to Immune ID for PhlP-seq analysis along with samples from three CO VID Recovered patients (1/3 CR from initial convalescent cohort in Fig 1; 2/3 new CR patients). All blood samples were collected under the Emory University Institutional Review Board-approved protocols. (2) MENSA Preparation
Medium enriched for newly synthesized antibodies (MENSA) was generated by isolating, washing, and culturing antibody- secreting cells (ASC)-containing peripheral blood mono-nuclear cells (PBMC) from blood using a modified procedure previously described. Peripheral blood samples were collected in sodium heparin tubes and PBMC were isolated by centrifugation (1,000 xg; 10 min) using Lymphocyte Separation Media (Coming) and Leucosep tubes (Greiner Bio-One). Five washes with RPMI-1640 (Coming) were performed to remove serum immunoglobulins (800 x g; 5 min) with erythrocyte lysis (3 mL; 3 min), and harvested PBMCs were cultured at 106 cells/mL in R10 Medium (RPMI-1640, 10% Sigma FBS, 1% Gibco Antibiotic/ Anti-mycotic) for 24 h at 37° C and 5% CO2. After incubation, the cell suspension was centrifuged (800 xg; 5 min), and the supernatant (MENSA) was separated from the PBMC pellet, aliquoted, and stored at -80°C for testing.
(3) Serum Preparation
Whole blood was collected and incubated at room temperature for at least 30 minutes. The clot was discarded, and the remaining serum supernatant was centrifuged (800xg; 10 min), aliquoted and stored at -80 °C for testing.
(4) Antigen Selection and Multiplex Immunoassays
Antigens of interest were selected from literature, coupled to Luminex MagPlex Microspheres of spectrally distinct regions via carbodiimide coupling, and tested for antigen specific IgG reactivity against patient samples as previously described.
(5) SARS2 antigens
SARS-CoV-2 Spike S I Receptor Binding Domain (RBD; catalog no. Z03483; expressed in HEK293 cells) and Nucleocapsid protein (N; catalog no. Z03480; expressed in Escherichia coli), were purchased from GenScript. SI (catalog no. S1N-C52H3; HEK293), S2 (catalog no. S2N-C52H5; HEK293) and SI N-terminal domain (NTD; catalog no. S1D- C52H6; HEK293) were purchased from ACROBiosystems. The C-terminus sequence of ORF3a (Accession: QHD43417.1, amino acids 134-275 plus N-terminal His6-Tag) was sent to Genscript for custom protein expression in E. coli. Each protein was expressed with an N-terminal His6-Tag to facilitate purification, at least 90% pure, and appeared as a predominant single band on SDS-PAGE analysis.
(6) EBV, CMV, and HSV2 antigens
EBV, CMV, and HSV2 antigens were carefully selected for antigenicity based on previous reports. The following proteins were used: EBV EBNA1 protein from Abeam (produced in E. coli, N-Terminus His Tag, CAT#abl38345); EBV VCA pl 8 from RayBiotech (produced in E. coli, CAT#227-20127); EBV gp350 protein from AcroBiosystems (produced in HEK293 cells, His Tag, MALS verified, CAT#GP0-E52H6); CMV glycoprotein B from AcroBiosystems (strain AD169, expressed from HEK293 cells, His Tag, MALS verified, CAT#CMB-V52H4); CMV gH pentamer complex, consisting of gH, gL, UL128, UL130 and UL131A proteins, produced in mammalian HEK293 cells from The Native Antigen Company (CAT#CMV-PENT); HSV2 envelope glycoprotein D from AcroBiosystems (gD, expressed HEK293 cells, His Tag, MALS verified, CAT#GLD- V52H4).
(7) Serum and MENSA assays for SARS2 and other viruses
Serum samples were tested at 1:500 dilution in assay buffer (1XPBS, 1% BSA) while MENSA samples were tested neat with no dilution. Results were analyzed on a Luminex FLEXMAP 3D instrument. Median fluorescent intensity (MFI) using phycoerythrin-conjugated detection antibodies (Goat Anti-Human IgG-PE, Southern Biotech cat. #2040-09) was measured for each sample using the Luminex xPONENT software on Enhanced PMT setting. The background value of assay buffer or R10 media was subtracted from the serum or MENSA results, respectively, to obtain MFI minus background (Net MFI). All samples were tested in duplicate and the average of the two results were used for analysis. In the initial SARS2 assay, all SARS2 protein bound microparticles were run together as a six-bead SARS2 solution. Serum Co values of positivity were calculated as the average plus five standard deviations of the Healthy Control population (N=60) for each antigen (RBD: 1724; N: 682). MENSA Co positivity values were calculated as the average plus three standard deviations of the Contemporary Controls (COVID Recovered N=19 (CR) group described above) for each antigen (RBD: 570; N: 441).
Later, the assay was modified to contain only RBD, N, SI, and S2 from SARS2 (NTD and ORF3a dropped) and also included the addition of all EBV, CMV, and HSV2 antigens, for a combined multi viral 10-antigen bead assay. All new Cos were calculated based on the new assay data in Figure 7. MENSA Cos were calculated as the average plus three standard deviations of 22/23 CR samples (RBD: 404; N: 266; EBNA1: 475; VCA: 426; gp350: 523; gB: 298; Pentamer: 455; gD: 308). One sample was excluded from the MENSA Co calculation due to multiple antigen reactivity measuring greater than 10 times the median value of the entire group. SARS2 Serum Co was calculated as the average plus three standard deviations of the 16 Healthy Donor samples collected prior to SARS2 exposure. Since the majority of the population is expected to be positive for EBV, CMV, and/or HSV2 antibodies in their serum, we obtained de-identified clinically confirmed negative sera from the Emory clinical laboratory. For each virus, three confirmed negative serum samples were used. The average Net MFI plus three standard deviations was calculated for each antigen and used as the Co threshold for positivity (RBD: 410; N: 564; EBNA1: 3,578; VCA: 593; gp350: 228; gB: 778; Pentamer: 769; gD: 858).
(8) Phage immunoprecipitation sequencing and analysis
We constructed a custom T7 bacteriophage library consisting of 149,259 peptides tiling all protein-coding sequences from viruses with human hosts. Viral sequences were downloaded from Uniprot, collapsed on 90% identity, and bioinformatically parsed into 90 amino-acid peptide tiles with 45 amino-acid overlaps between adjacent tiles. Healthy and Covid patients’ plasma or serum and matched MENSA reactivities were profiled using phage immunoprecipitation and sequencing (PhlP-seq). MENSA samples were profiled in duplicate and plasma/serum samples in triplicate. PhlP-seq was performed as previously described with some modifications. T7 bacteriophage libraries were aliquoted into 96-well plates and incubated with 20pl each of protein A and G Dynabeads on a rotator for 4 h at room temperature. Next, plates were placed on a magnet and supernatants were transferred to a fresh 96-well plate, to which we added patient plasma containing 2 pg of total IgG, and continued with the immunoprecipitation and washing steps, as previously described. Following the washes, protein A and protein G Dynabeads were resuspended in PCR master mix, amplified with 16 rounds of PCR, SPRI cleaned to remove primers, and indexed for sequencing with 8 rounds of PCR with primers containing Illumina p5 and p7 barcodes. NGS libraries were quantified on a Tapestation4200 and normalized for sequencing on Illumina Nextseq 2000 or Novaseq 6000 instruments. Each sequencing library received a minimum of 3M reads.
PhlP-seq single-end DNA sequences were aligned to a library of reference DNA sequences (149,259 75bp for viral) with the bowtie2 aligner (v2.0) using end-to-end matching. Read counts were summarized using samtools (vl.14) and collated into a counts matrix. The raw counts were converted to counts per million (CPM) using the ‘cpm‘ function from the R package edgeR (v3.36.0). CPM values for healthy controls were summarized by computing the peptide-wise mean and standard deviation across all healthy control samples. CPM values for each patient sample were collapsed by computing the peptide-wise minimum across technical replicates. Peptide-wise z-scores were then computed as: where Zi,j is the z-score for patient i, peptide j; Ci,j is the minimum CPM for patient i, peptide j; is the mean of peptide j in the healthy control samples, and 07 is the standard deviation of peptide j in the healthy control samples. For each patient, hits were identified as those peptides with cij > 10 AND ZIJ > 10.
VII. References
Bedran, D., Bedran, G. & Kote, S. A Comprehensive Review of Neurodegenerative Manifestations of SARS-CoV-2. Vaccines (Basel) 12(2024).
Belshe, R.B., et al. Correlate of immune protection against HSV-1 genital disease in vaccinated women. The Journal of infectious diseases 209, 828-836 (2014).
Carter, M.J., Mitchell, R.M., Meyer Sauteur, P.M., Kelly, D.F. & Truck, J. The Antibody- Secreting Cell Response to Infection: Kinetics and Clinical Applications. Front Immunol 8, 630 (2017).
Cervia-Hasler, C., et al. Persistent complement dysregulation with signs of thromboinflammation in active Long Covid. Science 383, eadg7942 (2024).
Chang, R., et al. Risk of autoimmune diseases in patients with CO VID- 19: A retrospective cohort study. EClinicalMedicine 56, 101783 (2023).
El Sahly, H.M., et al. Efficacy of the mRNA-1273 SARS-CoV-2 Vaccine at Completion of Blinded Phase. N Engl J Med 385, 1774-1785 (2021).
Grant, R.A., et al. Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID- 19. JCI Insight 9(2024).
Haddad, N.S., et al. Circulating antibody-secreting cells are a biomarker for early diagnosis in patients with Lyme disease. PloS one 18, e0293203 (2023).
Haddad, N.S., et al. Detection of Newly Secreted Antibodies Predicts Non-recurrence in Primary Clostridioides difficile Infection. J Clin Microbiol, jcm0220121 (2022).
Haddad, N.S., et al. Novel immunoassay for diagnosis of ongoing Clostridioides difficile infections using serum and medium enriched for newly synthesized antibodies (MENSA). J Immunol Methods 492, 112932 (2021).
Haddad, N.S., et al. One-Stop Serum Assay Identifies COVID-19 Disease Severity and Vaccination Responses. Immunohorizons 5, 322-335 (2021).
Halliley, J.L., et al. Long-Lived Plasma Cells Are Contained within the CD19(- )CD38(hi)CD138(+) Subset in Human Bone Marrow. Immunity 43, 132-145 (2015).
Karsten, C.B., et al. Evolution of functional antibodies following acute Epstein-Barr virus infection. PLoS pathogens 18, el010738 (2022).
Klein, J., et al. Distinguishing features of long COVID identified through immune profiling. Nature 623, 139-148 (2023).
Koren, I., et al. The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C- Terminal Degrons. Cell 173, 1622-1635 el614 (2018).
Kumar, S., Thambiraja, T.S., Karuppanan, K. & Subramaniam, G. Omicron and Delta variant of SARS-CoV-2: A comparative computational study of spike protein. J Med Virol 94, 1641-1649 (2022).
Kyu, S., et al. Diagnosis of Streptococcus pneumoniae infection using circulating antibody secreting cells. PLoS One 16, e0259644 (2021).
Lanz, T.V., et al. Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM. Nature 603, 321-327 (2022).
Larman, H.B., et al. Autoantigen discovery with a synthetic human peptidome. Nat Biotechnol 29, 535-541 (2011).
Lee, F.E., et al. Circulating human antibody-secreting cells during vaccinations and respiratory viral infections are characterized by high specificity and lack of bystander effect. J Immunol 186, 5514-5521 (2011).
Lee, F.E., Falsey, A.R., Halliley, J.L., Sanz, I. & Walsh, E.E. Circulating antibody- secreting cells during acute respiratory syncytial virus infection in adults. J Infect Dis 202, 1659— 1666 (2010).
Liew, F., et al. Large-scale phenotyping of patients with long CO VID post-hospitalization reveals mechanistic subtypes of disease. Nat Immunol 25, 607-621 (2024).
Lilleri, D., et al. Fetal human cytomegalovirus transmission correlates with delayed maternal antibodies to gH/gL/pUL128-130-131 complex during primary infection. PloS one 8, e59863 (2013).
Macagno, A., et al. Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex. Journal of virology 84, 1005-1013 (2010).
Magri, G., et al. Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals. Immunity 47, 118-134 ell8 (2017).
Middeldorp, J.M. Epstein-Barr Virus-Specific Humoral Immune Responses in Health and Disease. Current topics in microbiology and immunology 391, 289-323 (2015).
Mizrahi, B., et al. Long covid outcomes at one year after mild SARS-CoV-2 infection: nationwide cohort study. BMJ 380, e072529 (2023). Muthukrishnan, G., et al. A Bioinformatic Approach to Utilize a Patient’s Antibody- Secreting Cells against Staphylococcus aureus to Detect Challenging Musculoskeletal Infections. Immunohorizons 4, 339-351 (2020).
Nakagawa, S. & Takahashi, M.U. gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes. Database (Oxford) 2016(2016).
Nguyen, D.C., et al. Majority of human circulating IgG plasmablasts stop blasting in a cell- free pro-survival culture. Scientific reports 14, 3616 (2024).
Oh, I., et al. Tracking Anti-Staphylococcus aureus Antibodies Produced In Vivo and Ex Vivo during Foot Salvage Therapy for Diabetic Foot Infections Reveals Prognostic Insights and Evidence of Diversified Humoral Immunity. Infect Immun 86(2018).
Omer, S.B., Malani, P. & Del Rio, C. The COVID-19 Pandemic in the US: A Clinical Update. JAMA 323, 1767-1768 (2020).
Organization, W.H. COVID- 19 weekly epidemiological update, 29 December 2020. (2020).
Panel, C.-T.G. Coronavirus Disease 2019 (COVID-19) Treatment Guidelines. (National Institutes of Health, 2020).
Peluso, M.J., et al. Chronic viral coinfections differentially affect the likelihood of developing long CO VID. J Clin Invest 133(2023).
Phad, G.E., et al. Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts. Nat Immunol 23, 1076-1085 (2022).
Post COVID-19 condition (Long COVID).
Rippee-Brooks, M.D., et al. Viral Infections, Are They a Trigger and Risk Factor of Alzheimer’s Disease? Pathogens 13(2024).
Stein, S.R., et al. SARS-CoV-2 infection and persistence in the human body and brain at autopsy. Nature 612, 758-763 (2022).
Su, Y., et al. Multiple early factors anticipate post-acute COVID-19 sequelae. Cell 185, 881-895 e820 (2022).
Swank, Z., et al. Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae. Clin Infect Dis 76, e487-e490 (2023).
Syed, U., et al. Incidence of immune-mediated inflammatory diseases following CO VID- 19: a matched cohort study in UK primary care. BMC Med 21, 363 (2023).
Tesch, F., et al. Incident autoimmune diseases in association with SARS-CoV-2 infection: a matched cohort study. Clin Rheumatol 42, 2905-2914 (2023).
Thaweethai, T., et al. Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection. JAMA 329, 1934-1946 (2023). Walsh, E.E., et al. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. The New England journal of medicine 383, 2439-2450 (2020).
Wiegers, A.K., Sticht, H., Winkler, T.H., Britt, W.J. & Mach, M. Identification of a neutralizing epitope within antigenic domain 5 of glycoprotein B of human cytomegalovirus. Journal of virology 89, 361-372 (2015).
Wong, A.C., et al. Serotonin reduction in post-acute sequelae of viral infection. Cell 186, 4851-4867 e4820 (2023).
Woodruff, M., et al. Critically ill SARS-CoV-2 patients display lupus-like hallmarks of extrafollicular B cell activation. medRxiv (2020).
Woodruff, M.C., et al. Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID. Nat Commun 14, 4201 (2023).
Wrammert, J., et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. The Journal of experimental medicine 208, 181-193 (2011).
Wrammert, J., et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667-671 (2008).
Xie, Y. & Al-Aly, Z. Risks and burdens of incident diabetes in long COVID: a cohort study. Lancet Diabetes Endocrinol 10, 311-321 (2022).
Xie, Y., Xu, E., Bowe, B. & Al-Aly, Z. Long-term cardiovascular outcomes of CO VID- 19. Nat Med 28, 583-590 (2022).
Xu, G.J., et al. Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome. Science 348, aaa0698 (2015).

Claims

VIII. CLAIMS What is claimed is:
1. A kit for detecting the presence of a persistent or latent viral infection in a subject comprising one or more viral antigens from a persistent or latent virus; wherein the latent or persistent virus is selected from the group consisting of Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus, avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)- 229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)- Coronavirus (CoV)-2 (SARS-CoV-2) middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.
2. The kit of claim 1 , wherein the kit further comprises a luminex microsphere.
3. The kit of claim 1 or 2, wherein the kit comprises a SARS-CoV-2 antigen.
4. The kit of claim 3, wherein the SARS-CoV-2 antigen comprises the SARS-CoV-2 S I Receptor Binding Domain (RBD), Nucleocapsid protein, S I, S2, SI N-terminal domain (NTD), and/or the C-terminus of ORF3a.
5. The kit of claim of any one of claims 1-4, wherein the kit comprises an EBV antigen.
6. The kit of claim 5, wherein the EBV antigen comprises EBNA1, VCA pl8, and/or gp350.
7. The kit of any one of claims 1 -6, wherein the kit comprises a CMV antigen.
8. The kit of claim 7, wherein the CMV antigen comprises glycoprotein B, and/or gH pentamer.
9. The kit of any one of claims 1-8, wherein the kit comprises an HSV-2 antigen.
10. The kit of claim 9, wherein the HSV-2 antigen comprises glycoprotein D.
11. The kit of claim of any one of claims 1-10, further comprising one or more reagents to obtain whole blood or PBMC, one or more reagents to separate newly proliferated ASC from plasma, media to incubate the newly proliferated ASC, one or more antigens, and/or one or more reagents for detecting the presence of antibodies secreted by the newly proliferated ASC.
12. The kit of claim 11, wherein the one or more reagents to separate newly proliferated ASC from plasma comprises one or more antibodies specific one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and/or Ki67.
13. A method of detecting a persistent or latent viral infection in a subject comprising: a) obtaining media enriched by newly synthesized antibodies (MENSA) from the subject; b) contacting one or more antigens from a persistent or latent virus with the MENSA; and c) detecting the presence of MENSA bound to the antigen wherein the presence of MENSA bound to an antigen indicates the presence of a persistent or latent viral infection.
14. The method of claim 13, wherein the persistent or latent viral infection comprises an infection with Herpes Simplex virus- 1 (HSV-1 ), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus, avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoVj(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2), and middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2.
15. The method of claim 13 or 14, wherein the one or more antigens are antigens from a Herpes Simplex virus- 1 (HSV-1), Herpes Simplex virus-2 (HSV-2, Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Variola virus, Hepatitis B virus, Hepatitis C virus, Coronavirus, avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea vims (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2, middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV), Measles virus, Polyomavirus, Human Papillomavirus, Adenovirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.
16. The method of any one of claims 13-15, wherein the antigen is a SARS-CoV-2 antigen.
17. The method of claim 16, wherein the SARS-CoV-2 antigen comprises the SARS- CoV-2 SI Receptor Binding Domain (RBD), Nucleocapsid protein, SI, S2, SI N-terminal domain (NTD), and/or the C-terminus of ORF3a
18. The method of any one of claims 13-17, wherein the antigen is an EBV antigen.
19. The method of any one of claims 18, wherein the EBV antigen comprises EBNA1,
VCA pl8, and/or gp350.
20. The method of any one of claims 13-19, wherein the antigen is a CMV antigen.
21. The method of any one of claims 20, wherein the CMV antigen comprises glycoprotein B, and/or gH pentamer.
22. The method of any one of claims 13-21, wherein the antigen is an HSV-2 antigen.
23. The method of claim 22, wherein the wherein the HSV-2 antigen comprises glycoprotein D.
24. The method of any one of claims 13-23, wherein the MENSA is obtained by obtaining whole blood or PBMC from a subject; separating plasma from the whole blood or PBMC to produce separated cells; isolating newly proliferated ASC from the separated cells; washing the newly proliferated ASC; and incubating the washed newly proliferated ASC in a media that supports maintenance of ASC and antibody secretion to thereby producing MENSA; wherein the MENSA comprises at least a 106 or 107-fold reduction of contaminating pre-existing plasma antibodies relative to whole blood, plasma, or PBMC.
25. The method of claim 24, wherein the newly proliferated ASC are isolated by magnetic bead separation, ficoll gradient separation, elutriation, or FACS.
26. The method of claim 24 or 25, wherein newly proliferated ASC are separated and isolated through use of antibodies specific for one or more cell surface markers comprising CD38, CD27, CD19, CD138, IgD, and Ki67.
27. The method of any one of claims 13-26, wherein the MENSA is detected by immunoassay.
28. The method of claim 27, wherein MENSA is detected by plasma cell ELIspot, ELISA, flow cytometry, immunohistochemistry, chemiluminescence, surface plasmon resonance, and hospital modular analyzers, or multiplex assay.
29. The method of any one of claims 13-28, wherein the method further comprises measuring serum antibody levels specific for the same antigens as assayed for MENSA.
30. The method of any one of claims 13-28, wherein when a persistent or latent viral infection is detected, the method further comprises the administration of a therapeutically effective amount of an antiviral therapy to the subject.
31. A methods of detecting a subsequent acute viral infection comprising a) obtaining media enriched by newly synthesized antibodies (MENSA) from the subject; b) contacting an antigen from a virus with the MENSA; and c) detecting the presence of MENSA bound to the antigen; wherein the presence of MENSA bound to an antigen indicates the presence of an acute viral infection.
32. The method of detecting a subsequent acute viral infection of claim 31, wherein the acute viral infection is from a virus selected from the group consisting of Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)- Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2), middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Hantavirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.
33. The method of detecting a subsequent acute viral infection of claim 31 or 32, wherein the one or more antigens are antigens from a Herpes Simplex virus-1 (HSV-1), Herpes Simplex virus-2 (HSV-2), Varicella-Zoster virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpes virus-6, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)(SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2), middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS- CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Hantavirus, Human T-cell Leukemia virus type-1, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2.
34. The method of detecting a subsequent acute viral infection of any one of claims 31- 33, wherein the MENSA is detected by immunoassay.
35. The method of detecting a subsequent acute viral infection of claim 34, wherein the immunoassay comprises ELISA, immunohistochemistry, chemiluminescence, ELIspot, surface plasmon resonance, and hospital modular analyzers, flow cytometry, or multiplex assay.
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Citations (2)

* Cited by examiner, † Cited by third party
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US20200072836A1 (en) * 2014-05-05 2020-03-05 Microbplex, Inc. Media elaborated with newly synthesized antibodies (mensa) and uses thereof
US20210181199A1 (en) * 2017-05-08 2021-06-17 University Of Pittsburgh--Of The Commonwealth System Of Higher Education Methods and compositions for the detection of flavivirus infections

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200072836A1 (en) * 2014-05-05 2020-03-05 Microbplex, Inc. Media elaborated with newly synthesized antibodies (mensa) and uses thereof
US20210181199A1 (en) * 2017-05-08 2021-06-17 University Of Pittsburgh--Of The Commonwealth System Of Higher Education Methods and compositions for the detection of flavivirus infections

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