WO2023034933A1 - Methods and systems for assessing adaptive immunity to coronavirus - Google Patents

Methods and systems for assessing adaptive immunity to coronavirus Download PDF

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Publication number
WO2023034933A1
WO2023034933A1 PCT/US2022/075861 US2022075861W WO2023034933A1 WO 2023034933 A1 WO2023034933 A1 WO 2023034933A1 US 2022075861 W US2022075861 W US 2022075861W WO 2023034933 A1 WO2023034933 A1 WO 2023034933A1
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protein
peptide
coronavirus
subject
biological sample
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PCT/US2022/075861
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French (fr)
Inventor
Jerome P. Lapointe
Andrew Senyei
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Nonigenex, Inc.
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Publication of WO2023034933A1 publication Critical patent/WO2023034933A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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

Definitions

  • Viral infections on a cellular level are mediated by viruses binding to receptors expressed on the surface of a target cell.
  • the spike glycoprotein of a coronavirus binds to the angiotensin-converting enzyme 2 (ACE2) receptor, and binding between the receptor-binding domain (RBD) of the spike protein and ACE2 precedes entry of the coronavirus into the cell.
  • ACE2 angiotensin-converting enzyme 2
  • RBD receptor-binding domain
  • Individuals who are exposed to a new virus e.g., coronavirus
  • This adaptive immune response significantly reduces incidences of a second infection by the same virus.
  • the coronavirus disease of 2019 is an ongoingpandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Post-COVID syndrome which includes cardiac, neuro, and respiratory complications, can persist indefinitely.
  • Onethird of recovered COVID-19 hospitalized patients are readmitted, and 1 in 8 die within 5 months.
  • regulatory agencies and vaccine developers may look to surrogate markers of protection against future infections by SARS-CoV-2 until proof of protection is available.
  • the data being collected is so scattered that there’ s little in sight into which h ealth care workers, or first responders, have been among the people getting the initial vaccines, as intended — or how many doses instead have gone to people who should be much further down the list.
  • thermostability of vaccines means that any delay during distribution poses a risk that the vaccines will have reduced potency upon arrival to its destination.
  • delays in distribution due to unpredictable and extreme weather conditions across the United States are a source of major concern. Addressing this challenge for vaccines against SARS-CoV-2 is made even more difficult with the limited number of facilities around the world manufacturing the vaccines requiring longer delivery routes.
  • a method of assessing adaptive immunity against a coronavirus in subject comprising: (a) bringing a biological sample of the subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from the coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample when the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is a signal from the binding complex, SN is a signal expected for complete neutralization of binding between the first peptid
  • ACE2 Angiotensin-converting enzyme 2
  • the biological sample has an average volume of less than 1 milliliter when obtained from the subject. In some embodiments, the biological sample is not a liquid when it is provided in (a). In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the one or more control subjects received a vaccine against the coronavirus within one month of comparing in (d). In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. In some embodiments, the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus. In some embodiments, the SARS virus is SARS-CoV-2 or a variant thereof.
  • SARS Severe acute respiratory syndrome-related
  • the subject is a human subject that has an age comprising more than or equal to about 50 years of age.
  • the method further comprises eluting the at least the portion of the biological sample from a dried blood spot card.
  • the subject is unvaccinated against the coronavirus.
  • a method comprising: identifying a subject as having adaptive immunity against a coronavirus relative to a control subject that is vaccinated against the coronavirus, wherein the identifying comprises: (a) bringing a biological sample obtained from the subject into contact with (1) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (2) a second peptide or protein comprising a spike protein or a portion thereof derived from the corona virus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring a percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample; and (c) comparingthe percentinhibition of the bindingto a percentinhibition of binding of the first peptide or protein to the second peptide or protein
  • the biological sample has an average volume of less than 1 milliliter when obtained from the subject. In some embodiments, the biological sample comprises capillary blood . In some emb odiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the control subject received a vaccine against the coronavirus within one month of comparing in (c). In some embodiments, the method further comprises: (d) identifying the adaptive immunity against the coronavirus of the subject as being lower than the adaptive immunity of the control or the index; and (e) administering a vaccine against the coronavirus to the subject. In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
  • the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus.
  • SARS virus is SARS-CoV-2, or a variant thereof.
  • the human subject has an age comprising greater than or equal to about 50 years of age.
  • the first peptide or protein or the second peptide or protein is immobilized on a substrate
  • the method further comprises detectingthe binding complex using a lateral flow assay.
  • detectingthe binding complex in comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device.
  • the method further comprises eluting the biological sample from a dried blood spot card.
  • the subject is unvaccinated against the coronavirus.
  • a method of analyzing a biological sample comprising: bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an accuracy of at least about 80%.
  • ACE2 Angiotensin-converting enzyme 2
  • the subject is vaccinated against the coronavirus. In some embodiments, the subject is unvaccinated against the coronavirus.
  • the coronavirus comprises SARS-CoV-2 or a variant thereof.
  • the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide.
  • the second protein or peptide comprises the S1 subunit of the spike protein.
  • the biological sample has an average volume of less than 1 milliliter when obtained. In some embodiments, the biological sample comprises capillary blood. In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture.
  • the method further comprises administering a vaccine against the coronavirus to the subject based on measuring in (b).
  • the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
  • the subject is an age comprising greater than or equal to about 50 years of age.
  • the first peptide or protein or the second peptide or protein is immobilized on a substrate
  • the method further comprises detecting the binding complex using a lateral flow assay.
  • detecting the binding complex in (b) comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device.
  • the method further comprises eluting the at least the portion of the biological sample from a dried blood spot card.
  • the accuracy is measured by: (a) introducing a reference sample with (i) the first peptide or protein; and (ii) the second peptide or protein, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC 50 ; and (b) detecting a binding complex between the first peptide or protein and the second peptide or protein; and (c) determining an IC 50 for the neutralizing antibody, wherein the IC 50 is approximately the known IC 50 .
  • a method of analyzing a biological sample comprising: bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an sensitivity of at least about 80%.
  • ACE2 Angiotensin -converting enzyme 2
  • the subject is vaccinated against the coronavirus. In some embodiments, the subject is unvaccinated against the coronavirus.
  • the coronavirus comprises SARS-CoV-2 or a variant thereof.
  • the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof.
  • the second protein or peptide comprises the S1 subunit of the spike protein.
  • the biological sample has an average volume of less than 1 milliliter when obtained. In some embodiments, the biological sample comprises capillary blood. In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture.
  • the method further comprises administering a vaccine against the coronavirus to the subject based on measuring in (b).
  • the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
  • the subject is an age comprising greater than or equal to about 50 years of age.
  • the method further comprises eluting the at least the portion of the biological sample from a dried blood spot card.
  • the first peptide or protein or the second peptide or protein is immobilized on a substrate
  • the method further comprises detecting the binding complex using a lateral flow assay.
  • detecting the binding complex in (b) comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device.
  • a method of analyzing a biological sample comprising: bringing at least a portion of a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with percent coefficient of variation of less than 10%.
  • ACE2 Angiotensin-converting enzyme 2
  • the subject is vaccinated against the coronavirus. In some embodiments, the subject is unvaccinated against the coronavirus.
  • the coronavirus comprises SARS-CoV-2 or a variant thereof .
  • the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof.
  • the second protein or peptide comprises the S1 subunit of the spike protein.
  • the biological sample has an average volume of less than 1 milliliter when obtained. In some embodiments, the biological sample comprises capillary blood. In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture.
  • the method further comprises administering a vaccine against the coronavirus to the subject based on measuring in (b).
  • the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
  • the subject is an age comprising greater than or equal to about 50 years of age.
  • the method further comprises eluting the biological sample from a dried blood spot card.
  • the percent inhibition is predictive of a clinical endpoint when the subject received a pre-approved or licensed vaccine against the coronavirus.
  • the pre-approved or licensed vaccine comprises Ad26.COV.S, mRNA-1273 or BNT162b2, or any combination thereof.
  • the percent inhibition is predictive of individual protection against another infection by the coronavirus.
  • the method further comprises administering to the subject a vaccine booster against the coronavirus or a second dose of a vaccine against the coronavirus, when the percent inhibition is below low relative to a control of the index derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus.
  • the subject has an underlying immune-compromising condition.
  • the underlying immune-comprising condition comprises a pregnancy, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof.
  • the subject is breastfeeding.
  • the method further comprises detecting the binding complex using a lateral flow assay.
  • the one or detectable moieties comprises a nanoparticle, a fluorescent metal, a fluorescent dye, an enzymatic label, or a colorimetric label, or any combination thereof.
  • the method further comprises determining that the biological sample comprises about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell -based pseudovirion neutralization assay (PNA), when the percent neutralization measured is about 62%. In some embodiments, the method further comprises determining that the subject is adequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of about 62%. In some embodiments, the method further comprises determining that the biological sample comprises less than about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell -based pseudovirion neutralization assay (PNA), when the percent neutralization measured is less than about 62%.
  • PNA cell -based pseudovirion neutralization assay
  • the method further comprises determining that the subject is inadequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of less than about 62%.
  • the method is performed without a wash step to wash away the first peptide or protein or the second peptide or protein that are unbound following the bringing in (a).
  • the bringing the biological sample into contact with the first peptide or protein and the second peptide or protein is performed in a container.
  • the container is optically clear.
  • the container is a disposable cartridge.
  • the method further comprises repeating the method on a plurality of biological samples obtained from the subject, wherein at least two biological samples of the plurality of biological samples are obtained at different time points.
  • the subject is receiving an immunosuppressant therapy.
  • the method further comprises: (a) identifying the subject as needing a dose of a vaccine against the coronavirus, provided the percent neutralization is lower than 62%; and (b) suspending a treatment regimen comprising the immunosuppressant therapy for a period of time during which period of time, the subject is administered the dose of the vaccine.
  • the subject is a pregnant subject. INCORPORATION BY REFERENCE
  • FIG. 1 shows an exemplary assay measuring binding between an immobilized human angiotensin-converting enzyme 2 (ACE2) receptor and a peptide-conjugate derived from a spike glycoprotein of a SARS-CoV-2 in a biological sample obtained from a patient that has notbeen exposed to SARS-CoV-2.
  • ACE2 angiotensin-converting enzyme 2
  • FIG. 2 shows an exemplary assay measuring binding between an immobilized human ACE2 receptor and a peptide-conjugate derived from the spike glycoprotein of SARS-CoV-2 in a biological sample obtained from a patient exposed to SARS-CoV-2.
  • FIG. 3 shows an exemplary lateral flow assay to measure binding between human ACE2 receptor and a peptide-conjugate derived from the spike glycoprotein of SARS-CoV-2 in a biological sample obtained from a patient that has notbeen exposed to SARS-CoV-2.
  • FIG. 4 shows an exemplary lateral flow assay to measure binding between human ACE2 receptor and a peptide-conjugate derived from the spike glycoprotein of SARS-CoV-2 in a biological sample obtainedfrom a patient that was exposed to SARS-CoV-2.
  • FIG. 5 shows an exemplary system according to some embodiments.
  • FIG. 6 shows a computing device; in this case, a device with one or more processors, memory, storage, and a network interface, in accordance with some embodiments.
  • FIG. 7 shows an exemplary assay in FIG. 2 in an image-free system; results from the exemplary assay is visible and can be interpreted by the naked eye.
  • FIG. 8A-8D shows an exemplary assay assembly to measure inhibition of binding between the receptor binding domain (RBD) of SARS-CoV-2 and its cognate receptor, ACE2.
  • FIG. 8A illustrates assay with a first RBD (RBD 1 ) with a patient sample that does not contain neutralizing antibodies that functionally inhibitRBDl -ACE2 binding.
  • FIG. 8B shows the same assay from FIG. 8A with a patient sample that contains neutralizing antibodies that functionally inhibit RBD 1-ACE2 binding.
  • FIG. 8C shows a neutralization assay that measures a presence of neutralizing antibodies against at least two different RBD peptides (RBD1 and RBD2) in the absence of neutralizing antibodies (FIG. 8C) and in the presence of neutralizing antibodies (FIG. 8D).
  • FIG. 9A-9D illustrate that the assay assembly from FIG. 8A-8D may be performed in solution (e.g., not on a solid surface) using fluorescence resonance energy transfer (FRET).
  • FIG. 9 A shows electron transfer between the donor fluoroph ore and the acceptor fluorophore, indicating an absence of neutralizing antibodies that block binding between RBD and ACE2.
  • FIG. 9B shows no electron transfer between the donor fluorophore and the acceptor fluorophore, indicating a presence of neutralizing antibodies that b lockbinding between RBD.
  • FRET fluorescence resonance energy transfer
  • FIG. 9C shows a neutralization assay in solution in which electron transfer occurs between two different donor fluorophore and the acceptor fluorophore pairs, indicating an absence of neutralizing antibodies thatblockbindingbetween firstRBD, RBD 1, and second RBD, RBD2.
  • FIG. 9D shows a neutralization assay in solution in which no electron transfer occurs between two different donor fluorophore and the acceptor fluorophore pairs, indicating a presence of neutralizing antib odies that block binding b etween RBD 1 and RBD2.
  • FIG. 10A-10E illustrate homogenous solution with reaction components, where the homogenous solution is applied to a surface with surface-bound capture molecules.
  • FIG. 10A illustrates a homogenous solution comprising the reaction components of FIG. 8A being applied to surface-bound capture molecules of FIG. 8A.
  • the capture molecule captures an epitope tag conjugated to RBD, which forms a complex with ACE2 conjugated to the detective moiety.
  • FIG. 10B illustrates a homogenous solution comprising reaction components being applied to surface-bound capture molecules of FIG. 8A.
  • the capture molecule captures an epitope tag conjugated to ACE2, which forms a complex with RBD conjugated to the detective moiety.
  • FIG. 10C illustrates a homogenous solution comprising the reaction components of FIG.
  • FIG. 10D illustrates a homogenous solution comprisingthe reaction components of FIG. 8C being applied to surfacebound capture molecules of FIG. 8C.
  • FIG. 10E illustrates a homogenous solution comprising the reaction components of FIG. 8D being applied to surface-bound capture molecules of FIG. 8D
  • FIS. 11A-11B illustrate one of the many advantages of utilizing the neutralizing antibody test disclosed herein to reduce risk posed by individuals who may not know if they are adequately protected by their vaccine.
  • FIG. 11 A illustrates two vaccines having different levels of efficacy in a population of individuals, and the risks and benefits associated therewith.
  • FIG. 11B illustrates that the methods provided herein, in some embodiments, can reduce or remove the risk associated with usage of a commercialized vaccine.
  • FIG. 12A-12C illustrate a biological sample medium (BSM) capable of storing one or more biological samples in both a closed (FIG. 12A) and open (FIG. 12B) configuration.
  • FIG 12C illustrates a survey printed on the BSM.
  • BSM biological sample medium
  • FIG 13 illustrates a system accordingto some embodiments disclosed herein.
  • FIG. 14 illustrates a workflow of the automated systems disclosed herein accordingto some embodiments.
  • FIG. 15 shows results neutralizing antibody inhibition of SARS-CoV-2 S1 bindingto ACE2 from a time-resolved fluorescence resonance energy transfer (TR-FRET) assay run on 384 well plate (left) and a 1536 well plate (right).
  • TR-FRET time-resolved fluorescence resonance energy transfer
  • FIG. 16 shows that, unexpectedly, neutralization levels against SARS-CoV-2 can vary widely in subjects who are vaccinated against SARS-CoV-2 using.
  • FIG. 16 shows the percent of neutralizing antibody response in 7 vaccinated individuals.
  • FIG. 17 shows the repeatability of IC 50 measurements using systems described herein.
  • FIGS. 18A-18C shows results from three-time separate TR-FRET assays (also referenced as Runs 1 , 2 and 3) using different reagent lots and performed on difference days. The linear trends observed when run 1 was plotted against run 2 (FIG. 18A) and run 1 against run 3 (FIG. 18B) indicate that no batch effects were observed.
  • FIG. 18C is a plot of two additional TR-FRET assays on different sera than FIGS. 18A-18B, which also show a linear trend.
  • FIG. 19 shows that the neutralizing antibody test disclosed herein correlates with the cell-based Pseudovirion Neutralization Assay (PNA) neutralization assay up to a titer of IC50 of about 1,000.
  • PNA Pseudovirion Neutralization Assay
  • the inventors of the instant disclosure discovered an urgent and unmet need for a confirmatory diagnostic test that determines whether an allowed or approved vaccine for the treatmentof a disease caused by SARS-CoV-2 is effective to confer adaptive immunity against SARS-CoV-2 outside the clinical trial context.
  • the inventors also discovered that existingtests for antibodies against SARS-CoV-2 fail to measure the functional inhibition (e.g., blocking) of binding between the spike protein and its cognate receptor (ACE2), which mediates infection by SARS-CoV-2. This means that existing tests are not able to determine whether a subject has sufficient adaptive immunity against SARS-CoV-2 induced by the vaccine following vaccine administration.
  • the inventors of the instant disclosure developed a neutralizing antibody test that measures the functional inhibition between the spike protein and ACE2, which can be used to determine whether a vaccine is effective to confer an adaptive immune response sufficient to protect a subject from a future infection.
  • the neutralizing antibody tests described herein can also be used to measure the durability of a vaccine over time following a prior administration of the vaccine, which indicates whether a booster of the vaccine may be needed.
  • the neutralizing antibody tests described herein can be used to screen a population of individuals.
  • the neutralizing antibody tests described herein can be used to identify individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine.
  • the neutralizing antibody tests described herein can be used to minimize or reduce the risk associated with vaccine that has been commercialized for usage in a population (e.g., by identifying individuals that do not respond to the vaccine).
  • the neutralizing antibody tests described herein can provide a surrogate marker of protection from a vaccine.
  • the neutralizing antibody tests described herein can be used to classify vaccines by % efficacy in a population. With over 300 million people in the United States alone in need of vaccination, the neutralizing antibody tests described herein are both scalable and cost- efficient.
  • SARS-CoV-2 tests that detect a presence of antibodies against SARS-CoV-2 lack specificity because they cannot assess the amount of cross reactivity between the antibodies in a patient sample and SARS-CoV-2 versus more benign (e.g., seasonal) coronaviruses.
  • SARS-CoV-2 tests thatdetectbindingbetween an antibody in a patient sample and the spike protein exist they do not necessarily indicate when a sufficient adaptive immune response to SARS-CoV-2 has occurred. Even direct antibody binding to the receptor binding domain of the spike protein does not always inhibit viral entry into a cell initiating an infection.
  • the gold standard test for assessing clinical immunity to a virus utilizes a cell-based assay.
  • the cell-based assay quantitatively measures neutralizing antibody titers in patient sera by adding patient serum and a solution containing the virus to viral -susceptible cells, and analyzing the cells to determine if the virus can no longer infect the cells.
  • a reduction in viral induced cytotoxicity is a measure of neutralization activity in the sera.
  • the strength of neutralization is reported in two ways: (i) the IC50 (e.g., half the cells are killed) or (ii) highest dilution at which neutralization activity disappears.
  • This cell -based assay has limited clinical utility, because it is difficult to scale up, is cost and time intensive, and require technical training to perform.
  • the inability to scale these cell-based assays means clinical utility for larger populations of individuals is limited.
  • the time, cost, and technical expertise required means clinical utility of cell-based assay at the point of need is limited. Accordingly, there is an urgent need for a scalable assay that can also be used at the point of need to measure clinical immunity.
  • the testing devices and systems described herein directly detect the functional inhibition of binding between the receptor binding domain of the spike protein and its cognate receptor (e.g., ACE2) by neutralizing antibodies in a patient sample.
  • the devices and systems described herein have increased specificity to neutralizing antibodies to SARS-CoV-2, because they can (i) discriminate neutralizing antibodies from nonneutralizing antibodies, and (ii) assess cross reactivity between antibodies in the patient sample and benign coronaviruses.
  • the testing devices and systems described herein are designed to indicate when a sufficient adaptive immune response to SARS-CoV-2 has occurred, by detecting the functional inhibition of binding between ACE2 and the spike protein when in a presence of antibodies in a patient sample.
  • Testing devices and systems described herein are portable, do not require technical expertise to use or perform, and are far less costly than the gold standard test (e.g., cell-based assay) for assessing clinical immunity. For at least these reasons, the devices and systems described herein are an ideal test at the point of need.
  • the gold standard test e.g., cell-based assay
  • testing devices and systems described herein are easily scalable, at least because the testing device (i) has an assay assembly that is simple (e.g., lateral flow, solution, or solid phase capture), (ii) does not require complex imaging devices, like f or e.g., BioTek® Cytation plate reader, and (iii) does not require use of a cell (or cell line).
  • the testing device is manufactured before the discovery of a new strain of virus.
  • the assay assembly of the testing device is modified by replacing one component of the assay assembly (e.g., the recombinant RBD peptide unique to the new strain of SARS- CoV).
  • the new strain may comprise one or more mutations to SEQ ID NO: 5.
  • the new strain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1.
  • the assay assembly is configured for centralized and automated processing, enabling high throughput performance with significantly lower reagent amounts. Methods of utilizing the testing devices and systems described herein, in some cases, leverage robotics or other means of automation that can perform upwards of 60,000 tests per day using less than one-quarter of the reagents required for existing tests.
  • the high throughput systems and methods for analyzing a biological sample described herein may analyze more than one biological sample (e.g., hundreds or thousands of biological samples) in a single day.
  • the high throughput performance may include between about 5,000 to about 20,000 unique processed biological samples every 8 hours.
  • the high throughputperforman ce may be achievedby utilizing a biological sample medium (BSM) 1201, as shown in FIG. 12A-12B, which contains a biological sample obtained from a subject (e.g., a finger prick) and, in some embodiments, integrates the biological sample into an automated or semi-automated system for sample processing and assayingthe biological sample.
  • the BSM may be a dried blood spot card (DBS).
  • DBS dried blood spot card
  • the automated or semi-automated systems 1300 described herein comprise a biological sample processor 1302 and an assay assembly block 1309.
  • serial dilutions e.g., dilutions of 1 :20, 1 : 160, 1 :320, 1 :640 sample to solution
  • assay e.g., FRET, ELISA
  • the testing devices described herein measure a presence or quantity of all antibodies(e.g., neutralizing or otherwise) againstthe pathogen (e.g, SARS-CoV-2) that are unique to a subject.
  • the testing devices differentiate between antibody classes, such as immunoglobulin G (IgG) and immunoglobulin M (IgM).
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • a presence or a quantity of IgG, IgM, or a combination thereof, in a patient sample is indicative of an acute infection.
  • the testing device described herein can determine whether a subject is suffering from an acute infection by the pathogen (e.g, SARS-CoV-2), and whether the subject has developed a sufficient adaptive immunity to the pathogen such that they are immune from a future infection.
  • the testing devices and systems described here are capable of adapting to rapidly evolving viral outbreaks in a manner that is considerably faster than existing technologies.
  • the testing devices test for more than one strain of a virus.
  • the testing devices and systems described herein are capable of identifying whether an individual is immune to human 2002-2003 and 2003-2004 SARS-CoV, with S proteins corresponding to GenBank accession no. AY274119 and GenBank accessionno. AY525636, respectively.
  • the testing devices and systems utilize recombinant RBD peptides that each have a different epitope region unique to the strain of SARS-CoV. Proper epitope presentation is accomplishedby engineering the recombinant peptides to exhibit the same tertiary and quaternary structures as the corresponding pathogenic virus.
  • testing devices and systems for measuring adaptive immunity to a pathogen in a subject are point of need or point of care devices.
  • the testing devices are configured to perform an assay on a biological sample of the subject to detect sufficient antibody titers to confer adaptive immunity to a pathogen of interest in the subject.
  • the biological sample includes cell, tissue, or bodily fluid obtained from the subject.
  • biological samples include aqueous humour, vitreous humour, bile, breast milk, cerebrospinal fluid, endolymph, perilymph, female ejaculate, amniotic fluid, gastric juice, menses, mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, urine, feces, whole blood, blood serum, blood plasma, sputum, cerebrospinal fluid, synovial fluid, lymphatic fluid, nasal swab, or cheek swab.
  • the biological sample comprises blood, urine, saliva, or feces.
  • the blood is capillary blood.
  • the capillary blood is obtained by a finger prick.
  • the capillary blood sample comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 drops of blood.
  • the capillary blood sample comprises a volume of blood less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ⁇ L.
  • the subject pricks a finger, obtains a drop of blood from the finger prick, and places the drop of blood on a blood card to be stored.
  • the subject may do this 3-4 times for the same blood card.
  • the subject mails the blood card to the laboratory for processing.
  • the assay is a competition assay comprising one or more capture molecules, optionally coupled to a surface, and a detectable peptide-conjugate.
  • a signal from the detectable peptide-conjugate is detected by capturing an image of a detection zone of the device by an imaging device.
  • the imaging device is a smartphone.
  • the assay is a lateral flow assay (LFA).
  • the pathogen of interest is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • the detectable peptide-conjugate comprises a peptide derived from a spike glycoprotein of a coronavirus, and the one or more capture molecules is derived from an angiotensin-converting enzyme 2 (ACE2) receptor, or a surrogate thereof (e.g., heparin or a fragment thereof).
  • the detectable peptide-conjugate comprises a peptide derived from the ACE2 receptor, and the one or more capture molecules is derived from the spike glycoprotein of a coronavirus.
  • the coronavirus is a severe acute respiratory syndrome coronavirus (SARS-CoV).
  • the SARS-CoV is SARS-CoV-2.
  • the ACE2 receptor is derived from a human ACE2 receptor.
  • more than one detectable peptide-conjugate is utilized to measure neutralizing antibodies against more than one strain of SARS-CoV.
  • Systems described herein comprise the testing device and the imaging device.
  • systems further comprise a computing device with an application (e.g., web or mobile) comprising a data analytics module for receiving an analyzing data from the imaging device to provide a result.
  • the imaging device is the computing device (e.g., smartphone).
  • the imaging device is notthe computing device.
  • the result is a positive result indicating that a subject is immune to an infection by the pathogen.
  • the result is a negative result indicating that the subject is not immune to an infection by the pathogen.
  • the application further comprises a communication module configured to display via a graphical user interface (GUI) one or more results to a user.
  • systems comprise a data store configured to store and retrieve data from the imaging device, the external device, or both.
  • GUI graphical user interface
  • systems comprise an external device, such as a wearable tracking device (e.g., Aurora®, Fitbit ®, Apple Watch).
  • the data analytics module receives and analyzes external data from the external device.
  • external data comprise body temperature, heart rate, heart rate variability, sleep quality, or sleep quantify of the subject.
  • data analytics module is configured to analyze the external data in combination with the data received from the imaging device to identify the subject as being infected with a specific pathogen.
  • systems described herein comprise multiple users with multiple computing devices. Within a geographical location of interest, systems described herein are designed to determine whether a population of users has become immune to an infection by the pathogen (e.g., SARS-CoV-2). In some instances, global position system (GPS) tracking of the personal computing device, the imaging devices, or both, can be used to produce a geofence surrounding the geographical location of interest.
  • pathogen e.g., SARS-CoV-2
  • GPS global position system
  • testing devices that can be deployed at the point of need to determine whether a patient is immune to an infection by the pathogen.
  • devices comprise an assay assembly capable of assaying a biological sample obtained from the patient.
  • the testing devices comprise one or more components, such as a housing, a sample receiver, a sample processor, a sample purifier, or a detection zone.
  • the testing systems described herein comprise one or more testing devices described herein and a biological sample medium (BSM) that stores the biological sample.
  • BSM biological sample medium
  • the one or more components of the testing devices or systems described herein elutes the biological sample from the BSM prior to assaying the sample using the assay assembly ofthe testing device or system.
  • devices comprising an assay assembly that is capable of detecting a target analyte.
  • the target analyte is an antibody specific to a pathogen of interest.
  • the antibody specific to the pathogen of interest functionally blocks binding between the pathogen to its cognate receptor (e.g., a “neutralizing antibody”).
  • the neutralizing antibody is elicited in a subject by a vaccine or an infection by the pathogen.
  • the testing devices comprise two assay assemblies, the first assay assembly capable of detecting an antibody specific to a pathogen of interest, and the second assay assembly capable of detecting a neutralizing antibody that functionally blocks binding between the pathogen and its cognate (host) receptor.
  • the target analyte is a biomarker. It is contemplated that any combination of assay assemblies described herein may be used in combination, depending on the desired result. In some embodiments, the combination is housed in a single testing device. In some embodiments, the combination is housed in more than one testing device.
  • the pathogen comprises a virus, a bacteria, a parasite, a fungus, or a combination thereof.
  • the virus is a coronavirus.
  • the coronavirus is a severe acute respiratory syndrome coronavirus (SARS- CoV).
  • the SARS-CoV is SARS-CoV-2.
  • the coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV).
  • the coronavirus is an alpha coronavirus (e.g., 229E, NL63).
  • the coronavirus is a beta coronavirus (e.g., OC43, HKU1).
  • the virus is an influenza virus.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequences, or portions thereof, provided in SEQ ID NOS: 1-116.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO : 12, or a portion thereof.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, 47-48, 71-72, 95-96, or 115, or a portion thereof.
  • the coronavirus comprises a viral genome comprising a sequence that is greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7-10, 13-20, 43, 67, or 91 , or a portion thereof.
  • the pathogen ofinterest is a variant of the pathogen, for example, a pathogen that has evolved through mutations in the genes encoding the pathogen.
  • the variant virus may have a mutation in an amino acid sequence of a viral protein, such as an envelope protein, nucleocapsid protein, membrane protein, hemaggluitinin, neuraminidase, or spike protein.
  • the virus e.g., coronavirus
  • the mutation does not affect, or minimally affects, host receptor binding by the spike protein of the coronavirus.
  • the mutation is an insertion, a deletion, or a substitution at an amino acid (or a plurality of amino acids) of one or more sequences provided in SEQ ID NOS: 1-116 for coronaviruses (e.g., SARS, SARS-CoV, and NL63).
  • SEQ ID NOS: 1-116 for coronaviruses e.g., SARS, SARS-CoV, and NL63.
  • the Non-limiting examples of mutations in an amino acid sequence of the spike protein of SARS-CoV-2 (SEQ ID NO: 5) are provided in Long et al., Molecular Architecture of Early Dissemination andMassive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area, Am. Soc. M.
  • the variant of SARS-CoV-2 comprises a spike protein having an amino acid sequence comprising a substitution of an aspartic acid (Asp) to a glycine (Gly) at amino acid position 614 (Asp614Gly) with reference to SEQ ID NO: 5 (SARS-CoV-2).
  • the variant of SARS- CoV-2 comprises a spike protein having an amino acid sequence comprising one or more substitution selected from Ala442Val, Ala448Val, Ala553Pro or Vai, Gly682Arg, Ser758Pro, and Cys812Phe, with reference to SEQ ID NO: 5 (SARS-CoV-2).
  • the antibody specific to coronavirus is specific to the receptor binding domain of the spike protein of the coronavirus, such that the antibody blocks binding of the spike protein to its cognate receptor (e.g., ACE2).
  • the analyte is a complex comprising the spike protein bound to the antibody at the receptor binding region of the spike protein.
  • the antibody specific to the pathogen of interest belongs to an immunoglobulin class comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, or immunoglobulin D.
  • the target analyte (or second target analyte) is a biomarker.
  • the assay assembly is configured to detect at least one, two, three, four, five, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1,000 or more biomarkers in the biological sample.
  • the biomarker comprises a non -peptide coding nucleic acid sequence.
  • the biomarker comprise a peptide coding nucleic acid sequence such as mRNA or cDNA.
  • the biomarker comprises a peptide or a protein.
  • the biomarker is derived from the pathogen of interest (e.g., coronavirus).
  • the assay assembly is adapted to detect a region of the pathogen of interest.
  • the biomarker comprises orfla, orflab, spike protein (S protein), 3a, 3b, envelope protein (E protein), matrix protein (M protein), p6, 7a, 7b, 8b, 9b, nucleocapsid protein (N protein), orf 14, nspl (leader protein), nsp2, nsp3, nsp4, nsp5 (3C-like proteinase), nsp6, nsp7, nsp8, nsp9, nspl 0 (growth -factor-like protein), nspl2 (RNA- dependent RNA polymerase, or RdRp), nspl 3 (RNA 5 '-triphosphatase), nspl 4 (3'-to-to
  • the biomarker is a proinflammatory marker.
  • the proinflammatory marker comprises 4-1BBL, acylation stimulating protein, adipokine, albinterferon, APRIL, Arh, BAFF, Bcl-6, CCL1, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L/CD178,
  • the biomarkers includes GM-CSF, IL-1 ⁇ , IL-5, IL-7, IL-12/23 p40, IL-15, IL- 16, IL-17A, TNF- ⁇ , VEGF, INF-y, IL-1 ⁇ , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p70, IL-13, TNF- ⁇ , Eotaxin, MIP-1 ⁇ , Eotaxin-3, TARC, IP-10, MIP-1 ⁇ , IL-8, MCP-1, MDC, MCP-4, IFN-g, or VEGF-A, or a combination thereof.
  • the assay assembly comprises one or more capture molecules.
  • Non-limiting examples of capture molecules include a nucleic acid molecule, peptide, protein, or fragments thereof.
  • the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule.
  • the nucleic acid molecule or peptide is an aptamer that binds to an analyte described herein.
  • the peptide or protein is derived from the receptor binding domain (RBD) or the spike protein from a coronavirus.
  • the peptide or protein is derived from the ACE2 receptor surfaces for biomolecular detection (e.g., human ACE2).
  • the peptide or protein is an antibody or antigen-binding fragment that directly or indirectly binds the RBD of the spike protein or ACE2.
  • the capture molecule may include heparin, or a fragment thereof.
  • the one or more capture molecules is coupled to a solid surface. In some embodiments, the one or more capture molecules is not coupled to a solid surface. In some embodiments, the solid surface is made of silicon, glass (silicon dioxide), nitrocellulose, gold, silver, polystyrene, graphene, or crystal. In some embodiments, the solid surface is ahead or a plate.
  • the plate comprises reaction wells, such as in a 96-well plate, a 384-well plate, or a 1536-well plate.
  • the bead is sub stantially spherical in shape.
  • the surface is passivated.
  • the surface comprises a polymer coating comprising a polymer selected from polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly glutamic acid (PGA), poly -lysine, polyglucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyridine)
  • PVP poly(vinyl pyrrolidone)
  • PAA poly(acrylic acid)
  • PIPAM polyacrylamide
  • PMA
  • the one or more capture molecules is coupled to the surface directly or indirectly.
  • Capture molecules coupled indirectly to the surface may, for example, be coupled to the surface by a linker.
  • the linker is a chemical linker, a peptide link, a polymer linker, or a combination thereof.
  • an epitope tag is conjugated to the one or more capture molecules.
  • Non -limiting examples of an epitope tag include MBP, His-tag, V5 tag, c-my, HA, S-tag, strep-tag, strep-MAB, VSV-G, GST, GFP, mCherry, CFP, BFP, and DYKDDDDK (SEQ ID NO: 116).
  • the one or more capture molecules is bound by a primary capture antibody that is bound to the surface covalently or non-covalently.
  • the one or more capture molecules is conjugated to an epitope tag and the epitope tag is coupled to a primary capture antibody that is bound to the surface.
  • Capture molecules coupled directly to the surface may, for example, be covalently or non-covalently bound to the surface.
  • the one or more capture molecules described herein may be immobilized to the surface directly or indirectly using a suitable surface modification chemistry depending on the intended use.
  • suitable surface modification chemistries are provided in Sonawane el al., Surface Modification Chemistries ofMaterials used in Diagnostic Platforms with Biomolecules, Journal of Chemistry (2016), which is hereby incorporated by references in its entirety.
  • the one or more capture molecules is derived from an angiotensin-converting enzyme 2 (ACE2) receptor or a fragment thereof, or a surrogate of ACE2 (e.g., heparin) or a fragment thereof .
  • ACE2 angiotensin-converting enzyme 2
  • the fragment of ACE2 comprises an extracellular portion of ACE2 that binds to the spike protein. In some embodiments, the extracellular portion of ACE2 comprises the peptidase domain. In some embodiments, the fragment of ACE2 is a fragment of the extracellular portion of ACE2 that is sufficient for binding to the spike protein of a coronavirus, which are provided in Li et al., Structure of SARS coronavirus spike receptor -binding domain complexed with receptor. Science 309, 1864-1868 (2005); and Yan et al., Structural basis for the recognition ofSARS- CoV-2 by full-length human ACE2. Science.
  • ACE2 is human ACE2 (Entrez ID 59272).
  • the one or more capture molecules comprises a portion of the ACE2 polypeptide provided in SEQ ID NO: 1.
  • the one or more capture molecules comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the human ACE2 polypeptide (SEQ ID NO: 1).
  • the one or more capture molecules comprisesan amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence from amino acid 19 to amino acid 615 of SEQ ID NO: 1.
  • the one or more capture molecules is a peptide or protein comprising an amino acid sequence encoding a variant of the human ACE2 polypeptide (SEQ ID NO: 1), or a fragment thereof .
  • the one or more capture molecules comprises an amino acid sequence comprising a Serl9Pro or a Asp329Gly substitution with reference to SEQ ID NO: 1
  • Variants of ACE2 are provided in Hussain et al., Structural variants in human ACE2 may influence its binding with SARS-CoV-2 spike protein, J. Med. Virol. 2020; 92:1580- 1586, which is incorporated by reference herein in its entirety.
  • the one or more capture molecules comprises at least 2, 3 , 4, 5, 6, 7, 8, 9, or 10 capture molecules.
  • the one or more capture molecules is a surrogate for ACE2.
  • a non-limiting example of a surrogate for ACE2 is heparin.
  • the one or more capture molecules comprises heparin, or a fragment thereof that is sufficient for binding to the spike protein.
  • Heparin may competitively bind to the spike protein of a SARS-CoV with an affinity that is similar to that of ACE2.
  • the affinity of binding between heparin and the spike protein is less than about 1E -7 , 1E -8 , 1E -9 , or 1E -10 Kd. In some cases, the binding affinity is from about 1E -9 to about 1E -10 Kd.
  • a surrogate of ACE2 (e.g., heparin) provided herein has a binding affinity to a spike protein of less than about 1E -7 , 1E -8 , 1E -9 , 1E -10 , or lE -11 Kd
  • the affinity of binding between heparin (or other surrogate of ACE2) and the spike protein is 1.20E -8 .
  • the affinity of binding between heparin (or other surrogate of ACE2) and the spike protein is 8.30E -10 .
  • the one or more capture molecules is a surrogate f or ACE2 with a binding affinity or avidity for the spike protein of less than about 1E -7 , 1E -8 , 1E -9 , 1E -10 , or 1E -11 Kd.
  • Suitable methods of identifying surrogates for ACE2 to comparable binding affinities to the spike protein are provided at least in Guan-Yu et al., The discovery of potential natural products for targeting SARS-CoV-2 spike protein by virtual screening, bioRxiv 2020.06.25.170639. Suitable methods of calculating the affinity of binding are provided in Vangone A, Bonvin A. PRODIGY : a contact-based predictor ofb in ding affinity in protein-protein complexes. Bio-Protocol. (2017) 7:2124.
  • the one or more capture molecules is derived from a spike glycoprotein of a coronavirus.
  • the coronavirus is a severe acute respiratory syndrome coronavirus (SARS-CoV).
  • the SARS-CoV is SARS-CoV-2.
  • the coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV).
  • the coronavirus is an alpha coronavirus (e.g., 229E, NL63).
  • the coronavirus is a beta coronavirus (e.g., OC43, HKU1).
  • the one or more capture molecules comprises a portion of the spike protein.
  • the portion comprises subunit 1 of the spike protein. In some embodiments, the portion comprises the receptor binding domain (RBD) of subunit 1 of the spike protein.
  • the one or more capture molecules comprises an amino acid sequence atleast70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the spike protein, the subunit 1 of the spike protein, or the RBD of the subunit 1 of the spike protein, or a combination thereof. In some embodiments, the one ormore capture molecules comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 capture molecules.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12.
  • the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24, 47-48, 71-72, 95-96, or 115
  • the one or more capture molecules comprises a peptide or a protein having an amino acid sequence atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 50 amino acids of SEQ ID NO: 1-6, 11-12, 24, 47-48, 71-72, 95-96, or 115.
  • the one or more capture molecules comprises a peptide or a protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 100 amino acids of SEQ ID NO: 1-6, 11-12, 24, 47-48, 71-72, 95-96, or 115.
  • the one or more capture molecules comprises a peptide or a protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 150 amino acids of SEQ ID NO: 1-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • the one or more capture molecules comprises an amino acid sequence comprising asparagine at amino acid positions 90, 322, 546, and 343 of the amino acid sequence of the spike protein, such as SEQ ID NO: 2, 5, or 11.
  • the spike protein has an amino acid sequence comprising one ormore of 344K,360F, 472L, 479N, 480D, and 487T with reference to SEQ ID NO: 2, 5, or 11 In some embodiments, the spike protein has an amino acid sequence comprising one or more of 344R, 360S, 472P, 479 N, 480G, and 487S with reference to SEQ ID NO: 2, 5, or 11 In some embodiments, the spike protein has an amino acid sequence comprising 344R, 360S, 472L, 479K, 480D, 487S with reference to SEQ ID NO: 2, 5, or 11
  • the spike protein is derived from a mammal. In some embodiments, the spike protein is derived from a human. In some embodiments, the spike protein is derived from a non -human mammal, such as a palm civet or a bat.
  • the one or more capture molecules is an antibody, or antibody fragment, specificto one or more antibodies against the pathogen described herein (e.g., IgM, IgG, IgA, IgE, IgD).
  • the one ormore capture molecules is a monoclonal antibody or a polyclonal antibody.
  • the one or more capture molecules comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 capture molecules.
  • the one ormore capture molecules is a fusion protein comprising a peptide directly or indirectly bound to the surface.
  • the peptide comprises a fragment crystallizable (Fc) region of a monoclonal antibody.
  • the Fc region is derived from an antibody belongingto an immunoglobulin class comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, or immunoglobulin D.
  • a biological sample obtained from a subject that has not been exposed to a coronavirus is assayed using the assay assembly described herein.
  • a labeled capture molecule (“CoV-2 S-Au”) comprises a peptide-conjugate comprising a detection agent (e.g., gold nanoparticle) and a peptide derived from the spike protein of SARS-CoV-2.
  • a fluid formulation comprising the peptide-conjugate is contacted with a biological sample from the subject.
  • the target analyte is an activity of one or more antibodies comprising blocking the binding between the spike protein of SARS- CoV-2 and ACE2.
  • the patient has not been exposed to the coronavirus, so a presence of the analyte is not expected to be detected.
  • an immobilized capture molecule is coupled to a solid surface.
  • the immobilized capture molecule is human ACE2 receptor, surrogate thereof (e.g., heparin), or a fragment thereof.
  • the liquid formulation is applied to the solid surface.
  • a high number of complexes between the immobilized human ACE2 and the detectable peptide-conjugate is detected, correlating with an absence or a low amount of analyte in the sample.
  • an image of the surface is captured with an imaging device.
  • the image is a video or still image.
  • the imaging device comprises a reflectance reader.
  • the imaging device is a personal electronic device, such as a smartphone.
  • a low signal When imaged from above the surface, a low signal indicates a high degree of binding between human ACE2 and the peptide-conjugate (therefore, a low amount of the analyte).
  • a high signal When imaged from below the surface, a high signal indicates a high degree of binding between the human ACE2 and the peptide-conjugate.
  • FIG. 2 a biological sample obtained from a subject that was exposed to a coronavirus assayed usingthe assay assembly described herein, is provided in FIG. 2.
  • a low number of complexes between the immobilized human ACE2 and the detectable peptide- conjugate is detected, which correlates with a presence absence or a high amount of analyte in the sample.
  • the surface of the image is not captured.
  • a biological sample obtained fromthe subject exposed to a coronavirus is assayed usingthe assay assembly described here.
  • a low number of complexes between the immobilized human ACE2 and the detectable peptide-conjugate is detectable by the human eye in the detection zone of the device.
  • the assay assembly is adapted for nucleic acid molecule detection, such as to detect a nucleic acid molecule encoding a biomarker or an antibody described herein.
  • the assay assembly is adapted to detect RNA (or cDNA) encoding a region of the pathogen of interest or a marker of an adaptive immune response, such as a cytokine.
  • the assay assembly performs reverse transcription polymerase chain reaction (RT-PCR).
  • the assay assembly is a singleplex (e.g., individual assays) or multiplexed (e.g., detection of more than one biomarker in a single reaction).
  • the assay assembly comprises a primer pair and a probe set to amplify and detect the biomarker.
  • the assay assembly comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 primer pairs.
  • the assay assembly comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 probes.
  • a “probe” is a nucleic acid molecule comprising greater than or equal to about 30 contiguous nucleobases adapted to hybridize to the nucleic acid sequence of the target region of the biomarker.
  • the RT -PCR is qualitative PCR (qPCR)
  • the probe comprises a detectable moiety.
  • the detectable moiety comprises TaqManTM, SYBR green, SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin, or a combination thereof.
  • theprobe may be a hydroly sableprobe comprising a fluorophore and quencher that is hydrolyzedby DNA polymerase when hybridizedto a target nucleic acid, such as for a TaqManTM assay assembly.
  • the assay assembly is an array comprising probes conjugated or otherwise immobilized on a surface described herein (e.g., a bead, multi-well plate), wherein the probes are configured to hybridize with the nucleic acid sequence of the target region.
  • the target region of the biomarker comprises a mutation, such as a single nucleotide variation or an indel.
  • the surface comprises an Affymetrix gene chip array, and the like.
  • the assays provided herein have an accuracy of at least 80%, for example, atleast 85%, atleast 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, atleast 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater.
  • the assays provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the assays provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, atleast 90%, atleast 91%, atleast 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the assays provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%.
  • the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the ref erence neutralizing antib ody has a known IC 50 ; and (b) detecting a binding complex b etween the detectable peptide and the one or more capture molecules; and (c) determine an IC 50 for the neutralizing antibody, wherein the IC 50 is approximately the known IC 50 .
  • accuracy is measured by comparingthe positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population.
  • true positive results both true positive and true negative
  • accuracy wouldbe measured by comparingthe true positives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) and true negatives (e.g., subjects that were exposed to the SARS-CoV-2 and did become infected).
  • the assay assembly is a solution -based assay and does not consist of a solid surface.
  • the solution-based assay comprises labeling any one of the capture molecules described herein by conjugating the capture molecule to a detectable moiety of any one of the moietiesdescribed herein.
  • the signal is detected when the capture molecule forms a complex between the capture molecule and the target analyte. In some embodiments, the signal is not detected when the capture molecule forms a complex between the capture molecule and the target analyte.
  • Exemplary solution-based assay comprising the labeled capture molecules includes Forster resonance energy transfer (FRET), fluorescence polarization (FP), radioligand binding assay, bioluminescent binding assay, and immunoprecipitation (e. g. ELISA and western blotting).
  • the solutionbased assay comprises unlabeled capture molecules, where the detection of the complex formed between the capture molecule and the target analyte is detected by a separate mechanism.
  • Exemplary solution-based assay comprising unlabeled capture molecules includes surface plasmon resonance (SPR), plasmon -waveguide resonance, whispering gallery microresonator (WGM), resonant waveguide grating (RWG), mass spectrometry, nuclear magnetic resonance, X-ray crystallography, thermal denaturation assays (TDA), and isothermal titration calorimetry (ITC).
  • SPR surface plasmon resonance
  • WGM whispering gallery microresonator
  • RWG resonant waveguide grating
  • mass spectrometry nuclear magnetic resonance
  • X-ray crystallography X-ray crystallography
  • TDA thermal denaturation assays
  • ITC isothermal titration calorimetry
  • the assay assembly is a (FRET) system.
  • the FRET system comprises coupling the FRET donor and FRET acceptor to any one of the capture molecules described herein.
  • the FRET donor and the FRET acceptor are fluorescent or luminescent molecules.
  • the luminescent molecule is a luminescent metal, such as lanthanide (e.g., terbium or europium).
  • the fluorescent molecule comprises an organic fluorescent dye, a fluorescent protein, semiconductor quantum dots, metal chelates, various noble metal and other nanoparticles, intrinsically fluorescent amino acids, biological cofactors, and polymers .
  • the FRET is lanthanide -based FRET (LRET).
  • a first peptide or protein derived from an antigenic component of a pathogen disclosed herein e.g. coronavirus
  • a first detectable moiety such as the FRET donor or FRET acceptor.
  • the second peptide or protein derived from a cognate receptor of the pathogen e.g., ACE2 receptor
  • a second detectable moiety such as a FRET acceptor or FRET donor.
  • the FRET donor and FRET acceptor emit detectable signals that differ from one another, such that when the FRET donor and the FRET acceptor are in close proximity, they emit a combined signal that differs from either of them alone.
  • performing the FRET assay comprises mixing a biological sample with the first peptide or protein (coupled to the FRET donor) and the second peptide or protein (coupled to the FRET acceptor) under conditions sufficient for the first peptide or protein and the second peptide or protein to form a binding complex in absence of neutralizing antibodies in the biological sample, and measuring a ratio of the signal emitted from the binding complex to the signal emitted from either of the FRET acceptor or the FRET donor alone.
  • this FRET assay is al so possible, wherein the first peptide or protein is coupled to the FRET acceptor and the second peptide or protein is coupled to the FRET donor.
  • the ratio of the signal for either of the FRET acceptor of the FRET donor will be larger in relation to the signal emitted from the binding complex.
  • the antigenic component of the pathogen is a portion of the spike protein in a coronavirus, such as SARS-CoV-2, and the cognate receptor is ACE2, such as human ACE2.
  • ACE2 such as human ACE2.
  • FIG. 9A-9D illustrates how FRET is utilized in solution to identify a presence or a quantity of neutralizing antibodies that functionally block binding between the RBD of the spike protein derived from SARS-CoV and the cognate receptor, ACE2.
  • FRET relies on the excitation energy of a donor fluorescent or luminescent molecule to a nearby acceptor fluorescent or luminescent molecule.
  • the firstpeptide or protein (e.g., RBD peptide, or portion thereof) is coupled to a donor fluorophore
  • the second peptide or protein (e.g., ACE2, or portion thereof) is coupled to an acceptor fluorophore
  • the donor or the acceptor fluorophore may be any suitable fluorescent protein.
  • the fluorescent proteins comprise a color selected from, blue, cyan, green, and yellow.
  • FIG. 9A when a biological sample that is obtained from an individual that does not have neutralizing antibodies against the RBD, excitation of the acceptor fluorophore is observed. Whereas, no excitation of the acceptor fluorophore is observed if the biological sample contains neutralizing antibodies against RBD, as shown in FIG. 9B.
  • FIG. 9C-9D provides an assay assembly in solution (e.g., not involving a solid surface) using FRET.
  • the FRET assay may be performed in a vessel, such as a container or a test tube.
  • the FRET assay is performed in an assay plate, such as, a 96 -well plate, a 384-well plate, or a 1536-well plate.
  • the FRET assay is performed in a high-throughput method to decrease the amount of reagents and the cost of performing the assay.
  • the FRET assay utilizes small volumes of reagents, for example, less than 100 nL, less than 90 nL, less than 80 nL, less than 70 nL, less than 60 nL, or less than 50 nL of reagents.
  • the biological sample (e.g., blood from a finger prick) is stored in or on a biological sample medium (BSM), such as a dried blood spot card (DBS), described herein.
  • BSM biological sample medium
  • DBS dried blood spot card
  • the biological sample is removed from the BSM and placed into the vessel (e.g., a well of a 96-well plate) to be eluted from the BSM.
  • the vessel e.g., a well of a 96-well plate
  • the eluted biological sample is assayed using the FRET system deposited on a solid surface described herein.
  • the FRET assay confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’ s biological sample. In some instances, the FRET assay prognoses a subject’s immunity to any one of the pathogen described herein. In some embodiments, the FRET assay prognoses a subject’s immunity to coronavirus. In some embodiments, the FRET assay prognoses a subject’s immunity to SARS-CoV-2. In some embodiments, the FRET assay measuresthe subject’s susceptibility to an infection by any one of the pathogens described herein. In some embodiments, the FRET assay measures the subject’s susceptibility to an infection by coronavirus.
  • the FRET assay measures the subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the FRET assay identifies individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the FRET assay minimizes or reduces the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the FRET assay identifies neutralizing antibodies as a surrogate marker of protection from a vaccine (e.g., in place of traditional, randomized, placebo-controlled trials). In some embodiments, the FRET assay classifies vaccines by % efficacy in a population.
  • the FRET assays provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the FRET assays provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, atleast 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the FRET assays provided herein have a specificity of atleast 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the FRET assays provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%.
  • the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the ref erence neutralizing antib ody has a known IC 50 ; and (b) detecting a binding complex b etween the detectable peptide and the one or more capture molecules; and (c) determine an IC 50 for the neutralizing antibody, wherein the IC 50 is approximately the known IC 50 .
  • accuracy is measured by comparing the positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population.
  • true positive results both true positive and true negative
  • accuracy would be measured by comparingthe true positives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) andtrue negatives (e.g., subjects th at were exposed to the SARS-CoV-2 and did become infected).
  • the FRET assay is performedin solution, such that neither of the first peptide or protein or the second peptide or protein are coupled to a solid phase.
  • the FRET assay may be performed in a container.
  • the container is optically clear.
  • the container is a cartridge.
  • the cartridge is disposable, such as a single-use disposable cartridge.
  • the cartridge is made of a material comprising a plastic, Polyethylene Terephthalate (PET or PETE), High-Density Polyethylene (HDPE), Polyvinyl Chloride(PVC or Vinyl), Low -Density Polyethylene (LDPE), Polypropylene (PP), or Polystyrene (PS or Styrofoam), or any combination thereof.
  • PET Polyethylene Terephthalate
  • HDPE High-Density Polyethylene
  • PVC or Vinyl Polyvinyl Chloride
  • LDPE Low -Density Polyethylene
  • PP Polypropylene
  • PS or Styrofoam Polystyrene
  • the FRET assay is performed under conditions sufficient for either the first peptide or protein or the second peptide or protein are coupled to a surface of a solid-phase. For example, if the first peptide or protein is coupled to the surface, it will capture (or bind) to the second peptide or protein in solution under otherwise sufficient conditions for their binding when there is an absence of neutralizing antibodies in the solution. Whereas the firstpeptide or protein coupledto the surface will capture fewer ofthe secondpeptideor protein in the solution in the presence of neutralizing antibodies in the solution. The opposite configuration is also possible, where the second peptide or protein is coupled to the surface, which captures the first peptide or protein in solution.
  • the first peptide or protein or the second peptide or protein are coupledto a surface of a solid -phase directly or indirectly by way of a linker or primary capture molecule (e.g., antibody, antigen -binding fragment) specific to the first peptide or protein or the second peptide or protein.
  • a linker or primary capture molecule e.g., antibody, antigen -binding fragment
  • the surface is an interior surface of a capillary, such as a capillary of a capillary flow cell or other micro fluidic device.
  • the interior of the flow cell is passivated.
  • the passivated surface comprises a polymer layer coupled thereto comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (acrylic acid) (PAA), polyacrylamide, poly(N -isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethyleneglycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyrrolidone)
  • PAA poly (acrylic acid)
  • PIPAM polyacrylamide
  • PMA poly(N -isoprop
  • the surface is a waveguide, such as a planar waveguide.
  • the surface compri ses a particle, such as ahead or nanoparticle.
  • the particle is made of a material comprising metal, agarose, acrylic, or plastic, or any combination thereof.
  • the particle is magnetic. [0100] In some embodiments, whether the FRET assay is performed in solution or solid-phase, the FRET assay does not comprise a wash step to wash away the first peptide or protein or the second peptide or protein that are unbound.
  • the signal to background noise is resolved using a software tool kit described herein such as FRET-Image Background- subtracted Ratiometric Analysis (IBRA).
  • signal to background noise is resolved using FRET -IBRA using the methodologies disclosed in Munglani, Gautam et al. “Fast and flexible processing of large FRET image stacks usingthe FRET -IBRA toolkit.” PLoS computational biology vol. 18,4 e1009242. 4 Apr. 2022, doi:10. 1371/journal.pcbi.1009242, which is incorporated by reference in its entirety.
  • the signal from the binding complex (between the first peptide or protein and the second peptide or protein) and the signals from the first peptide or protein and the second peptide or protein that are unbound are detected with an optical sensor in optical communication therewith.
  • the optical sensor is a planar waveguide optical sensor.
  • the optical sensor comprises a photomultiplier tube (PMT) detector, a charge-coupled device (CCD) detector, or complementary metal-oxide semiconductor (CMOS) detector.
  • PMT photomultiplier tube
  • CCD charge-coupled device
  • CMOS complementary metal-oxide semiconductor
  • methods further comprise calculating the percent neutralization in the biological sample from the ratio of the signals detected from the FRET assay.
  • the percentneutralization is calculated with a formula comprising: [(S T - S N )/(S 0 - S N )* 100], wherein S T is the signal corresponding to the binding complex, S N is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S 0 is a signal expected for zero neutralization ofbindingbetween the first peptide or protein and the second peptide or protein.
  • percent neutralization of about 62% correlates to about 400 reciprocal titers when measured using a cell-based pseudovirion neutralization assay (PNA).
  • PNA cell-based pseudovirion neutralization assay
  • a percent neutralization of 0% correlates with 0 antibody titers using the cell-based PNA.
  • a percent neutralization of 100% correlates with above 800 antibody titers usingthe cell -based PNA, or any combination thereof.
  • a non-limiting example of a PNA is provided in Example 23.
  • the assay assembly is a lateral flow assay (LFA).
  • the surface is a membrane comprising porous paper, a polymer structure, a sintered polymer, or a combination thereof.
  • the LFA assembly has one or more zones situated laterally, including a detectable zone. The detectable zone comprise at least a control region and a test region.
  • the LFA assembly further comprises a sample receptor (e.g., a sample pad) configured to receive a biological sample.
  • the sample receptor comprises a filter designed to separate a component of the biological sample to be tested. For instance, if a biological sample were blood, the sample component would be blood serum.
  • the LFA assembly further comprises an absorbent pad.
  • the target analyte moves without the assistance of external forces, e.g., by capillary action.
  • the target analyte moves with assistance of external forces, e.g., by facilitation of capillary action by movement of the lateral flow assembly (e.g, shaking, turning, centrifuging, applying an electrical field or magnetic field, applying a pump, applying a vacuum, or rocking).
  • lateral flow test strip detection format Any suitable lateral flow test strip detection format known to those of skill in the art is contemplated for use in an assay assembly of the present disclosure.
  • Lateral flow test strip detection formats are well known and have been described in the literature.
  • Lateral flow test strip assay formats are generally described by, e.g., Sharma et al., (2015) Biosensors 5:577- 601, incorporated by reference herein in its entirety.
  • Detection of nucleic acids using lateral flow test strip sandwich assay formats is described by, e.g., U.S. Pat. No. 9, 121 ,849, “Lateral Flow Assays,” incorporated by reference herein in its entirety.
  • the imagingdevice is a personal electronic device (e.g, smartphone or tablet).
  • the imaging device comprises a fluorescence reader, a colorimeter, or a sensor.
  • the peptide -conjugate described herein comprises detection reagent or a label.
  • a detection reagent include a fluorophore, a chemical, a nanoparticle, an antibody, a peptide, and a nucleic acid probe.
  • the nanoparticle comprises a material selected from agarose, plastic, acrylic, or metal.
  • the nanoparticle is a microsphere.
  • the nanoparticle is magnetic.
  • the imaging device detects color, reflectance, fluorescence, bioluminescence, chemiluminescence, light, or an electrical signal.
  • the LFA assembly is in a sandwich format, a competitive format
  • the detected signal is directly proportional to the amount of the target analyte present in the sample, so that increasing amounts of the target analyte lead to increasing signal intensity.
  • the detected signal has an inverse relationship with the amount of analyte present, and increasing amounts of analyte lead to decreasing signal intensity.
  • sample pad a sample receptor
  • the conjugate pad comprises a labeled capture molecule, e.g., an antibody or aptamer labeled with e.g., a dye, enzyme, or nanoparticle.
  • a complex between the capture molecule and the target analyte is formed if the target analyte is present in the test sample.
  • This complex then flows to a first test zone or sector (e.g., a test line) comprising an immobilized second capture molecule which is specific to the target analyte, thereby trapping any labeled capture molecule -target analyte complexes.
  • a first test zone or sector e.g., a test line
  • the intensity or magnitude of signal, e.g., color, fluorescence, reflectance, at the first test zone or sector is used to indicate the presence or absence, quantity, or presence and quantity of target analyte in the test sample.
  • the assay assembly comprises a second test zone or sector comprising a third capture molecule that binds to excess labeled capture molecule.
  • the applied test sample comprises the target analyte
  • little or no excess labeled capture molecule will be present on the test strip following capture of the target analyte by the labeled capture molecule on the conjugate pad. Consequently, the second test zone or sector will not bind any labeled capture molecule, and little or no signal (e.g., color, fluorescence, reflectance) at the second test zone or sector is expected to be observed.
  • the absence of signal at the second test zone or sector thus provides assurance that signal ob servedin the firsttest zone or sector is due to the pres ence of the target analyte.
  • the sandwich assay is configured to receive a biological sample disclosed herein and retain sample components (e.g., target analyte).
  • the sandwich assay is configured to receive a flow solution that flushes unwanted cellular components (other than the analyte) of the biological sample, leavingthe target analyte behind.
  • the sandwich assay comprises a membrane that binds the target analyte to help retain the target analyte when the flow solution is applied.
  • a membrane that binds a target analyte includes chitosan modified nitrocellulose.
  • the assay assembly comprises a sandwich assay.
  • the target analyte is an antibody specific to a pathogen of interest (e.g., SARS- CoV-2).
  • capture molecule is a peptide derived from a spike protein from a coronavirus (e.g., SARS-CoV-2).
  • immobilized capture molecule is an antibody specific to the target analyte, such as an antibody or antigen -binding fragment.
  • the capture molecule is labeled, and a signal correspondingto the presence of the target analyte is visualized.
  • a labeled secondary molecule e.g, antibody or antibody fragment
  • a signal correspondingto the presence of a complex between the target analyte, the capture molecule, and the secondary molecule is visualized.
  • signal is observed at the first test zone comprising color, fluorescence, or reflectance emitted from the labeled capture molecule.
  • the labeled capture molecule is a peptide -conjugate comprising a peptide derived from a spike protein from a coronavirus, and a label comprising a nanoparticle (e.g., microsphere), an enzymatic label, or a fluorescent dye.
  • the assay assembly detects more than one target analyte.
  • the assay assembly provides two or more capture molecules that are specific to two or more antibodies specific to a pathogen of interest, such as, for example IgG, IgM, or IgA antibodies against SARS-CoV-2.
  • the two or more capture molecules are labeled.
  • the two or more capture molecules are not labeled.
  • two or more labeled secondary molecules specific to the two or more capture molecules are added to the assay assembly, and visualized as described herein.
  • a lateral flow competitive format also referred to as a “competitive assay”
  • the test sample is applied to a sample pad at one end of the test strip, and the target analyte binds to a labeled capture molecule to form a complex between the target analyte and the labeled capture molecule in a conjugate pad downstream of the sample application pad.
  • the first test zone comprises an immobilized capture molecule specific to second capture molecule that is labeled. In the absence of the target analyte, the immobilized capture molecule and the labeled second capture molecule form a detectable complex.
  • the intensity or magnitude of signal, e.g., color, fluorescence, reflectance, at the first test zone or sector is inversely proportionate to the presence or absence, quantity, or presence and quantity of the target analyte in the test sample.
  • the assay assembly comprises a competitive assay.
  • the labeled capture molecule comprises a peptide-conjugate comprising a detection agent (e.g., gold nanoparticle) and a peptide derived from the spike protein of a coronavirus.
  • a fluid formulation comprising the peptide-conjugate is contacted with a biological sample from a patient and human ACE2, wherein the peptide derived from the spike protein of the coronavirus comprises a receptor binding domain (RBD) specific to the human ACE2.
  • the target analyte is one or more antibodies against the spike protein of the coronavirus.
  • the patient has not been exposed to the coronavirus, so a presence of the analyte is not expected to be detected.
  • the liquid formulation comprises at least one detectable complex comprising the peptide-conjugate RBD and the human ACE2, because the biological sample doesnot consist of antibodies against the RBD of the peptide.
  • the liquid formulation comprisingthe peptide- conjugate, the biological sample, and the human ACE2 is applied to a solid surface at least partially enclosed in a housing (“test cartridge”).
  • the liquid formulation is applied to the distal end of the solid surface at a sample pad, and flows unidirectionally over the solid surface towards the opposite distal end of the solid surface.
  • the biological sample is blood, and the sample pad separates serum from blood, permitting only the serum to flow across the solid surface.
  • the solid surface is a nitrocellulose membrane.
  • the first test zone comprises an immobilized antibody specific to the human ACE2.
  • the second test zone comprises an immobilized antibody specific to the RBD of the spike protein.
  • a high signal is observed at the first test zone, because there is an absence of target analyte in the biological sample. In some embodiments, a high signal is observed at the second test zone (positive control), for the same reason.
  • the immobilized capture molecule in the first test zone is human ACE2 receptor, and the fluid formulation does not contain human ACE2.
  • the liquid formulation comprises the peptide-conjugate and the biological sample. No complexes form between the peptide-conjugate in the absence of the target analyte (antibodies against the RBD of the spike protein).
  • the liquid formulation is applied to the distal end of the solid surface at a sample pad, and flows unidirectionally over the solid surface towards the opposite distal end of the solid surface.
  • a high signal is observed in the absence of the test analyte.
  • the same competition assay is performed on biological sample obtained from a subject that has been exposed to the coronavirus.
  • the assay assembly measures analyte in the biological sample.
  • the assay assembly measures one analyte (e.g. an neutralizing antibody) in the biological sample.
  • one capture molecule is used, for example RBD.
  • FIG. 8A exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that does not have neutralizing antibodies against RBS.
  • the peptide-conjugate e.g., ACE2 conjugated to a detectable moiety binds to the RBD in the absence of neutralizing antibodies.
  • the detectable moiety is visualized usingthe naked eye, or an imaging device described herein.
  • 8B exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that has neutralizing antibody against RBD.
  • the peptide-conjugate e.g., ACE2 conjugated to a detectable moiety does not bind to the RBD in the presence of neutralizing antibody in the patient sample.
  • the assay assembly measures more than one analyte (e.g., neutralizing antibody) in the biological sample.
  • more than one capture molecule is used, for example RBD1 and RBD2.
  • two or more capture molecules are used.
  • the two or more capture molecules comprise a first capture molecule and a second capture molecule, wherein the first capture molecule comprises a first variant RBD and the second capture molecules comprises a second variant RBD.
  • at least 3, 4, 5, 6, 7, 8, 9, or 10 capture molecules are used.
  • one of the capture molecules are the same.
  • FIG. 8C exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that does nothave neutralizing antibodies against either ofRBDl andRBD2.
  • the peptide -conjugate (e.g., ACE2) conjugated to a detectable moiety binds to the RBD1 and RBD2 in the absence of neutralizing antibodies. The detectable moiety is visualized using the naked eye, or an imaging device described herein.
  • FIG. 8D exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that has neutralizing antibodies against both RBD1 and RBD2.
  • the peptide-conjugate (e.g., ACE2) conjugated to a detectable moiety does not bind to the RBDl and RBD2 in the presence of neutralizing antibodies in the patient sample.
  • a lateral flow test strip detection format more than one target analyte is detected using the test strip through the use of additional test zones or sectors comprising, e.g., probes specific for each of the target analytes.
  • the lateral flow device is a layered lateral flow device, comprising zones or sectors that are present in layers situated medially, e.g., above or below each other. In some instances, one or more zones or sectors are present in a given layer. In some instances, each zone or sector is present in an individual layer. In some instances, a layer comprises multiple zones or sectors. In some instances, the layers are laminated.
  • processes controlled by diffusion and directed by the concentration gradient are possible driving forces. For example, multilayer analytical elements for fluorometric assay or fluorometric quantitative analysis of an analyte contained in a sample liquid are described in EP0097952, “Multilayer analytical element,” incorporated by reference herein.
  • a lateral flow device comprises one or more functional zones or sectors.
  • the test assembly comprises 1 to 20 functional zones or sectors.
  • the functional zones or sectors comprise at least one sample purification zone or sector, at least one target analyte amplification zone or sector, at least one target analyte detection zone or sector, and at least one target analyte detection zone or sector.
  • the assay assembly comprises a detection zone made up of at least the first and the second test zones (test, and control). In some embodiments, the assay assembly comprises more than one test zone. In some embodiments, the assay assembly comprises a firsttestzone and a second test zone, whereinthe firsttestzone indicatesa presence or a quantity of neutralizing antibodies that competitively bind to the peptide-conjugate; and the second test zone indicates a presence or a quantity of antibodies against the pathogen (neutralizing or otherwise). In some embodiments, the assay assembly comprises a third test zone. In some embodiments, the second test zone detects IgG antibodies against the pathogen. In some embodiments, the third test zone detects IgM antibodies against the pathogen.
  • the assay assembly comprises a plurality of test zones, wherein each test zone of the plurality measures a different analyte.
  • an image of the detection zone is captured with an imaging device.
  • the image is a video or still image.
  • the imaging device comprises a fluorescence reader, a colorimeter, or a sensor.
  • the imaging device is a personal electronic device, such as a smartphone.
  • the assay assembly is a lab -on-chip system (e.g., Maverik®).
  • the solid surface comprises a silicon chip.
  • the imaging device comprises photonic biosensors that measure changes in refractive index caused by binding between the analyte and the peptide-conjugate to form complexes, as describedin Iqbal M., et al. (2010) Label -Free Biosensor Arrays Based on Silicon Ring Resonators and High-Speed Optical Scanning Instrumentation. IEEE J Sei Quantum Elec 16.
  • the lateral flow assay assembly confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample.
  • a subject may be a member of a variety of races.
  • the subject may be of the White, Black, Hispanic, or Asian race.
  • a subject may be of a variety of ages.
  • the subject may be age 0 to age 18, age 18 to age 65, or age 65 and above.
  • a subject may be a male or a female.
  • a subject may be pregnant, breastfeeding, or may have priorly beeninfected with a coronavirus.
  • a subject may be a high risk subject and have one or more risk factors related to a coronavirus. In some embodiments, a subject may be obese. In some instances, the lateral flow assay assembly prognoses a subject’s immunity to any one of the pathogens described herein. In some embodiments, the lateral flow assay assembly prognoses a subject’s immunity to coronavirus. In some embodiments, the lateral flow assay assembly prognoses a subject’s immunity to SARS-CoV-2. In some embodiments, the lateral flow assay assembly measures the subject’s susceptibility to an infection by any one of the pathogens described herein .
  • the lateral flow assay assembly measures the subjects susceptibility to an infection by coronavirus. In some embodiments, the lateral flow assay assembly measures the subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the lateral flow assay assembly identifies individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the lateral flow assay assembly minimizes or reduces the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine).
  • the lateral flow assay assembly identifies neutralizing antibodies as a surrogate marker of protection from a vaccine (e.g., in place of traditional, randomized, placebo-controlled trials). In some embodiments, the lateral flow assay assembly classifies vaccines by % efficacy in a population. [0127] In some embodiments, the lateral flow assays provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the lateral flow assays provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, atleast 88%, atleast 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the lateral flow assays provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the lateral flow assays provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%.
  • the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC 50 ; and (b) detecting a binding complex between the detectable peptide and the one or more capture molecules; and (c) determine an IC 50 for the neutralizing antibody, wherein the IC 50 is approximately the known IC 50 .
  • accuracy is measured by comparing the positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population.
  • true positive results both true positive and true negative
  • accuracy wouldbe measured by comparingthe truepositives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) and true negatives (e.g., subjects that were exposed to the SARS-CoV-2 and did become infected).
  • the assay assembly comprises an assembly for agglutination assay.
  • Agglutination assay relies on agglutination of particles or nanoparticles due to a capture molecule or peptide-conjugate forming a complex with the target analyte in the sample, thus indicating the presence of the target analyte in the sample.
  • the agglutination assay comprises separating free and bound label by chromatographically separating agglutinated and non -agglutinated antibody -coated colored particles along a membrane.
  • the agglutination assay comprises a colored latex agglutination reaction, where agglutinated and non -agglutinated particles are separated by a capillary which allows non -agglutinated latex beads through but traps the aggregated latex beads.
  • the agglutination assay comprises multivalent analytes whereby in the absence of analyte label passes through a filter, but in the presence of analyte an agglutinate is formed which is trapped.
  • the agglutination assay comprises a system, where a test mixture is formed by contacting the sample with colored particles having analyte-specific receptors on the surface of the particles.
  • the agglutination assay comprises alateral flow assay without immobilizingantibodyforverylarge analytes.
  • the agglutination assay comprises a 2-zone system, where one zone having large pores and one zone having small pores, such that analyte passes through the large pores but becomes trapped by the zone of small pores. Such arrangement is used in conjunction with a small label (e.g. gold particle) which passes through both zones.
  • the agglutination assay is a non-capillary agglutination assay.
  • the agglutination assay comprises covalently or non -covalently linking a particle or nanoparticle with at least one capture molecule, at least one detectable moiety, and/or at least one peptide-conjugate.
  • the particle or nanoparticle comprises macromolecule, colloidal metal (such as gold or silver) particle, bead (e.g. latex bead), charcoal, kaolinite, or bentonite.
  • the particle or nanoparticle also function as detectable moiety.
  • the particle or nanoparticle comprises of the same material as the detectable moiety.
  • the particle, nanoparticle, or detectable moiety comprises material that is agglutinable, with other (preferably similar or identical) particle, nanoparticle, or detectable moiety.
  • the ability of the particle, nanoparticle, and detectable moiety to agglutinate enables the formation of larger agglutinates, resulting in increased stability or the detectable signal generated from the agglutinated particle, nanoparticle, or detectable moiety.
  • the detectable moiety comprises detectable material such as a fluorescent dye, an enzymatic label, or a colorimetric label.
  • Detectable moiety includes enzymatic label (e.g., horseradish peroxidase (HRP), beta -galactosidase, alkaline phosphatase, etc), fluorescent dye, luminescent moiety, radioactive moiety, colorimetric label, colored latex particle or nanoparticle, and metal-conjugated moiety such as metallic nanolayer, metallic nanoparticle, or metallic nanoshell-conjugated moiety.
  • HRP horseradish peroxidase
  • beta -galactosidase alkaline phosphatase
  • metal-conjugated moiety such as metallic nanolayer, metallic nanoparticle, or metallic nanoshell-conjugated moiety.
  • Suitable metallic nanoparticle or metallic nanoshell moiety includes gold particle, silver particle, copper particle, platinum particle, cadmium particle, composite particle, gold hollow sphere, gold -coated silica nanoshell, and silica-coated gold shell.
  • Metallic nanolayer suitable for detectable moiety includes nanolayer comprising cadmium, zinc, mercury, gold, silver, copper, and platinum.
  • the detectable moiety is directly or indirectly tagged for a colorimetric assay (e.g., for detection of HRP or beta -galactosidase activity), visual inspection using light microscopy, immunofluorescence microscopy, confocal microscopy, by flow cytometry (FACS), autoradiography electron microscopy, immuno staining, or subcellular fractionation.
  • the detectable moiety is directly incorporated into the capture molecule.
  • a radioactive amino acid is inserted into the capture molecule comprising a peptide.
  • the detectable moiety is part of the peptide- conjugate.
  • the detection method comprises visually examining for signs of agglutination.
  • the detection method comprises visibly examining the agglutination fora color change oraphysical-chemical change. Physical -chemical changes can occur with oxidation reactions or other chemical reactions, which is detected both visually or via a spectrophotometer.
  • the agglutination of the particles or nanoparticles due to formation of a complex formed between: capture molecule and target analyte; capture molecule and peptide-conjugate; and/or target analyte and peptide-conjugate is visually examined.
  • the complex formed is examined using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • the target analyte is a monoclonal or polyclonal antibody against a protein of any one of the pathogen described herein.
  • the target comprises a neutralizing antibody.
  • the target analyte is a monoclonal or polyclonal neutralizing antibody recognizing a spike glycoprotein of a coronavirus.
  • the target analyte is a monoclonal or polyclonal neutralizing antibody recognizing a spike glycoprotein of SARS- CoV-2.
  • the target analyte is targeted and captured by the capture molecules described herein.
  • agglutination assay assembly comprises particle or nanoparticle linked to capture molecules comprisingboth ACE2 and spike glycoprotein. If the target analyte comprisingthe neutralizing antibody recognizing spike glycoprotein is absent in a biological sample, agglutination is ob served due to the complex formed by the spike glycoprotein binding to ACE2. In such case, the observed agglutination or signal of the agglutination is indicative of the absence of the target analyte in the biological sample. Conversely, if the target analyte comprising the neutralizing antibody recognizing the spike glycoprotein is present in the biological sample, the binding of the target analyte to the spike glycoprotein prevents the complex formation between spike glycoprotein and ACE2.
  • the absence of agglutination or signal of the agglutination is indicative of the presence of the target analyte in the biological sample.
  • the agglutination assay assembly confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample.
  • the agglutination assay assembly prognoses a subject’s immunity to any one of the pathogen describedherein.
  • the agglutination assay assembly measures the subj ect’ s susceptibility to an inf ection by any one of the pathogen described herein.
  • the assay assembly comprises an assembly for enzyme -linked immunosorbent assay (ELISA).
  • one or more capture molecules are attached or immobilized on a solid support disclosed herein.
  • the peptide- conjugate is attached or immobilized on the solid support.
  • the one or more capture molecules comprise a first peptide or protein and a second peptide or protein.
  • the first peptide or protein is derived from an antigenic component of a pathogen of interest.
  • the second peptide or protein is derived from the cognate receptor of the pathogen of interest.
  • the first peptide or protein or the second peptide or protein is attached or immobilized to the solid support.
  • the target analyte is attached or immobilized on the solid support.
  • the solid support is a bead such as colloidal particle, metallic nanoparticle, nanoshell, or latex bead; a flow path in a lateral flow immunoassay device (e.g., a porous membrane); a blot (e.g., Western blot, a slot blot, or dot blot); a flow path in an analytical or centrifugal rotor, or a tube or well (e.g., in a plate suitable for an ELISA or microarray).
  • the solid support is a plate such as a microtiter plate with multiple wells.
  • the one or more capture molecules or one or more peptide conjugates is biotinylated, or conjugated to streptavidin, avidin, or neutravidin.
  • the target analyte is attached to the surface of the well of the microtiter plate via direct binding to the well of the microtiter plate. In some embodiments, the target analyte is attached to the well via binding to one or more capture molecules that are attached to the well of the microtiter plate.
  • the target analyte is attached or immobilized to the well via binding to one or more peptide-conjugates that are attached or immobilized to the well of the microtiter plate. In some embodiments, the target analyte is attached to the well via binding to one or more peptide-conjugates that form a complex with the one or more capture molecules that are attached or immobilized to the well of the microtiter plate. Residual or nonspecific protein-binding on the solid support can then blocked with an blocking agent, such as bovine serum albumin (BSA) or heat-inactivated normal goat serum (NGS).
  • BSA bovine serum albumin
  • NGS heat-inactivated normal goat serum
  • the solid support is incubated with a biological sample suspected of containing the target analyte.
  • the sample is applied and/or attached to the solid support undiluted or diluted.
  • the solid support is washed to remove unbound protein and then incubated with an optimal concentration of the one or more capture molecules conjugated to a detectable moiety or to a substrate of the detectable moiety.
  • one or more capture molecules conjugated to the detectable moiety comprises secondary antibody, which binds to the neutralizing antibody in the target analyte.
  • the target analyte comprises any one of the antibody or any one of the neutralizing antibody described herein.
  • the detectable moiety comprises an enzyme, including HRP, beta-galactosidase, alkaline phosphatase (ALP), and glucose oxidase. Sufficient time is allowed for specific binding between the one or more capture molecules and the target analyte to occur. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally (measured at an appropriate wave length) via the use of a spectrophotometer.
  • the ELISA assay assembly confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some instances, the ELISA assay assembly prognoses a subject’s immunity to any one of the pathogen described herein. In some embodiments, the ELISA assay assembly measures the subject’s susceptibility to an infection by any one of the pathogen described herein.
  • the ELISA assays provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the ELISA assay s provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the ELISA assays provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, atleast 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater.
  • the ELISA provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%.
  • the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the ref erence neutralizing antib ody has a known IC 50 ; and (b) detecting a binding complex b etween the detectable peptide and the one or more capture molecules; and (c) determine an IC 50 for the neutralizing antibody, wherein the IC 50 is approximately the known IC 50 .
  • accuracy is measured by comparing the positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population.
  • true positive results both true positive and true negative
  • accuracy would be measured by comparing the true positives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) andtrue negatives (e.g., subjects th at were exposed to the SARS-CoV-2 and did become infected).
  • testing devices comprising a housing.
  • a testing device is at least partially enclosed by the housing.
  • the testing device is fully enclosed by the housing.
  • the housing comprises a synthetic polymer material, such as plastic.
  • the housing is configured to provide information to an imaging device describedherein.
  • Information can include, butis notlimited to, information to normalize an image of the testing device and identifying information (e.g., barcode, RFID chip).
  • the identifying information comprises test parameters, test result interpretation instructions, expected values for imaging controls, and the like.
  • testing devices comprising a sample receptor.
  • the sample receptor is a sample receiver, a sample processor, a sample purifier, or a combination thereof.
  • the sample receptor is a sample receiver configured to receive and retain a biological sample obtained from a subject.
  • the sample receptor is a sample processor configured to remove a component of the sample or separate the sample into multiple fractions (e.g., blood cell fraction and plasma or serum).
  • Useful separation materials may include specific binding moieties that bind to or associate with the substance. Binding is covalent or noncovalent. Any suitable binding moiety known in the art for removing a particular substance is used. For example, antibodies and fragments thereof are commonly used for protein removal from samples.
  • a sample purifier disclosed herein comprises a binding moiety that binds a nucleic acid, protein, cell surface marker, or microvesicle surface marker in the biological sample.
  • the binding moiety comprises an antibody, antigen binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof.
  • the sample receptor is a sample purifier, configured to remove an unwanted substance or non-target component of a biological sample.
  • unwanted substances can include, but are not limited to, proteins (e.g., antibodies, hormones, enzymes, serum albumin, lipoproteins), free amino acids and other metabolites, microvesicles, nucleic acids, lipids, electrolytes, urea, urobilin, pharmaceutical drugs, mucous, bacteria, and other microorganisms, and combinations thereof.
  • the sample purifier separates components of a biological sample disclosed herein.
  • the sample purifier disclosed herein removes one or more components of a sample that would inhibit, interfere with or otherwise be detrimental to the analyses of the target analyte.
  • the resulting modified sample is enriched for the target analyte. This is considered indirect enrichment of target analytes. Alternatively, or additionally, target analytes may be captured directly, which is considered direct enrichment of target analytes.
  • sample purifiers disclosed herein comprise a filter.
  • sample purifiers disclosed herein comprise a membrane.
  • the filter or membrane is capable of separating or removing cells, cell particles, cell fragments, blood components other than cell-free nucleic acids, or a combination thereof, from the biological samples disclosed herein.
  • the sample purifier facilitates separation of plasma or seium from cellular components of a blood sample.
  • the sample purifier facilitates separation of plasma or serum from cellular components of a blood sample before starting a molecular amplification reaction or a sequencing reaction. Plasma or serum separation is achieved by several different methods such as centrifugation, sedimentation or filtration.
  • the sample purifier comprises a filter matrix for receiving whole blood, the filter matrix having a pore size that is prohibitive for cells to pass through, while plasma or serum can pass through the filter matrix uninhibited.
  • the filter matrix combines a large pore size at the top with a small pore size at the bottom of the filter, which leads to very gentle treatment of the cells preventing cell degradation or lysis, during the filtration process. This is advantageous because cell degradation or lysis would result in release of nucleic acids from blood cells or maternal cells that would contaminate target cell-free nucleic acids.
  • Non-limiting examples of such filters include Pall VividTM GR membrane, Munktell Ahlstrom filter paper, and TeraPore filters.
  • a vertical filtration system is used to facilitate separation of plasma or serum from a cellular component of a blood sample.
  • the filtration is driven by capillary force to separate a component or fraction from a sample (e.g., plasma from blood).
  • vertical filtration may comprise gravitation assisted plasma separation.
  • a high-efficiency superhydrophobic plasma separator is described, e.g, by Liu et al., A High Efficiency Superhydrophobic Plasma Separation, Lab Chip 2015.
  • the sample purifier comprises a lateral filter (e.g., sample does not move in a gravitational direction or the sample moves perpendicular to a gravitational direction).
  • the sample purifier may comprise a vertical filter (e.g., sample moves in a gravitational direction).
  • the sample purifier may comprise vertical filter and a lateral filter.
  • the sample purifier may be configured to receive a sample or portion thereof with a vertical filter, followed by a lateral filter.
  • the sample purifier may be configured to receive a sample or portion thereof with a lateral filter, followed by a vertical filter.
  • a vertical filter comprises a filter matrix.
  • the filter matrix of the vertical filter comprises a pore with a pore size that is prohibitive for cells to pass through, while plasma can pass the filter matrix uninhibited.
  • the filter matrix comprises a membrane that is especially suited for this application because it combines a large pore size at the top with a small pore size at the bottom of the filter, which leads to very gentle treatment of the cells preventing cell degradation during the filtration process.
  • the filter comprises a material that moves, draws, pushes, or pulls the biological sample through the filter.
  • the material is a wicking material.
  • the separation material is a hydrophobic filter, for example a glass fiber filter, a composite filter, for example Cytosep (e.g., Ahlstrom Filtration or Pall Specialty Materials, Port Washington, NY), or a hydrophilic filter, for example cellulose (e.g., Pall Specialty Materials).
  • whole blood is fractionated into red blood cells, white blood cells and serum components for further processing according to the methods of the present disclosure using a commercially available kit (e.g., Arrayit Blood Card Serum Isolation Kit, Cat. ABCS, Arrayit Corporation, Sunnyvale, CA).
  • kit e.g., Arrayit Blood Card Serum Isolation Kit, Cat. ABCS, Arrayit Corporation, Sunnyvale, CA.
  • the sample purifier comprises at least one filter or at least one membrane characterized by at least one pore size.
  • at least one pore size of at least one filter is about 0.05 microns to about 10 microns.
  • the pore size is about 0.05 microns to about 8 microns.
  • the pore size is about 0.05 microns to about 6 microns.
  • the pore size is about 0.05 microns to about4 microns.
  • the pore size is about 0.05 microns to about 2 microns.
  • the pore size is about 0.05 microns to about 1 micron.
  • At least one pore size of at least one filter is about 0.1 microns to about 10 microns. In some embodiments, the pore size is about 0.1 microns to about 8 microns. In some embodiments, the pore size is about 0.1 microns to about 6 microns. In some embodiments, the pore size is about 0.1 microns to about 4 microns. In some embodiments, the pore size is about 0.1 microns to about 2 microns. In some embodiments, the pore size is about 0.1 microns to about 1 micron.
  • the sample processor is configured to separate blood cells from whole blood. In some embodiments, the sample processor is configured to isolate plasma from whole blood. In some embodiments, the sample processor is configured to isolate serum from whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 1 milliliter of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 1 milliliter of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 500 microliters ( ⁇ L) of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 400 ⁇ L of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 300 ⁇ L of whole blood.
  • the sample processor is configured to isolate plasma or serum from less than 200 ⁇ L of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 150 ⁇ L of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 100 ⁇ L of whole blood.
  • the detection zone comprises a test region and a control region.
  • the imaging device captures an image of the detection zone.
  • the control region is a positive control.
  • the control region is a negative control.
  • the control region comprises a positive and a negative control.
  • the detection zone comprises a first test region comprising a competition assay assembly to measure a presence of neutralizing antibodies in a patient sample against a first SARS-CoV-2 spike protein receptor binding domain (RBD).
  • the first test region in some embodiments, comprises a first capture molecule described herein.
  • the first capture molecule comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3.
  • the competition assay assembly further comprises a peptide- conjugate comprising a peptide derived from a cognate receptor of the spike protein (e.g., human ACE2) conjugated to a detectable moiety.
  • the detection zone comprises a second test region comprising a second competition assay assembly to measure a presenceofneutralizingantibodiesin a patient sample against a second SARS-CoV-2 spike protein RBD that differs from the first SARS- CoV-2 spike protein RBD by at least 1 amino acid.
  • the first SARS- CoV-2 spike protein RBD and the second SARS-CoV-2 spike protein RBD differ by at least 2 amino acids.
  • the first SARS-CoV-2 spike protein RBD and the second SARS-CoV-2 spike protein RBD differ by at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the second test region comprises a second assay assembly configured to measure total antibodies (e.g., neutralizing or otherwise) against SARS-CoV-2 or another virus.
  • the second assay assembly comprises a capture molecule that binds to anti-nucleocapsid IgG or IgM, anti-spike RBD IgG or IgM, anti-Spike S1 S2 IgG or IgM, anti-spike S2 IgG or IgM, anti-spike S1 IgG or IgM, or a combination thereof.
  • the capture molecule binds to anti-nucleocapsid IgG or IgM for SARS-CoV, anti-Sl IgG or IgM for MERS, anti-H1 IgG or IgM for influenza and anti-H3 IgG or IgM for influenza.
  • the testing device comprises multiple detection zones.
  • the testing device comprises a first detection zone and a second detection zone.
  • the first detection zone comprises the first and second testing regions described herein.
  • the first and second detection zones are partially enclosed in a single housing.
  • the first and second detection zones are not enclosed in a single housing.
  • the testing device of the present disclosure may comprise any number of detection zones or testing regions, depending on the desired output.
  • the testing device comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 detection zones.
  • at least one of the detection zones comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 testing regions.
  • the detection zones are partially enclosed in a single housing. In some embodiments, the detection zones are not enclosed in a single housing
  • a biological sample medium may be used to store a biological sample to be assayed using the testing devices, systems or methods described herein.
  • the BSM is a dried blood spot card (DBS).
  • the BSM 1201 may comprise one or more regions 1205 for containing the one or more biological sample(s).
  • the one or more regions may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more regions.
  • the one or more regions may comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 regions.
  • the one or more regions may comprise 2 to 5 regions (e.g., 2, 3, 4, or 5).
  • the one or more regions 1205 may comprise spatially distinct adjacent regions.
  • the biological sample(s) stored on the BSM may comprise two or more biological samples.
  • the one ormore regions may comprise atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, or more samples.
  • the one or more regions may comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 samples.
  • the one or more regions may comprise 2 to 5 samples (e.g., 2, 3, 4, or 5).
  • the BSM may comprise a sealable member 1207 to protect the one or more regions of one or more deposited biological sample 1205 during transport of the BSM. As shown in FIG.
  • the BSM may comprise subject identifying information associated with a barcode 1202, numeric 1203, text-based characters 1204, RFID 1206, or any combination thereof to identify the subject from whom the one or more biological samples originated.
  • the BSM may comprise a fillable region 1208 for a subject to provide answers to a survey related to the health of the subject, including for example symptoms related to an infection by a pathogen of interest (e.g., SARS-CoV-2).
  • a pathogen of interest e.g., SARS-CoV-2
  • the fillable region is positions on a dried spot card (DSC).
  • the survey may include one ormore clinical questions 1210 pertaining to a symptom associated with an infection by a pathogen of interest.
  • the BSM may comprise a survey specific to coronavirus symptoms.
  • the associated symptoms may include cough, nasal congestion, chest pain, headache, diarrhea, vomiting, difficulty breathing or any combination thereof.
  • the survey may comprise a response severity scale 1211 of associated symptoms.
  • said severity scale may include terminology to indicate symptom s never present, sometimes present, often present, or present every day.
  • clinical questions may include indicating whether or not the subject has been exposed to a second subject who has confirmed contracting one or more viruses or microbes.
  • clinical questions may further comprise a unique computer recognizable code 1209 (e.g. QR code, alpha numeric character, RFID or any combination thereof) that a computer software image processing algorithm may recognize, and associate said medical differential diagnosis survey response with.
  • a computer software image processing algorithm may further provide a combined interpretation of a subject’s assay results in view of survey severity scale responses.
  • the systems and methods described herein may perform steps on a BSM including: (a) providing a stack of uniformly oriented BSMs 1401; (b) reading the BSM barcode 1402, (c) opening the BSM to expose biological sample(s) 1403, (d) reading BSM patient info 1404, (e) identifying a region on the BSM 1405, (f) punchingthe biological sample spot 1406, (g) droppingthe punch into a well of a multi-well microtiter plate 1407, (h) confirming the position of punch sample in the well 1408, (i) repeating steps (a)-(h) until sufficient biological samples have been punched 1409, (j) stackingthe multi-well microtiter plates 1410, (k) transferring the stackedmulti -well microtiter plates to the automated centralizing assay system 1411, (l) foldingthe BSM for storage 1412 or any combination thereof.
  • One or more of the steps performed by the system may be automated.
  • the systems described herein are automated high throughput systems.
  • the systems described may comprise an automated system capable of : (a) receiving and processing a biological sample in preparation for assaying, and (b) conductingthe assay .
  • one ormore of (a) or (b) is automated.
  • (a) and (b) are automated.
  • an automated system may comprise system components operated by one or more robots.
  • the system components are otherwise in mechanical, electrical, fluidic or optical communication with one another such that human intervention is not necessary to perform the disclosed functions .
  • Such automated systems may be capable of completingthe methods disclosedherein with or without the intervention of a human user.
  • the systems utilize a BSM comprising the biological sample to be assayed with the system.
  • the systems disclosed herein comprise a biological sample (BS) processor 1302 and an assay assembly block 1309.
  • the BS processor comprises: (a) a BS receiver 1303; (b) a BS scanner 1304; (c) a BS separator 1305; (d) BS imager 1306; (e) BS chemical extractor 1307; and (f) a BS purifier 1308.
  • the biological sample may be contained within a BSM and further processed by the high throughput system.
  • the BS receiver 1303 may mechanically manipulate the BS through linear translation or rotation of the BS to properly position the BS for further processing.
  • theBS receiver comprises a receptacle for a container (e.g, a vial, 96 well plate).
  • BS receiver is a crate with multiple receptacles for containers.
  • theBS receiver comprises a mechanical arm with a distal hand-like member configuredto hold the container and transferthe container to theBS scanner to be scanned.
  • the BS processor 1302 may comprise a BS scanner 1304 in electrical communication with the BS processor 1302.
  • the BS scanner 1304 may interpret and record subject identifying information on the BS.
  • the BS scanner 1304 may scan identifying information providedin, for example, barcode 1202, numeric 1203, text-based characters 1204, RFID 1206 or any combination thereof, as shown in FIG. 12A.
  • the BS processor 1302 may further comprise a BS separator 1305 capable of mechanically separating a segment of the BS.
  • the BS separator 1305 may comprise a cutting tool that isolates a segment of the BSM containing the BS.
  • the cutting tool may comprise a knife or a punch .
  • the punch comprises a plurality of diameters.
  • the punch outer-diameter may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more millimeters.
  • the punch outer-diameter may comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 millimeters.
  • the one or more punched segments of BS may be placed into an assay chamber for further processing
  • the BS separator may be a syringe or a needle for isolating a portion of the BS to be assayed.
  • the BS separator 1305 may aspirate a segment of the BS using a needle.
  • the needle may be 18 AWG, 19AWG, 20AWG, 21AWG, 22AWG, 23AWG, 24AWG, 25 AWG, 26AWG, 27AWG, or 28 AWG.
  • the one or more aspirated segment of BS may be placed into an assay chamber for further processing the BS.
  • the BS processor 1302 may further comprise a BS imager 1306 that may provide quality control feedback position of the BS in the assay chamber to ensure proper processing. After proper placement of one or more segments of BS in the assay chamber, the BS may then be catalogued and stored to be referenced in the future.
  • the assay chamber may comprise a well plate.
  • the well plate may be a 96-well, 384-well, or a 1536-well plate.
  • the well plate may be a microtiter well plate.
  • the assay chamber comprises a unique identifier that may be recognized as it moves between the biological sample processor 1302 and the assay assembly block 1309 described herein.
  • the BS may be further processed by the biological sample processor 1302 in preparation for assaying
  • Biological Sample Preparation is accomplished by a (a) BS chemical extractor 1307 and (b) a BS purifier 1308.
  • Suitable buffers that may be used to elute the BS from the BSM includes phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the BS chemical extractor 1307 may be capable of eluting the BS to isolate the BS (e.g., from a biological sample medium).
  • the chemical extractor elutes the BS from a biological sample medium in the assay chamber.
  • the BS chemical extractor 1307 may be in fluidic communication with one or more reservoirs of elution buffers andthe assay chamber to dispense elution buffer as needed into the assay chamber.
  • the assay chamber containing the elution buffer and BS may be agitated by mechanical shaking to facilitate the elution process.
  • the eluted BS may be purified by the BS purifier 1308 priorto transferring the assay chamber to the assay assembly block 1309.
  • the purification process may comprise centrifugation to isolate BS by density.
  • the biological sample processor 1302 may mechanically transfer the assay chamber to the assay assembly block 1309.
  • Assaying the Biological Sample may be accomplished by an assay assembly block 1309.
  • the assay assembly block comprises: (a) BS Diluter 1310 and (b) BS assay conductor 1311.
  • theBS diluter 1310 may be in fluidic communication with the assay chamber to prepare the serial dilutions of the BS.
  • assay chamber identification may be interpreted by the assay assembly block to indicate which assay to conduct on which BS.
  • the assay results 1312 upon completion of the assay by the BS assay conductor 1311, the assay results 1312 will be saved with respect to the identification of the assay chamber and more broadly the subject providing the biological sample assayed.
  • the system comprises the use of a preprogramed and automated robotics to perform one or more of the steps above, including the assays described herein.
  • the automated centralizing assay system comprises the use of preprogramed and automated robotics to perform the assay comprising a lateral flow assay, agglutination assay, ELISA, or FRET assay as described herein.
  • the system is capable of performing at least or about 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or more than 100,000 tests per day.
  • the system utilizes small volumes of reagents, such as less than 100 nL, less than 90 nL, less than 80 nL, less than 70 nL, less than 60 nL, or less than 50 nL of reagents.
  • the system performs the assay (e.g., a solution-based assay) in a vessel, such as a container or a test tube.
  • the system performs the assay (e.g., a solution -based assay) in an assay plate, such as a 96-well plate, a 384-well plate, or a 1536-well plate.
  • the biological sample (e.g., blood from a finger prick) is stored in or on a BSM, such as a DSC, described herein.
  • the biological sample is removed from the BSM and placed into the vessel (e.g., a well of a 96 -well plate) to be eluted from the BSM.
  • the vessel e.g., a well of a 96 -well plate
  • the eluted biological sample is assayed using an assay (e.g., FRET, ELISA) described herein deposited on a solid surface.
  • an assay e.g., FRET, ELISA
  • biological samples are collected from the subjects via the use of blood or sample collection cards.
  • the biological samples is tested by the automated centralizing assay system.
  • the blood or sample collection card comprises multiple slots for the blood or sample collection.
  • the biological sample from each slot is tested separately by a different assay method.
  • a blood collection card i.e. a blood spot card
  • the automated centralizing assay system comprises five slots that is tested by the automated centralizing assay system performing fives assays such as lateral flow assay, an ELISA, a biomarker panel, a qPCR, and a neutralizingantibody titer assay.
  • the same assay is performedmultiple times (e.g., duplicates) to ensure accuracy of the results.
  • the assay is performed at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.
  • multiple dilutions are tested, for e.g., 1 :40, 1 :80, 1 :160, and 1 :320 dilutions are tested at the same time.
  • the dilutions tested are one or more of 1 :5, 1 : 10, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, 1 :50, 1 :55, or 1 :60, 1 :65, 1 :70, 1 :75, 1 :80, 1 :85, 1 :90, 1 :95, 1 :100, 1 : 100, 1 : 105, 1 :110, 1 : 115, 1 :120, 1 :125, 1 : 130, 1 :135, 1 :140, 1 : 145, 1 :150, 1 :155, 1 : 160, 1 : 165, 1 :170, 1 :175, 1 : 180, 1 :185, 1 :190, 1 : 195, 1 :200, 1 :205, 1 :210, 1 :215, 1 :220, 1 :225, 1 :60, 1
  • the automated centralizing assay system confirms the efficacy or effectiveness of a vaccine in a population.
  • the biological samples are collected from a population before and after the population is vaccinated.
  • additional biological samples are collected at any time frame or frequency described herein to confirm presence and sufficient quantity of neutralizing antibody (induced by the vaccine) in the population.
  • the automated centralizing assay system can prognose immunity or measure susceptibility of the population to any one of the pathogen described herein by confirming the absence or insufficient quantity of neutralizing antibody in the population.
  • the automated centralizing assay system confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample.
  • the automated centralizing assay system prognoses a subject’s immunity to any one of the pathogen described herein. In some embodiments, the automated centralizing assay system prognoses a subject’s immunity to coronavirus. In some embodiments, the automated centralizing assay system prognoses a subject’s immunity to SARS-CoV-2. In some embodiments, the automated centralizing assay system measures the subject’s susceptibility to an infection by any one of the pathogens described herein. In some embodiments, the automated centralizing assay system measures the subject’s susceptibility to an infection by coronavirus. In some embodiments, the automated centralizing assay system measures the subject’s susceptibility to an infection by SARS-CoV- 2.
  • the automated centralizing assay system identifies individual s in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the automated centralizing assay system minimizes or reduces the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the automated centralizing assay system identifies neutralizing antibodies as a surrogate marker of protection from a vaccine (e.g., in place of traditional, randomized, placebo-controlled trials). In some embodiments, the automated centralizing assay system classifies vaccines by % efficacy in a population.
  • the automated centralizing assay systems provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the automated centralizing assay systems provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater.
  • the automated centralizing assay systems provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the system 500 comprises, in some embodiments, a testing device, an imaging device 501, and a computing device, for determining whether a subject is immune to an infection by a pathogen of interest (e.g., SARS-CoV-2).
  • the imaging device and/or the computing device are configured to receive and analyze data generated by the testing device and/or one or more external devices, to provide a result to the subject.
  • the result is provided to the subject via a graphical user interface (GUI) by the imaging device and/or computing device using an application (web application or mobile application).
  • GUI graphical user interface
  • Systemsfurthercompriseone ormore data store for storing and retrieving the data.
  • the data are structured or unstructured.
  • the data are generated by the imaging device when an image is captured of the detection zone of the testing devices described herein.
  • the data are external data generated from an external device.
  • the external device comprises a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • the external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, or sleep quantity, or a combination thereof.
  • imaging devices for capturing an image of the detection zone of the testing devices described herein.
  • the imaging device is a camera.
  • the imaging device is a computing device with a camera, such as a smartphone, laptop, or tablet.
  • the imaging device is not a computing device, but is in communication with the computing device via a communication network.
  • the communication network is wireless, such as wireless Internet or Bluetooth.
  • the system 500 comprises an imaging device 501, an external device 502, a data store 505, all in communication via a communication network 503, equipped with cloud-based computing executed by the data analytics module 507 and communications module 508.
  • the imaging device transmits data from an image captured of the detection zone of the testing device described herein, via the communication network, to the data analytics module 507 in the cloud 506 to be analyzed.
  • the data analytics module 507 transmits the result to the communications module 508 to be packaged for display to the user.
  • theresult is displayed on the imaging device 501, whichis a personal electronic device belongingto the user (e.g., the subject in this example).
  • the data store 505 is a remote server in this example.
  • the data store is a cloud-based data store.
  • computing devices comprising a computing system configured to analyze data described herein to provide a result.
  • FIG. 6 a block diagram is shown depicting an exemplary computing device that includes a computing system 600 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and/or methodologies for static code scheduling of the present disclosure.
  • the components in FIG. 6 are examples only and do not limit the scope ofuse or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.
  • Computer system 600 can include one or more processors 601, a memory 603, and a storage 608 that communicate with each other, and with other components, via a bus 640.
  • the bus 640 can also link a display 632, one or more input devices 633 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 634, one or more storage devices 635, and various tangible storage media 636. All of these elements can interface directly or via one or more interfaces or adaptors to the bus 640.
  • the various tangible storage media 636 can interface with the bus 640 via storage medium interface 626.
  • Computer system 600 can have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.
  • ICs integrated circuits
  • PCBs printed circuit boards
  • mobile handheld devices such as mobile telephones
  • Computer system 600 includes one or more processor(s) 601 (e.g., central processing units (CPUs) or general purpose graphics processing units (GPGPUs)) that carry out functions.
  • processor(s) 601 optionally contains a cache memory unit 602 for temporary local storage of instructions, data, or computer addresses.
  • Processor(s) 601 are configured to assist in execution of computer readable instructions.
  • Computer system 600 can provide functionality for the components depicted in FIG. 6 as a result of the processor(s) 601 executing non -transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 603, storage 608, storage devices 635, and/or storage medium 636.
  • the computer-readable media can store software that implements particular embodiments, and processor(s) 601 can execute the software.
  • Memory 603 can read the software from one or more other computer-readable media (such as mass storage device(s) 635, 636) or from one or more other sources through a suitable interface, such as network interface 620.
  • the software can cause processor(s) 601 to carry out one or more processes or one or more steps of one or more processes described or illustrated herein. Carrying out such processesor steps can include defining data structures stored in memory 603 and modifying the data structures as directed by the software.
  • the memory 603 can include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 604) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phase-change random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 605), and any combinations thereof.
  • ROM 605 can act to communicate data and instructions unidirectionally to processor(s) 601
  • RAM 604 can act to communicate data and instructions bidirectionally with processor(s) 601.
  • ROM 605 and RAM 604 can include any suitable tangible computer-readable media described below.
  • a basic input/output system 606 (BIOS) including basic routines that help to transfer information between elements within computer system 600, such as during start-up, is stored in the memory 603
  • Fixed storage 608 is connected bidirectionally to processors) 601, optionally through storage control unit 607.
  • Fixed storage 608 provides additional data storage capacity and can also include any suitable tangible computer-readable media described herein.
  • Storage 608 is used to store operating system 609, executable(s) 610, data 611, applications 612 (application programs), and the like.
  • Storage 608 can also include an optical disk drive, a solid-state memory device (e.g., flash -based systems), or a combination of any of the above.
  • Information in storage 608 may, in appropriate cases, be incorporated as virtual memory in memory 603.
  • storage device(s) 635 is removably interfaced with computer system 600 (e.g., via an external port connector (not shown)) via a storage device interface 625.
  • storage device(s) 635 and an associated machine-readable medium can provide non-volatile and/or volatile storage of machine-readable instructions, data structures, program modules, and/or other data for the computer system 600.
  • software can reside, completely or partially, within a machine -readable medium on storage device(s) 635.
  • software can reside, completely or partially, within processor(s) 601.
  • Bus 640 connects a wide variety of subsystems.
  • reference to a bus can encompass one or more digital signal lines serving a common function, where appropriate.
  • Bus 640 is any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures.
  • such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.
  • ISA Industry Standard Architecture
  • EISA Enhanced ISA
  • MCA Micro Channel Architecture
  • VLB Video Electronics Standards Association local bus
  • PCI Peripheral Component Interconnect
  • PCI-X PCI-Express
  • AGP Accelerated Graphics Port
  • HTTP HyperTransport
  • SATA serial advanced technology attachment
  • Computer system 600 can also include an input device 633.
  • a user of computer system 600 can enter commands and/or other information into the computer system 600 via input device(s) 633.
  • Examples of an input device(s) 633 include, but are not limited to, an alpha-num eric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, atouch screen, a multi-touch screen, ajoystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof.
  • an alpha-num eric input device e.g., a keyboard
  • a pointing device e.g., a mouse or touchpad
  • a touchpad e.g., a touch screen
  • a multi-touch screen e.g.,
  • the input device is a Kinect, Leap Motion, or the like.
  • Input device(s) 633 is interfaced to bus 640 via any of a variety of input interfaces 623 (e.g., input interface 623) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.
  • computer system 600 when computer system 600 is connected to network 630, computer system 600 can communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 630. Communications to and from computer system 600 is sent through network interface 620.
  • network interface 620 can receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 630, and computer system 600 can store the incoming communications in memory 603 for processing
  • IP Internet Protocol
  • Computer system 600 can similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 603 and communicated to network 630 from network interface 620.
  • Processor(s) 601 can access these communication packets stored in memory 603 for processing.
  • Examples of the network interface 620 include, but are not limited to, a network interface card, a modem, and any combination thereof.
  • Examples of a network 630 or network segment630 include, butarenotlimited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof.
  • a network, such as network 630 can employ a wired and/or a wireless mode of communication. In general, any network topology is used.
  • a display 632 includes, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof.
  • the display 632 can interface to the processor(s) 601, memory 603, and fixed storage 608, as well as other devices, such as input device(s) 633, via the bus 640.
  • the display 632 is linked to the bus 640 via a video interface 622, and transport of data between the display 632 and the bus 640 is controlled via the graphics control 621.
  • the display is a video projector.
  • the display is a head-mounted display (HMD) such as a VR headset.
  • suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like.
  • the display is a combination of devices such as those disclosed herein.
  • computer system 600 can include one or more other peripheral outputdevices 634including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof.
  • peripheral output devices is connected to the bus 640 via an outputinterface 624.
  • Examples of an outputinterface624 include,butarenotlimited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.
  • computer system 600 can provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which can operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein.
  • Reference to software in this disclosure can encompass logic, and reference to logic can encompass software.
  • reference to a computer-readable medium can encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate.
  • the present disclosure encompasses any suitable combination of hardware, software, or both.
  • DSP digital signal processor
  • ASIC application specific integrated circuit
  • FPGA field programmable gate array
  • a general purpose processor is a microprocessor, but in the alternative, the processor is any conventional processor, controller, microcontroller, or state machine.
  • a processor can also be implemented as a combination of computing devices, e.g, a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.
  • a software module can reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art.
  • An exemplary storage medium is coupled to the processor such that the processor can read information from, and write information to, the storage medium.
  • the storage medium is integral to the processor.
  • the processor and the storage medium can reside in an ASIC.
  • the ASIC can reside in a user terminal.
  • the processor and the storage medium can reside as discrete components in a user terminal.
  • suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, smart phones, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • server computers desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, smart phones, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • Suitable tablet computers, invariousembodiments include those with booklet, slate, and convertible configurations, known to those of skill in the art.
  • the smartphone is an Apple iPhone or an androiddevice (e.g., Samsung Galaxy).
  • the computing device includes an operating system configured to perform executable instructions.
  • the operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications.
  • suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®.
  • suitable personal computer operating systems include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®.
  • the operating system is provided by cloud computing.
  • suitable mobile smartphone operating systems include, by way of non -limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.
  • suitable media streaming device operating systems include, by way of non -limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®.
  • video game console operating systems include, by way of non -limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.
  • the data processor is a mobile processor.
  • the data processor is configured to receive data from an imaging device, an external device, or a data store, or a combination thereof.
  • data processor analyzes the data to provide a result.
  • the imaging device and the data processor are housed in the same device, such as a smartphone.
  • the data processor is configured to provide a computer program or application, comprising a data analytics module.
  • the application is a web application.
  • the application is a mobile application.
  • the data analytics module is configured to receive data from an imaging device and analyze the data to provide a result.
  • the data analytics module is configured to receive external data from an external device.
  • the external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, or sleep quantity, or a combination thereof.
  • the external device comprises a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • the health tracking device is the Aurora®, Fitbit®, or Apple Watch.
  • systems comprise multiple external devices.
  • the data analytics module is configured to analyze the external data to provide a result.
  • the data analytics module is configured to analyze the external data and the data received from the imaging device to provide a result.
  • the mobile application comprises a communication module.
  • the communication module is configured to communicate the result to the subject.
  • the communication module is configured to display the result to the subject via a graphical user interface (GUI) of an electronic device.
  • GUI graphical user interface
  • the electronic device is the imaging device described herein, the computing device described herein, or a combination thereof.
  • the electronic device is a smartphone, such as those described herein.
  • the data processor is configured to run a computer program.
  • a computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task.
  • Computer readable instructions is implemented as program modules, such as functions, obj ects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types.
  • program modules such as functions, obj ects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types.
  • a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof
  • the computer programs described herein are configured to perform any one of the methods described herein.
  • the computer program is configured to analyze data obtained from an assay described herein (e.g., lateral flow assay, FRET assay) and provide a result based on the analysis of the data.
  • an assay described herein e.g., lateral flow assay, FRET assay
  • the computer program is configured to confirm the presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some instances, the computer program is configured to prognose a subject’s immunity to any one of the pathogen described herein. In some embodiments, the computer program is configured to prognose a subject’s immunity to coronavirus. In some embodiments, the computer program is configured to prognose a subject’s immunity to SARS-CoV-2. In some embodiments, the computer program is configured to measure a subject’s susceptibility to an infection by any one of the pathogen described herein. In some embodiments, computer program is configured to measure a subject’s susceptibility to an infection by coronavirus.
  • the computer program is configured to measure a subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the computer program is configured to identify individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the computer program is configured to identify neutralizing antibodies as a surrogate marker of protection from a vaccine. In some embodiments, the computer program is configured to classify a vaccine by % efficacy in a population. In some embodiments, the computer program is configured to minimize or reduce the risk associated with vaccine usage following market entry (e.g., by identifying individuals that do not respond to the vaccine).
  • Non-transitory computer readable storage medium
  • the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device.
  • a computer readable storage medium is a tangible component of a computing device.
  • a computer readable storage medium is optionally removable from a computing device.
  • a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like.
  • the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media
  • a web application in various embodiments, utilizes one or more software frameworks and one or more database systems.
  • a web application is created upon a software framework such as Microsoft®.NET or Ruby on Rails (RoR).
  • a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems.
  • suitable relational database systems include, by way of non -limiting examples, Microsoft® SQL Server, my SQLTM, and Oracle®.
  • a web application in various embodiments, is written in one or more versions of one or more languages.
  • a web application is written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof.
  • a web application is written to some extent in a markup language such as HypertextMarkupLanguage (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML).
  • a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS).
  • CSS Cascading Style Sheets
  • a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash® Actionscript, Javascript, or Silverlight®.
  • AJAX Asynchronous Javascript and XML
  • Flash® Actionscript Javascript
  • Javascript or Silverlight®
  • a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, JavaTM, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), PythonTM, Ruby, Tel, Smalltalk, WebDNA®, or Groovy.
  • a web application is written to some extent in a database query language such as Structured Query Language (SQL).
  • SQL Structured Query Language
  • a web application integrates enterprise server products such as IBM® Lotus Domino®.
  • a web application includes a media player element.
  • a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, JavaTM, and Unity®
  • data processors comprising one or more mobile applications.
  • the mobile application is provided to a mobile digital processing device at the time it is manufactured.
  • the mobile application is provided to a mobile digital processing device via the computer network described herein.
  • Mobile applications disclosed herein are configured to locate, encrypt, index, and/or access information.
  • Mobile applications disclosed herein are configured to acquire, encrypt, create, manipulate, index, and peruse data.
  • a mobile application is created by suitable techniques using hardware, languages, and development environments known to the art.
  • Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, JavaTM, Javascript, Pascal, Object Pascal, PythonTM, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.
  • Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non -limiting examples, Airplay SDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite,. NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non -limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non -limiting examples, iPhone and iPad (iOS) SDK, AndroidTM SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.
  • iOS iPhone and iPad
  • a standalone application is an independent computer process; not an add-on to an existing process, e.g., not a plug-in.
  • a compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, JavaTM, Lisp, PythonTM, Visual Basic, and VB.NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program.
  • a computer program includes one or more executable complied applications.
  • the data processor comprises a computer program configured with one or more software modules.
  • the one or more software module is a data analytics module.
  • the one or more software module is a communication module.
  • a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof.
  • a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof.
  • the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application.
  • software modules are in one computer program or application.
  • software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location [0208]
  • the computing system described herein comprise a data analytics module. In some embodiments, the data analytics module is configured to run one or more algorithms. In some embodiments, the one or more algorithms comprise a machine learning algorithm.
  • the machine learning algorithm is capable of supervised learning, unsupervised learning, reinforcement learning, semi-supervised learning, self-supervised learning, multiinstance learning, inductive learning, deductive inference, transduction learning, multi-task learning, active learning, online learning, transfer learning, or ensemble learning.
  • the data analytics module is configured with artificial intelligence (Al), such as a limited memory Al.
  • the data analytics module receives data from the testing device, and optionally, external data from one or more external devices, and analyzes the data to provide a result.
  • the data analytics module normalizes the resultby subtracting background signal intensity of the testing device (e.g., background plasma concentration, non-specific binding to the solid surface).
  • background signal intensity of the testing device e.g., background plasma concentration, non-specific binding to the solid surface.
  • the data analytics module identifies whether external data indicates symptoms to an acute infection by a pathogen.
  • the result is either positive or negative.
  • the positive result indicates an acute infection and negative result indicates a lack of an acute infection.
  • the positive result indicates that the subject is immune to a future infection by a pathogen of interest.
  • the data analytics module transmits the result to a communications module for display to the subject.
  • the communication module is configured to display one or more results to the subject via a graphical user interface (GUI).
  • GUI graphical user interface
  • the data store is a database suitable for the storage and retrieval of data.
  • suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, and XML databases. Further nonlimiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, and Sybase.
  • a database is internet-based.
  • a database is web -based.
  • a database is cloud computing-based.
  • a database is a distributed database.
  • a database is based on one or more local computer storage devices, such as a smartphone.
  • GUI graphical user interfaces
  • the user is the subject.
  • the GUI comprises one or more dashboards of an application (e.g., web application or mobile application).
  • the dashboard comprises relevant health information to a subject.
  • the GUI is a part of personal electronic device, such as a smartphone or tablet, belonging to the user.
  • a web portal providing a single access point for multiple users to access information about the immune status of a subject.
  • a web portal provides access to a subject, a subject’s doctor, or a healthcare worker responding to an urgent public health crisis.
  • the portal provides a single access point for a population of individuals, wherein the data is anonymized.
  • the web portal provides access to policy makers, health care professionals, governmental organization, and non-governmental organizations responding to a public health crisis.
  • the portal can indicate one or more geographical locations comprising subjects that are either acutely infected by a pathogen of interest, or immune to the pathogen of interest.
  • a subject may be a member of a variety of races.
  • the subject may be of the White, Black, Hispanic, or Asian race.
  • a subject may be of a variety of ages.
  • the subject may be age 0 to age 18, age 18 to age 65, or age 65 and above.
  • a subject may be a male or a female.
  • a subject may be pregnant, breastfeeding, or may have priorly been infected with a coronavirus.
  • a subject may be a high risk subject and have one or more risk factors related to a coronavirus. In some embodiments, a subject may be obese.
  • methods comprise utilizing the testing devices described herein. In some embodiments, methods further comprise analyzing data generatedby the testing device disclosed herein, and providing a result to a user of an electronic device. In some embodiments, providing the result comprises displayingthe result on a GUI of the electronic device. In some embodiments, the analyzing and displaying is performed by a single computing device (e.g., smartphone, tablet). In some embodiments, analyzing and displaying is performed at the point of need (e.g., at the time and space that the analyte is detected in the biological sample using the testing device described herein).
  • kits for processing a biological sample comprising: (a) providing a biological sample from a subject; (b) measuring a presence, an absence, or a level of a labeled complex between (i) a capture molecule and (ii) a peptide-conjugate comprising a peptide derived from a pathogen in the presence of the biological sample; (c) classifying the biological sample as having a presence or a level of the neutralizing antibody.
  • methods further comprise determining whether a subject suffers from an acute infection by the pathogen (e.g., SARS-CoV-2). In some embodiments, methods further comprise determining whether the subject is immune to an infection by the pathogen (e.g., SARS-CoV-2). In some embodiments, methods further comprise determining whether a vaccine administered to the subject is effective to immunize the subject against the pathogen. In some embodiments, the methods further comprise determining whether the biological sample is safe for transfusion into another subject (e.g., blood, or blood plasma transfusion).
  • another subject e.g., blood, or blood plasma transfusion
  • the methods further comprise determining whether the biological sample is safe as a convalescent plasma therapy for the prevention of an infection by the pathogen or treatment of a disease or condition associated with the pathogen. In some embodiments, the methods further comprise identifying the subject as having an acute infection by the pathogen of interest. In some embodiments, the methods further comprise identifying a subject in need of a vaccination to the pathogen of interest. The methods described herein may be performed at the point of care or point of need.
  • the pathogen comprises a coronavirus or an influenza virus.
  • the methods provided herein confirm presence, absence, or quantity of the neutralizing antibody described herein from a subject’ s biological sample. In some instances, the methods described herein prognose a subject’s immunity to any one of the pathogens described herein. In some embodiments, the methods described herein prognose a subject’s immunity to coronavirus. In some embodiments, the methods described herein prognose a subject’s immunity to SARS-CoV-2. In some embodiments, the methods provided herein measure the subject’s susceptibility to an infection by any one of the pathogens described herein. In some embodiments, the methods provided herein measure the subject’s susceptibility to an infection by coronavirus.
  • the methods provided herein measure the subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the methods provided herein identify individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the methods provided herein minimize or reduce the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the methods provided herein identify neutralizing antibodies as a surrogate marker of protection from a vaccine. In some embodiments, the methods provided herein classify vaccines by % efficacy in a population.
  • the methods provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or greater.
  • the methods provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the methods provided herein have a specificity of at least 80%, for example, atleast 85%, atleast 86%, atleast 87%, at least 88%, at least 89%, atleast 90%, atleast 91%, atleast 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
  • the pathogen is a virus, a bacterium, a fungus, or a parasite.
  • the virus is a DNA virus or an RNA virus.
  • the virus is a single stranded virus, or a double stranded virus.
  • the virus is a plus strand or a minus strand DNA or RNA virus.
  • the virus replicates through reverse transcription of an RNA intermediate.
  • the virus is a coronavirus.
  • the coronavirus is a severe acute respiratory syndrome coronavirus (SARS-CoV).
  • the SARS-CoV is SARS-CoV-2.
  • the coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV).
  • the coronavirus is an alpha coronavirus (e.g., 229E, NL63).
  • the coronavirus is a beta coronavirus (e.g., OC43, HKU1).
  • the virus is an influenza virus.
  • the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequences, or portions thereof, provided in SEQ ID NOS: 1-116.
  • the pathogen of interest is a variant of the pathogen, for example, a pathogen that has evolved through mutations in the genes encoding the pathogen.
  • the variant virus may have a mutation in an amino acid sequence of a viral protein, such as an envelope protein, nucleocapsid protein, membrane protein, hemagglutinin, neuraminidase, or spike protein.
  • the virus e.g., coronavirus
  • the mutation does not affect, or minimally affects, host receptor binding by the spike protein of the coronavirus.
  • the mutation is an insertion, a deletion, or a substitution at an amino acid (or a plurality of amino acids) of one or more sequences provided in SEQ ID NOS: 1-116.
  • the human is an animal, such as a mammal.
  • the mammal is a dog, cat, monkey, non-human primate, rodent, or a farm animal.
  • the rodent is a gerbil, hamster, chinchilla, rat, mouse, or guinea pig
  • the farm animal is a horse, a pig, a cow, a sheep, an alpaca, or a lama.
  • the mammal is a human subject.
  • the subject is pediatric (e.g., age 0-18). In some embodiments, the subject is not pediatric. In some embodiments, the subject is age 50 or older.
  • the subject is an age between 50-100, 55-95, 60-90, 65-85, or 70-80. In some embodiments, the subject is female or male. In some embodiments, the subject has been exposed to the pathogen. In some embodiments, the subject has not been exposed to the pathogen. In some embodiments, the subject exhibits one or more symptoms comprising a cough, fever, tiredness, or difficulty breathing. In some embodiments, the subject has an underlying health problem comprising high blood pressure, a heart problem, diabetes, immunodeficiency, autoimmune disease. In some embodiments, the subject is immunocompromised.
  • the subject has an underlying immune-comprising condition comprising a pregnancy, an immune- mediated disease, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof.
  • the subject is breastfeeding.
  • the subject is receiving an immunosuppressant therapy for treatment of a disease or a condition.
  • the immunosuppressant therapy comprises a biologic drug (e.g., adalimumab (Humira®) and infliximab (Remicade®)), calcineurin inhibitors (e.g., tacrolimus (Envarsus XR® or Protopic) and cyclosporine (Gengraf®, Neoral® or Sandimmune®)), corticosteroids (e.g., prednisone), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolate mofetil (CellCept®)), janus kinase inhibitors (e.g., tofacitinib (Xeljanz®)), mechanistic target of rapamycin (mTOR) inhibitors (e.g., sirolimus (Rapamune®)), or monoclonal antibodies (e.g, basiliximab (Simulect®)), or any combination thereof.
  • the population comprises subjects of any demographics, age, or geographical locations. In some embodiments, all subjects in the population are vaccinated against the pathogen. In some embodiments, some subjects in the population are vaccinated against the pathogen. In some embodiments, none of the subjects in the population are vaccinated against the pathogen. In some embodiments, the number of vaccinated subjects in the population is unknown. In some embodiments, all subjects in the population are previously infected by the pathogen. In some embodiments, some subjects in the population are previously infected by the pathogen. In some embodiments, none of the subjects in the population are previously infected by the pathogen.
  • the number of subjects previously infected by the pathogen is unknown. In some embodiments, all subjects in the population are currently infected by the pathogen. In some embodiments, some subjects in the population are currently infected by the pathogen. In some embodiments, none of the subjects in the population are currently infected by the pathogen. In some embodiments, the number of subjects currently infected by the pathogen is unknown. In some embodiments, the population is a general population. In some embodiments, the population is a population undergoing clinical trial, where all subjects of the population are vaccinated or unvaccinated. In some embodiments, the population is a population that is at higher risk of the infection.
  • the population that is at higher risk of the infection includes healthcare workers, essential workers, elderly, subjects with pre-existing condition, subjects with respiratory disease or disorder, or subjects with comorbidity or any of the health problems described herein.
  • the population comprises individuals who are vaccine responders (e.g., develop neutralizing antibodies as described herein in response to the vaccine).
  • the population comprises individuals who are vaccine nonresponders (e.g., do not develop neutralizing antibodies as described herein in response to the vaccine).
  • the population comprises individuals who have been infected and/or exposed to the natural pathogen.
  • obtaining the biological sample from the subject or population of subjects is direct or indirect. Indirectly obtaining a biological sample from a subject may include receiving it from a laboratory or processing/ storage facility by mail, or otherwise. In some embodiments, indirectly obtaining the biological sample from the subject or the population comprises the use of blood collection means such as blood card. In some embodiments, the blood card comprises multiple slots for collecting multiple samples of blood droplets for multiple testing.
  • a biological sample medium (e.g., a dried blood spot card) comprises multiple slots is tested by the automated centralizing assay system performing assays such as control assay to verify the quality of the biological sample comprisingthe blood, lateral flow assay, ELISA, biomarker panel, qPCR, western blotting and a neutralizing antibody titer assay.
  • biological samples include cell, tissue, or bodily fluid obtained from the subject.
  • Non-limiting examples of biological samples include aqueous humour, vitreous humour, bile, breastmilk, cerebrospinal fluid, endolymph, perilymph, female ejaculate, amniotic fluid, gastric juice, menses, mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, urine, feces, whole blood, blood serum, blood plasma, sputum, cerebrospinal fluid, synovial fluid, lymphatic fluid, nasal swab, or cheek swab.
  • the biological sample comprises blood, urine, saliva, or feces.
  • the blood is capillary blood.
  • the blood is not venous blood (e.g., from a phlebotomy).
  • methods disclosed herein comprise obtaining a blood sample by administering a finger prick.
  • the biological sample is a swab sample (e.g., buccal swab, nasopharyngeal swab).
  • Directly obtaining the biological sample from the subject is performed by a healthcare professional (e.g., nurse or doctor), laboratory technician, or the subject at the point of need or the point of care.
  • the target analyte comprises macromolecules such as peptides, proteins, nucleic acids, pathogens, or pathogen particles that can be detected for presence or quantity (i.e. concentration) in a biological sample.
  • the target analyte is targeted and formed complex with an antigen or with an antibody.
  • the target analyte comprises an antibody against a pathogen.
  • the antibody against the pathogen is an antibody against an antigenic peptide derived from the pathogen.
  • the antibody is neutralizing antibody induced by an infection . In some instances, the antibody is neutralizing antibody induced by vaccination.
  • the analyte may be an antibody against a portion of the spike protein derived from a coronavirus (e.g., SARS-CoV-2).
  • the analyte is the activity of the antibody.
  • the activity is blocking binding between the spike protein of a coronavirus and its cognate receptor (e.g., ACE2).
  • the analyte is a complex comprising the spike protein bound to the antibody at the receptor binding region of the spike protein.
  • the antibody belongs to an immunoglobulin class comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, or immunoglobulin D.
  • the detectable peptide-conjugate is the peptide-conjugate described herein comprising a detection agent and a peptide.
  • the peptide is an antigenic peptide.
  • the analyte e.g., antibody
  • the peptide comprises at last a portion of the spike glycoprotein of a coronavirus (e.g., SARS-CoV-2) described herein.
  • the peptide is a receptor to the antigenic peptide.
  • the receptor comprises ACE2 receptor described herein.
  • measuring comprises performing an assay on the biological sampleto detecta number of complexes formedbetweenthe analyte and the peptide -conjugate, or the peptide-conjugate and the capture molecule, or both.
  • the assay comprises an assay assembly described herein.
  • the assay is a lateral flow assay.
  • the lateral flow assay is a competition assay.
  • measuring a number of complexes formed between the analyte and the peptide- conjugate, or the peptide-conjugate and the capture molecule, or both can be performed in one step without the need for washing any portion of the assay assembly.
  • the index or the control is derived from a subject that has not been exposed to the pathogen (negative control). In some embodiments, the index or control is from a subject that has been exposed to the pathogen (positive control). In some embodiments, the index or control is obtained from a convalescent plasma donor. In some embodiments, the index or control is synthetically derived. In a non-limiting example of a control that is synthetically derived, the control can be sample with cells engineered to express a peptide or protein that is known to induce production of neutralizing antibodies against the pathogen to a known degree. In some embodiments, the control or index is a maximum absolute dilution of a reference sample in which neutralizing activity is observed in the reference sample.
  • neutralizing activity in the diluted reference sample is a correlate of the level of neutralizing antibodies titers in the reference sample.
  • the biological sample is classified as having a sufficient presence or level of neutralizing antibodies to confer adaptive immunity against a pathogen.
  • the biological sample is classified as having a sufficient presence or high level of neutralizing antibodies when the level of neutralizing antibodies correlates with a dilution that is above 1 :60, or above or equal to about 1 :65, 1 :70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245,1:250, 1:255, 1:260,1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:35
  • the dilution comprises four (4) dilutions equal to about 1:80, 1 :160, 1:320, and 1:640.
  • the biological sample is classified as having a low level or insufficient presence of neutralizing antibodies to confer adaptive immunity.
  • the biological sample is classified as having an insufficient presence or a low level of neutralizing antib odies when the level of the neutralizing antibodies correlates with a dilution that is below or equal to about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, or 1:60.
  • Methods described herein do not consist of utilizing a cell culture, such as an immortalized cell line expressing human ACE2.
  • methods do not consist of handling or administering to a cell or cell line a purified or isolated pathogen, such as a live virus of pseudovirus.
  • the methods comprise measuring the target analyte in a biological sample from a subject or in a population of subjects multiple times.
  • the analyte is measured at least once every 12 hours, 24 hours, 36 hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, one year, 18 months, two years, three years, four years, five years, six years, seven years, eight years, nine years, decade, or a combination thereof.
  • the analyte is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In some embodiments, the analyte is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months following an event.
  • the event is an exposure to the pathogen, a first symptom of a disease or condition caused by the pathogen, a first dose of a vaccine against the pathogen, or a first dose of an anti-viral therapy to treat an acute infection by the pathogen.
  • the analyte is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years following the event. In some embodiments, the analyte is measured before the event, such as, for example before a first exposure to the pathogen to identify a subject or population of subjects who do not have sufficient immunity against the pathogen and who may be in need of a vaccine.
  • convalescent plasma for therapy to prevent SARS-CoV-2 infection requires demonstration that the plasma contains sufficient titers of neutralizing antibodies to confer immunity to SARS-CoV-2.
  • Existing methods of detecting sufficient neutralizing antibody titers for this purpose are limited at the point of need because they require use of an immortalized cell line expressing human angiotensin converting enzyme II (ACE2), e.g., 293T cells.
  • ACE2 human angiotensin converting enzyme II
  • the testing devices described herein are portable and methods of using the testing devices described herein are cell-free (e.g., do not require use of a cell or cell line). In this manner, the testing devices are more cost effective, do not require a laboratory technician, and is scaled quickly to meet the growing demand of individuals in need of convalescent plasma therapeutic interventions.
  • a biological sample for use as a convalescent plasma therapy comprising: (a) providing a biological sample from a donor subject; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (ii) a neutralizing antibody against the pathogen; (c) classifying the biological sample as having a presence or a level of the neutralizing antibody; and (d) identifying the biological sample as safe or not safe as a convalescent therapy based at least in part on the classifying in (c).
  • the level is high relative to an index or a control.
  • the biological sample is identified as safe for convalescent therapy provided the presence or the level of the labeled complex between the peptide-conjugate and the neutralizing antibody against the pathogen is measured.
  • the biological sample is identified as unsafe for convalescent therapy provided the presence or the level of the labeled complex between the peptide-conjugate and the capture molecule is measured.
  • the method comprises: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; (c) measuring a presence, an absence or a level of an antibody against the pathogen (e.g., neutralizing or otherwise); (d) classifying the biological sample as having a presence or a level of the neutralizing antibody and/or a presence or a level of a type of antibody (e.g., IgG, IgM, IgA) against the pathogen; and (d)identifyingthe
  • the subject has a sufficient adaptive immunity to an infection by the pathogen. In some embodiments, if the number of the complexes between (i) and (ii) is high relative to an index or a control, then the subject has a sufficient adaptive immunity to the infection by the pathogen. In some embodiments, the methods described herein are performed using the testing device of the present disclosure. In some embodiments, measuring a presence, an absence or a level of an antibody against the pathogen (e.g., neutralizing or otherwise) can be performed in one step without the need for washing any portion of the testing device.
  • the capture molecule or peptide-conjugate may comprise a peptide or protein that is encoded by a nucleic acid sequence, or that has an amino acid sequence, that is at least 80% identical to any one of SEQ ID NOS: 21-23, 25-42, 44-46, 49-66, 68-70, 73-90, 92-94, or 97-114.
  • the presence, absence, or the level of the antibody against the pathogen may be measured by detecting a complex between the antibody (e.g., IgG, IgM, IgA) and a capture molecule that is directly or indirectly labeled.
  • a level of the antibody that is high, relative to an index or a control is indicative that the subject was exposed to the pathogen.
  • the level of the antibody is low relative to the index or control, the subject was not exposed to the pathogen.
  • a presence, absence or a level of a biomarker is measured in the biological sample.
  • the biomarker is a marker of inflammation, such as a cytokine (e.g., interleukin 6).
  • the presence, absence or level of multiple biomarkers is measured in addition to the total antibodies and neutralizing antibodies againstthe pathogen.
  • thepathogen is SARS-CoV-2, or avariantthereof.
  • a biological sample of neutralizing antibody to be tested is serial -diluted and mixed with a viral suspension. The mixture is applied to known number of cells for inoculation.
  • the neutralizing antibody titer is determined by calculatingthe highest dilution of biological sample of neutralizing antibody that prevents infection of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of replicate inoculations.
  • the neutralization titer is determined by calculatingthe highest dilution of the biological sample of neutralizing antibody that prevents infection at 50% of replicate inoculation.
  • the method of measuring neutralization titer comprises the use of viral plaque assay, fluorescent focus assay, and endpoint dilution assay. Each of these three assays relies on serial viral dilutions added to cells to measure neutralization titer.
  • Other exemplary measurements for determining neutralization titer include qPCR or ELISA for quantifying the amount of pathogen genome or particle necessary to infect a set number of cells in the presence of the neutralizing antibody.
  • the quantity of neutralizing antibody detected in a biological sample is sufficient in inducing adaptive immunity when the concentration of the neutralizing antibody in the biological sample prevents infection at 50% of replicate inoculation. In some embodiments, the quantity of neutralizing antibody detected in a biological sample is insufficient in inducing adaptive immunity when the concentration of the neutralizing antibody in the biological sample prevents infection below 50% of replicate inoculation.
  • the method comprises detecting presence, absence, or quantity of analyte comprising the neutralizing antibody in the population.
  • the method prognoses immunity or susceptibility to the infection based on the presence, absence, or quantity of the neutralizing antibody in the biological samples obtained from the subjects in the population.
  • the method prognoses the population to have adaptive immunity against the infection when the presence or sufficient quantity of the neutralizing antibody is detected in the biological samples.
  • the method prognoses the subjectto have susceptibility or inadequate immunity to the infection when the neutralizing antibody is absent or is detected at an insufficient quantity in biological samples. In some embodiments, the method identifies the subjects with absence or insufficient quantity of neutralizing antibody as subjects who are in need of vaccination. In some embodiments, the population is screened at any one of the time frames or frequencies described herein.
  • the method determines if the populationis in need of vaccination against the infection based on the presence, absence, or quantity of the neutralizing antibody in the biological samples. In some embodiments, the method determines that the population does not need to be vaccinated due to presence or sufficient quantity of the neutralizing antibody detected in the biological samples. In some embodiments, the method determines that the population does not need to be vaccinated due to herd immunity when the percentage of the subjects of the population with presence or sufficient quantity neutralizing antibody is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the method determines that the population or subjects within the population need to be vaccinated due to absence or insufficient quantity of the neutralizing antibody detected in the biological samples. Confirmatory Diagnostic for a Vaccine
  • methods of determining the efficacy or effectiveness of a vaccine described herein comprise detecting presence, absence, or quantity of neutralizing antibody against the pathogen in a biological sample from a subject that was administered the vaccine.
  • determining the efficacy or effectiveness of a vaccine against a pathogen comprising: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide- conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; (c) classifying the biological sample as having a presence or a level of the neutralizing antibody; and (d) identifying the vaccine as effective to induce a sufficient or insufficient adaptive immune response against the pathogen in the subject.
  • the level is high relative to an index or a control.
  • the vaccine is effective, provided the presence or the level of the labeled complex between the peptide-conjugate and the neutralizing antibody against the pathogen is measured. In some embodiments, the vaccine is not effective, provided the presence or the level of the labeled complex between the peptide-conjugate and the capture molecule is measured. In some embodiments, methods further comprise measuring a presence, absence or a level of a biomarker described herein. In some embodiments, the biomarker is interleukin 6.
  • measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide -conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen can be performed in one step without the need for washing.
  • methods confirm that the vaccine lacks efficacy or effectiveness in the subject, provided an absence of the neutralizing antibody is detected in the biological sample obtained from the subject that was administered the vaccine. In some embodiments, methods confirm that the vaccine lacks efficacy or effectiveness, provided the level of the neutralizing antibody detected is low relative to an index or a control level. An absence or a low level of the neutralizing antibody is identified when a presence of the labeled complex between the peptide-conjugate and the capture molecule is detected. In some embodiments, methods confirm that the vaccine is effective, provided a presence of the neutralizing antibody is detected in the biological sample obtained from the subject that was administered the vaccine. In some embodiments, methods confirm that the vaccine is effective provided a level of the neutralizing antibody that is detected is high relative to an index or a control level.
  • the subject is part of a general population, where not everyone in the general population was or is administered the vaccine.
  • the testing devices and methods described herein confirm which subjects of the general population are in need of the vaccine by detecting the absence or the low level of the neutralizing antibody in biological samples obtained from the subjects.
  • the testing devices and methods described herein measure neutralizing antibodies against a pathogen in a biological sample from a subject prior to enrolling the subject in the clinical trial.
  • the presence or level of the neutralizing antibody detected in the biological sample excludes the subject from participating in the clinical trial for the vaccine.
  • the absence or level of the neutralizing antibody detected in the biological sample is an inclusion criteria for the subject to enroll in the clinical trial for the vaccine.
  • the testing devices and methods described herein measure neutralizing antibodies against a pathogen in a biological sample from a subject during the clinical trial for the investigation of the efficacy of the vaccine.
  • a biological sample from the vaccinated subject is tested using the testing devices and methods described herein when an adverse event is observed during the clinical trial.
  • the adverse event is correlated with a presence, absence or level of neutralizing antibodies in the biological sample.
  • the testing devices and methods described herein confirm efficacy or effectiveness of the vaccine in a population of subjects.
  • the population being tested for efficacy or effectiveness of the vaccine is a group receiving the vaccine (as opposed to a different group receiving the placebo) in a clinical trial.
  • the populationbeingtested for efficacy or effectiveness ofthe vaccine is agro up that is outside the clinical trial context, such as after the vaccine is allowed or approved for use in preventing a disease caused by the pathogen.
  • the testing devices and methods described herein detect presence, absence, or quantity of the neutralizing antibody against a pathogen in a biological sample from each subject in the group before and after the group is administered the vaccine.
  • the testing devices and methods described herein confirm adaptive immunity in the vaccinated population based on presence or high level of the detected neutralizing antibody in the biological samples obtained from the vaccinated subjects in the population. In some embodiments, the testing devices and methods described herein confirm a lack of sufficient adaptive immunity in the vaccinated population based on absence or low level of the detected neutralizing antibody in the biological samples obtained from the vaccinated subjects in the population. In this instance, a booster of the vaccine may be recommended for at least one subject of the group, or the entire group.
  • neutralizing antibodies induced by the vaccine are measured multiple times after administration of the vaccine to determine whether the vaccine is effective to confer immunity against the pathogen (e.g., SARS-CoV-2).
  • the neutralizing antibodies are measured after administration of the vaccine at least once every 12 hours, 24 hours, 36 hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, one year, per 18 months, two years, three years, four years, five years, six years, seven years, eightyears, nine years, 10 years, or a combination thereof.
  • the neutralizing antibodies are measured atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 ormore times. In some embodiments, the neutralizing antibodies are measured atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months following administration of the vaccine to the subject. In some embodiments, the neutralizing antibodies are measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years following administration of the vaccine to the subject.
  • methods are provided using the systems, devices, and compositions described herein for identifying those subjects that respond to a vaccine (e.g., responders) and those subjects that do notrespond to a vaccine (e.g., non -responders).
  • the systems, devices, and compositions described herein may be used to test individuals in a population of individuals for the presence or absence of neutralizing antibodies to identify those individuals that responded to the vaccine (e.g., produced neutralizing antibodies), and those individuals that did not respond to the vaccine (e.g., did not produce neutralizing antibodies).
  • the population may be a group of individuals that received a vaccine against the pathogen (e.g., SARS-CoV-2).
  • the population may be a group of individuals that received a vaccine during a clinical trial to assess vaccine efficacy.
  • the population may be a group of individuals that received a vaccine as part of a large-scale, mass vaccination campaign.
  • the methods provided herein assess an individual’s particularized response to a vaccine.
  • the methods provided herein identify those individuals that did not respond to a particular vaccine, inform those individuals that they did not develop immunity to the pathogen, and inform those individuals that they should seek alternative interventions (e.g., alternative vaccines) and/or start or continue safety precautions (e.g., social distancing, the use of PPE, etc.).
  • the methods may involve administering a vaccine to a population of individuals, and testing each of those individuals for the presence or absence of neutralizing antibodies to assess whether the vaccine conferred immunity again st the pathogen (e.g. , SARS - CoV-2).
  • the individuals after receiving the vaccine, may submit one or more biological samples to be tested for the presence or absence of neutralizing antibodies as described herein (e.g., by submitting the sample to a centralized testing center).
  • each individual that receives a vaccine may be given a sample collection device (e.g., a blood spot card) for submittinga biological sample fortesting.
  • the sample collection device e.g., blood spot card
  • the sample collection device may be given to the individual at the time of vaccination.
  • the individual may collect one or more biological samples (e.g., a finger prick to collect a blood sample) and provide the one or more biological samples in the sample collection device (e.g., blood card). This may be done before and after vaccine administration.
  • the one or more biological samples may be collected at a time point before the individual has been vaccinated to establish a baseline level of neutralizing antibodies.
  • the one or more biological sample may be collected again in the same manner at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 2 months after vaccine administration to determine whether the vaccineinduced a sufficient neutralizing antibody titer to confer protection against the pathogen.
  • the one or more biological samples are taken after sufficient time has elapsed for the individual to develop neutralizing antibodies (e.g., if the individual responds to the vaccine).
  • one or more biological samples may be collected in the same manner at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, or more following the vaccine administration.
  • the individual may submit the one or more biological samples in the sample collection device (e.g., blood spot card) to a testing center.
  • the testing center may test the one or more biological samples for the presence or absence of neutralizing antibodies (e.g., using the sy stems, devices, and compositions describedherein), and may identify the individual as either a vaccine responder (e.g., the individual developed neutralizing antibodies) or as a vaccine nonresponder (e.g., the individual did not develop neutralizing antibodies).
  • the testing center may provide the individual with a report (or may otherwise inform the individual) indicating the individual’s status as a vaccine responder or as a vaccine non-responder.
  • the individual When the individual is identified as a vaccine responder, the individual may be understood to have developed immunity (e.g., the individual responded to the vaccine) to the pathogen (e.g., SARS-CoV-2).
  • the pathogen e.g., SARS-CoV-2
  • the individual When the individual is identified as a vaccine non-responder (e.g., the individual did not respond to the vaccine), the individual may be understood to not have developed immunity to thepathogen (e.g., SARS-CoV-2).
  • an alternative intervention e.g., an alternative vaccine
  • the vaccine has entered the market.
  • the vaccine is a vaccinein developmentoravaccinethathasnotyetbeen approvedby aregulatory entity, such as the FDA.
  • the vaccine is a vaccine that has been licensed for use by the regulatory entity.
  • methods are provided herein using the systems, devices, and compositions described herein to mitigate or reduce the risk associated with usage of a commercialized vaccine in a population.
  • the methods provided herein may assess the benefit and the risk of a particular vaccine by identifying the individuals who respond to the vaccine and the individuals who do not respond to the vaccine.
  • the systems, devices, and compositions described herein may be used to test individuals in a population for the presence or absence of neutralizing antibodies to identify those individuals that responded to a particular vaccine (e.g., produced neutralizing antibodies), and those individuals that did not respond to the vaccine (e.g., did not produce neutralizing antibodies).
  • the non -responders are identified and informed that they did not develop immunity, thereby removing the risk associated with a particular vaccine.
  • the methods provided herein allow a vaccine developer to leverage the benefits associated with a particular vaccine, including vaccines with limited efficacy, while minimizing or reducing the risks associated with the vaccine.
  • the methods provided herein may be particularly suited for emergent situations in which multiple vaccines are being developed on an accelerated timeline against a pathogen (and may notbe subject to the entire FDA review process).
  • Vaccine 1 may be deemed “efficacious” because the benefit outweighs the risk (e.g., the number of responders is greater than the number of non-responders).
  • Vaccine 1 is developed into a vaccine for widespread use against the pathogen (e.g., SARS-CoV-2).
  • the pathogen e.g., SARS-CoV-2
  • 40% of the individuals in a population may respond to vaccination with Vaccine 2 (e.g. , the “benefit”), and 60% of the individuals in the populationmay notrespondto vaccination with Vaccine 2 (e.g., the “risk”).
  • Vaccine 2 may be deemed “not efficacious” because the risk outweighs the benefit (e.g., the number of non-responders is greater than the number of responders).
  • Vaccine 2 is not used to prevent infection in a population by the pathogen (e.g., SARS-CoV-2).
  • the methods provided herein may be used to identify the risk of a particularvaccine and remove the risk (e.g., by informingthe non-responders of their status).
  • the methods provided herein identify the risk of Vaccine 1 (e.g., the 30% of the population who did not respond to Vaccine 1) and those individuals are informed that they did not develop immunity to the pathogen (SARS-CoV-2).
  • SARS-CoV-2 the pathogen
  • the methods provided herein identify the risk of Vaccine 2 (e.g., the 60% of the population who did not respond to Vaccine 2) and those individuals are informed that they did not develop immunity to the pathogen (e.g, SARS-CoV-2).
  • the risk is removed because the non-responders are informed that they do not have immunity to the pathogen (and can take precautionary measures such as social distancing, the use of PPE, and the like).
  • the individual may collect one or more biological samples (e.g., a finger prick to collect a blood sample) and provide the one or more biological samples in the sample collection device (e.g., blood card). This may be done before and after vaccine administration.
  • the one or more biological samples may be collected at a time point before the individual has been vaccinated to establish a baseline level of neutralizing antibodies.
  • the one or more biological sample may be collected again in the same manner at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 2 months after vaccine administration to determine whether the vaccineinduced a sufficient neutralizing antibody titer to confer protection against the pathogen.
  • the one or more biological samples are taken after sufficient time has elapsed for the individual to develop neutralizing antibodies (e.g., if the individual responds to the vaccine).
  • one or more biological samples may be collected in the same manner at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, or more following the vaccine administration.
  • the individual may submit the one or more biological samples in the sample collection device (e.g., blood spot card) to a testing center.
  • the testing center may test the one or more biological samples for the presence or absence of neutralizing antibodies (e.g., using the systems, devices, and compositions describedherein), and may identify the individual as either a vaccine responder (e.g., the individual developedneutralizing antibodies) or as a vaccine nonresponder (e.g., the individual did not develop neutralizing antibodies).
  • the testing center may provide the individual with a report (or may otherwise inform the individual) indicating the individual’s status as a vaccine responder or as a vaccine non-responder.
  • the individual When the individual is identified as a vaccine responder, the individual may be understood to have developed immunity (e.g., the individual responded to the vaccine) to the pathogen (e.g., SARS-CoV-2).
  • the pathogen e.g., SARS-CoV-2
  • the individual When the individual is identified as a vaccine non-responder (e.g., the individual did notrespond to the vaccine), the individual may be understood to not have developed immunity to the pathogen (e.g., SARS-CoV-2).
  • an alternative intervention such as an alternative vaccine under development, maybe recommended to the individual.
  • the vaccine has entered the market for use.
  • the vaccine is a vaccine in development or a vaccine that has notyet been approvedby a regulatory entity (e.g., the FDA).
  • the vaccine is a vaccine that has been licensed for use by the regulatory entity.
  • methods are provided using the systems, devices, and compositions described herein to classify a vaccine in a population of individuals.
  • the methods comprise (a) providing a biological sample from each individual of a population of individuals; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen in the biological sample; (c) classifying each biological sample as having a presence, an absence, or a level of the neutralizing antibody; and (d) classifying the vaccine based on the presence or the level of the neutralizing antibodies measured in (b).
  • the vaccine is classified by % efficacy in the population (e.g., the % of individuals in a population that responded (e.g., developed neutralizing antibodies)to the vaccine). In some embodiments, the vaccine is at least 50% efficacious in a population (e.g., at least 60%, at least 70%, at least 80%, atleast90%, atleast95%, atleast97%, atleast98%, atleast 99%). In some embodiments, the classification of the vaccine is specific to a particular population class, such as gender, age, and individuals with pre-existing conditions (e.g., immune-compromised).
  • the vaccine may be 50% efficacious in children ages 12 andyounger, whereas the vaccine maybe 75% efficacious in adults ages 65 and older.
  • measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen in the biological sample can be performed in one step without the need for washing.
  • methods are provided using the systems, devices, and compositions described herein to identify the presence and/or a high level of neutralizing antibodies in an individual after administration of a vaccine.
  • the methods utilize the presence and/or high level of neutralizing antibodies as a surrogate marker of protection in the individual.
  • the presence and/or high level of neutralizing antibodies in the individual is used to inform a vaccine developer that the individual developed immunity to the pathogen (e.g., SARS-CoV-2).
  • the presence and/or high level of neutralizing antibodies in the individual is used in place of traditional methods that test efficacy of a vaccine (e.g., a randomized, placebo-controlled clinical trial).
  • the methods comprise (a) providing a biological sample from a subject having been administered a vaccine; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide -conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; (c) classifying the biological sample as having a presence, an absence, or a level of the neutralizing antibody; and (d) identifying the subject as having immunity against the pathogen based on the presence and/or a high level of the neutralizing antibody.
  • the vaccine is for the active immunization for the prevention of disease caused by a pathogen.
  • the pathogen described herein in some embodiments, is any bacteria, virus, or fungus that causes infection in a mammal.
  • a pathogen is a virus of any one of the virus described herein.
  • the virus is a DNA virus or an RNA virus.
  • a DNA virus is a single-stranded (ss) DNA virus, a double-stranded (ds) DNA virus, ora DNA virus that contains both ss and ds DNA regions.
  • An RNA virus is a single -stranded (ss) RNA virus or a double-stranded (ds) RNA virus.
  • a ssRNA virus can further be classified into a positive-sense RNA virus or a negative-sense RNA virus.
  • the dsDNA virus is from the family: Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfaviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Mimiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae,
  • a ssDNA virus is from the family: Anelloviridae, Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae.
  • a DNA virus that contains both ss and ds DNA regions is from the group of pleolipoviruses.
  • the pleolipoviruses include Haloarcula hispanica pleomorphic virus 1, Halogeometricum pleomorphic virus 1, Halorubrum pleomorphic virus 1, Halorubrum pleomorphic virus 2, Halorubrum pleomorphic virus 3, and Halorubrum pleomorphic virus 6.
  • the dsRNA virus is from the family: Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megavimaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Rotavirus and Totiviridae.
  • a positive-sense ssRNA virus is from the family: Alphaflexiviridae, Alphatetraviridae, Alvemaviridae, Arteriviridae, Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae, Dicistroviridae, Flaviviridae, Gammaflexiviridae, Iflaviridae, Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae, Roniviridae, Secoviridae, Togaviridae, Tombusviridae, Tymoviridae, and Virgaviridae.
  • a negative -sense ssRNA virus is from the family: Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, and Orthomyxoviridae.
  • the vaccine described herein confers adaptive immunity against the pathogen when administered to a subject. In some embodiments, the vaccine described herein confers adaptive immunity to a subject against pathogenic (e.g., viral) infection caused by the pathogen. In some embodiments, the vaccine described herein induces neutralizing antibody against the pathogen in the subject after administration of the vaccine.
  • pathogenic e.g., viral infection caused by the pathogen.
  • the vaccine described herein induces neutralizing antibody against the pathogen in the subject after administration of the vaccine.
  • the vaccine confers adaptive immunity against a coronavirus.
  • the coronavirus is selected from the group consisting of: alphacoronavirus, betacoronavirus, deltacoronavirus, and gammacoronavirus.
  • alphacoronavirus include, but are not limited to, Bat coronavirus CDPHE15, Bat coronavirus HKU 10, Human coronavirus 229E, Human coronavirus NL63, Miniopterus bat coronavirus 1 , Miniopterus bat coronavirus HKU8, Mink coronavirus 1, Porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2, and Scotophilus bat coronavirus 512.
  • betacoronavirus examples include, but are not limited to, Betacoronavirus 1, Hedgehog coronavirus 1, Human coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus, Tylonycteris bat coronavirus HKU4.
  • deltacoronavirus examples include, but are not limited to, Bulbul coronavirus HKU11, Common moorhen coronavirus HKU21, Coronavirus HKU15, Munia coronavirus HKU13, Night heron coronavirus HKU19, Thrush coronavirus HKU12, White-eye coronavirus HKU16, Wigeon coronavirus HKU20.
  • gammacoronavirus examples include, but are not limited to, Avian coronavirus, Beluga whale coronavirus SW1. Additional examples of coronavirus include MERS-CoV, SARS-CoV, and SARS-CoV-2.
  • the vaccine comprises an inactivated virus of any one of the virus described herein. In some embodiments, the vaccine comprises an inactivated coronavirus. In some embodiments, the vaccine comprises an inactivated SARS-CoV-2. In some embodiments, the vaccine comprises formalin -inactivated SARS-CoV-2. In some cases, the vaccine comprises a live -attenuated virus of any one of the virus described herein. In some cases, the vaccine comprises a live-attenuated coronavirus. In some cases, the vaccine comprises a live-attenuated SARS-CoV-2. In some embodiments, the vaccine comprises chemical inactivated SARS-CoV-2.
  • the goal of chemical and physical inactivation of viruses may be to destroy the infectivity of the pathogen without destroying the immunogenicity of important epitopes.
  • inactivation processes may require confirmation of that the virus infectivity is destroyed.
  • the inactivation processes may require confirmation that the immunogenicity is intact.
  • the inactivation process may require that the inactivated pathogen does not pause a health risks to the subject.
  • chemical inactivation of the virus is complete using various inactivating chemical agents, such as for example formaldehyde, ⁇ -Propiolactone (BPL), hydrogen peroxide treatment, zinc-finger reactive treatment.
  • the vaccine comprises a physically inactivated SARS-CoV-2.
  • physical inactivation uses heating or pasteurizing of viruses, gamma irradiation, ultraviolet light, among other techniques.
  • viral inactivation can use chemical or physical methods or a combination of the two.
  • various methods may be employed to inactivate SARS-CoV-2 virus for vaccine production; these may include methods described in Sanders et al, 2015 (Vaccine Analysis: Strategies, Principles and Controls), which reference is hereby incorporated in its entirety.
  • antigenic peptide is derived from a protein of any one of the pathogens described herein.
  • the antigen is viral antigen derived from a viral protein, a fragment of a viral protein, or a nucleic acid encoding the viral protein or the fragment of the viral protein.
  • the viral antigen is a viral antigen of coronavirus.
  • the viral antigen is a viral antigen of SARS-CoV-2.
  • the vaccine comprises viral antigen for inducing adaptive immunity in a subject.
  • the viral antigen is a non -mutated antigen.
  • the viral antigen is derived from publicly disclosed information on the viral genetic information.
  • the viral antigen is derived from analysis of the viral genome to predict suitable epitopes for T cell activation.
  • the viral antigen is derived from analysis of the sequence of the viral genome in a MHC-peptide presentation prediction algorithm implemented in a computer processor.
  • the viral antigen is derived from analysis of the viral sequences in an MHC-peptide presentation prediction algorithm implemented in a computer processor that has been trained by a machine learning software, which predicts the likelihood of binding and presentation of an epitope by an MHC class I or an MHC class II antigen.
  • the MHC-peptide presentation predictor is neonmhc2.
  • the viral antigen is encoded by a nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS: 7-10 (SARS-CoV-2), 13-16 (SARS-CoV NL630), 17-20 (SARS Co V Tor2).
  • the viral antigen is encoded by a nucleic acid sequence that is 100% identical to any one of SEQ ID NOS: 7-10, 13-16, or 17-20.
  • the viral antigen comprises a peptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 2-6, or 11-12. In some instances, the viral antigen comprises a peptide sequence that is 100% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115.
  • the viral antigen comprises a peptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a fragment of any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. In some instances, the viral antigen comprises a peptide sequence that is 100% identical to a fragment of SEQ ID NOS: 2-4, or 11-12
  • the viral antigen comprisesan amino acid sequence provided in SEQ ID NO: 5, or a variant thereof. In some embodiments, the viral antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1.
  • the vaccine comprises a viral antigen of SARS-CoV-2.
  • the viral antigen is selected from the group consisting of: orfla, orflab, Spike glycoprotein (S protein), 3a, 3b, Envelope protein (E protein), Membrane protein (M protein), p6, 7a, 7b, 8b, 9b, Nucleocapsid protein (N protein), orfl4, nspl (leader protein), nsp2, nsp3, nsp4, nsp5 (3C-like proteinase), nsp6, nsp7, nsp8, nsp9, nsplO (growth -factor-like protein), nspl2 (RNA-dependentRNA polymerase, orRdRp), nspl3 (RNA 5 '-triphosphatase), nspl4 (3'-to-5' exonuclease), nspl5 (endo
  • the viral antigen comprises Spike glycoprotein (S protein) or a fragment of the Spike glycoprotein.
  • the Spike glycoprotein or a fragment thereof is a monomer or a trimer.
  • the Spike glycoprotein or a fragment thereof is prefusion stabilized form of Spike glycoprotein or fragment thereof.
  • the viral antigen of the Spike glycoprotein or a fragment thereof is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115.
  • the viral antigen of the Spike glycoprotein or a fragment thereof is 100% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • the viral antigen of the Spike glycoprotein or a fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1.
  • the viral antigen of the Spike glycoprotein or a fragment thereof comprise an amino acid length at least 5 amino acids, 10 amino acids, 20 amino acids, 25 amino acids, 50 amino acids, 100 amino acids, 200 amino acids, ormore.
  • the viral antigen comprising the Spike glycoprotein or a fragment thereof induces formation of neutralizing antibody againstthe Spike -glycoprotein or a fragmentthereof in a subject after administration of the vaccine.
  • the vaccine comprises one or more mutations in the viral antigen that confers increased stability of the viral antigen.
  • the one or more mutations are in a polypeptide sequence of the S1, S2, or in a region between the S1 and S2 subunits of the spike protein.
  • the one or more mutations are effective to reduce the likelihood of the dissociation of the S1 and S2 subunits that can happen when these subunits of the viral antigen of the spike protein are produced in vitro.
  • the one or more mutations arrests the S1 and S2 subunits in a pre-fusion trimer conformation. In some embodiments, the one or more mutations increases stable interaction of the subunits following furin cleavage of in vitro produced spike protein.
  • theone ormoremutations are sufficientto curtailthe challenges posed by singly (in vitro) produced S1 and Su subunits of the spike protein (lack of intact viral production of spike protein) and increase the stability, reduce likelihood of S1 and S2 dissociation, and force the S1 and S2 subunits in a pre-fusion trimer conformation.
  • the one or more mutations comprises one or more proline mutations in the S2 subunit of the spike protein (e.g., K986P and V987P).
  • the one or more protein mutations comprise two proline mutations, such as K986P and V987P.
  • the two or more proline mutations are between the two alpha helices between the S1 and S2 subunit of the spike protein.
  • the one or more mutations comprises a deletion of the furin cleavage site of the spike protein.
  • the vaccine comprises Ad26.COV.S.
  • the vaccine comprises mRNA-1273.
  • the vaccine comprises BNT162b2.
  • the vaccine comprises displaying, conjugating, or complexing the viral antigen with cell-based carrier, polymer(e.g. polyester), or extra cellular vesicles such as exosomes, microvesicles, retrovirus -like particles, apoptotic bodies, apoptosomes, oncosomes, exoph ers, enveloped viruses, exomeres, or other very large extracellular vesicles.
  • the vaccine comprises at least one heterologous polynucleotide encoding the viral antigen described herein.
  • the heterologous polynucleotide comprises a viral vector or a plasmid.
  • Non -limiting examples of heterologous polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), self -amplifying RNA, uridine containing RNA (uRNA), self-amplifyingmRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell -free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.
  • mRNA messenger RNA
  • uRNA uridine containing RNA
  • sequenceof nucleotides is interrupted by non-nucleotide components.
  • the viral antigen translated from the heterologous polynucleotide induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine.
  • the heterologous DNA sequence is transcribed into mRNA and then translated into the viral antigen after administration of the vaccine to the subject.
  • the viral antigen translated from the heterologous DNA sequence induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine.
  • the viral antigen comprising Spike glycoprotein or a fragment thereof is translated from the heterologous DNA sequence and induce formation of neutralizing antibody against the Spike glycoprotein or fragment thereof in a subject after administration of the vaccine.
  • the vaccine comprises mRNA encoding Spike glycoprotein or a fragment thereof.
  • the vaccine comprises the heterologous mRNA, where the mRNA is a self-amplifying mRNA (saRNA).
  • the vaccine comprises the heterologous mRNA, where the mRNA comprises uridine (uRNA).
  • the heterologous RNA sequence is translated into the viral antigen after administration of the vaccine to the subject.
  • the viral antigen translated from the heterologous RNA sequence induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine.
  • the viral antigen comprising Spike glycoprotein or a fragment thereof is translated from the heterologous RNA sequence and induce formation of neutralizing antibody against the Spike glycoprotein or fragment thereof in a subject after administration of the vaccine.
  • the vaccine is mRNA-1273, which encodes a full-length, prefusion stabilized Spike (S) protein.
  • the vaccine is BNT162 vaccine, comprising mRNA or modified mRNA to express the Spike (S) protein or a fragment thereof.
  • the BNT162 vaccine comprises nucleoside modified mRNA (modRNA), uridine containing mRNA (uRNA), or self -amplifying mRNA (saRNA),
  • the vaccine comprising the at least one heterologous polynucleotide encoding the viral antigen comprises one or more DNA or RNA vectors.
  • the DNA or RNA vectors is plasmids.
  • the DNA or RNA vectors is viral vector.
  • Viral vectors, and especially retroviral vectors are engineered to contain a nucleic acid molecule with a sequence encoding any one of the viral antigens described herein and to be delivered to a target tissue.
  • the viral vectors are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like.
  • Non-limiting examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAVs), pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vectors (HSVs).
  • the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome.
  • the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome.
  • AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype.
  • the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses.
  • the viral vector is a recombinant viral vector.
  • the vaccine is an Ad5 -nCoC vaccine comprising nucleic acid sequence encoding the Spike protein.
  • the vaccine is AZD-1222 vaccine comprising a replication-deficient chimpanzee adenovirus, ChAdOxl, which is engineered to express the Spike (S) protein.
  • the vaccine is INO-4800 vaccine comprising pGX DNA plasmid with nucleic acid encoding the Spike (S) protein.
  • the vaccine comprises adjuvant.
  • the vaccine comprises immune modulator selected from any one of the biomarkers described herein. Companion or Complementary Diagnostics
  • companion complementary diagnostic testing devices or system are companion complementary diagnostic testing devices or system and methods of their use.
  • the companion diagnostic testing device or system is used to determine the efficacy or effectiveness of a vaccine against a pathogen described herein.
  • the companion diagnostic testing device or system is used to identify a person in need of the vaccine.
  • a “companion diagnostic” as used herein refers to a diagnostic test that may be required for administration of a therapeutic agent for the treatment or prevention of a disease caused by a pathogen disclosed herein.
  • a “complementary diagnostic,” refers to a diagnostic testthatmay be optional for administration of the therapeutic agent.
  • the method determines the efficacy or effectiveness of a vaccine described herein by detecting presence, absence, or quantity of neutralizing antibody against the pathogen in a biological sample from a subject th at was administered the vaccine.
  • the companion diagnostic method confirms adaptive immunity against a pathogen in a subject or in a population of subjects. In some embodiments, the method confirms the need for vaccination against the pathogen for a subject or for a population of subjects. In some embodiments, the companion diagnostic method confirms the prevalent of infection by the pathogen in a population. In some embodiments, the companion diagnostic method identifies a subject or subjects in a population, who are most likely to benefit from being vaccinated with any one of the vaccine describedherein.
  • the companion diagnostic method identifies a subject or subjects in a population, who are likely to be at increased risk for serious side effects as a result of being vaccinated.
  • the companion diagnostic method monitors response to vaccination for the purpose of adjusting the dosage or the frequency of vaccination to achieve improved safety, efficacy, or effectiveness of the vaccine. For example, based on efficacy results, subjects aged 65 or older may receive a dosage that contains two times, three times, four times, five times, six times, seven times, eight times, nine times, or ten or more times the amount of an antigen than a dosage for subjects aged 64 or younger.
  • the companion diagnostic method comprises utilizing the biomarker panel describedherein.
  • Companion diagnostic methods comprise: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide -conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; and (c) classifying the biological sample as having a presence or quantity of the labeled complex.
  • the method further comprises identifying a vaccine administered to the subject as effective to induce a sufficient or insufficient adaptive immune response against the pathogen in the subject, provided the biological sample is classified as having the presence or the quantity of the labeled complex between (ii) and (iii) . In some embodiments, the method further comprises identifying the subject as in need for a vaccine against the pathogen, provided the biological sample is classified as having the presence or the quantity of the labeled complex between (i) and (ii). In some embodiments, performing (a)-(c) is required for administering the vaccine to the subject (or a population of subjects).
  • the method further comprises identifying a vaccine administered to the subject as not effective to induce a sufficient or insufficient adaptive immune response against the pathogen in the subject, provided the biological sample is classified as having the presence or the quantity of the labeled complex between (i) and (ii).
  • the level is high relative to an index or a control.
  • the vaccine is effective, provided the presence or the level of the labeled complex between the peptide-conjugate and the neutralizing antibody against the pathogen is measured. In some embodiments, the vaccine is not effective, provided the presence or the level of the labeled complex between the peptide -conjugate and the capture molecule is measured.
  • method described herein comprises administering a therapeutic agent to a subject in need thereof, provided the subject is identified as having an acute infection by a pathogen.
  • the therapeutic agent is effective to reduce or eliminate the acute infection by the pathogen, such as an anti-viral therapeutic agent.
  • the companion diagnostic method is performed prior to treating the subject with the therapeutic agent. In some embodiments, performing the companion diagnostic method is required prior to treating the subject with the therapeutic agent.
  • the therapeutic agent is an anti-viral therapeutic agent. In some embodiments, the anti-viral therapeutic agent treats a disease or condition associated with SARS-CoV-2.
  • the pathogen is a virus. In some embodiments, the virus is SARS-CoV-2.
  • the companion diagnostic method further comprises identifying the subject as having the acute infection comprises by determining the presence, absence, or a quantity of any one of the biomarkers described herein. In some embodiments, the companion diagnostic method comprises determining the presence, absence, or a quantity of both neutralizing antibody and biomarker. In some embodiments, the biomarker is interleukin 6 (IL- 6).
  • the complementary diagnostic method determines the benefit-risk decision-making about vaccinating the subject or vaccinating the population, where the difference in benefit-risk is clinically meaningful.
  • the complementary diagnostic method determines the need to vaccinate a subject or subjects of a population, when the status or prevalence of infection by any one of the pathogen described herein is unknown.
  • the complementary diagnostic method determines the need to vaccinate or treat the subject or subjects of a population, when the status or prevalence of infection by any one of the pathogen described herein is unknown. For example, a subject or subjects of a population have displayed symptoms of respiratory disease or disorder.
  • the complementary diagnostic method confirmsthe particular pathogen that is causing the respiratory disease or disorder in the absence of a reason to test for that particular pathogen. In some embodiments, the complementary diagnostic method determines the efficacy or effectiveness of a vaccine. For example, a vaccine designed for one disease is showingunexpected protective effect against a different disease caused by a different pathogen. In some embodiments, the complementary diagnostic method identifies a subject or subjects in a population, who are most likely to benefit from being vaccinated with any one of the vaccine described herein. In some embodiments, the complementary diagnostic method identifies a subject or subjects in a population, who are likely to be at increased risk for serious side effects as a result of being vaccinated.
  • the complementary diagnostic method monitors response to vaccination for the purpose of adjusting the dosage or the frequency of vaccination to achieve improved safety, efficacy, or effectiveness of the vaccine. For example, based onresponse results, subjects aged 65 or older may receive a dosage that contains two times, three times, four times, five times, six times, seven times, eight times, nine times, or ten or more times the amount of an antigen than a dosage for subjects aged 64 or younger.
  • the complementary diagnostic method comprises utilizing the biomarker panel described herein.
  • biomarkers are associated with infection by any one of the pathogens described herein.
  • the biomarkers are induced by infection by the pathogen.
  • the biomarkers are cytokines induced by infection.
  • the biomarkers are serological biomarkers. The quantity or activity of the biomarkers can increase or decrease after the infection.
  • the biomarkers may comprise a peptide or protein that is encoded by a nucleic acid sequence, or that has an amino acid sequence, that is at least 80% identical to any one of SEQ ID NOS: 21-23, 25-42, 44-46, 49-66, 68-70, 73-90, 92-94, or 97-114.
  • Biomarker panel methods comprise: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, an absence, level, or activity of at least one biomarker; (c) classifying the biological sample as having a presence or level of the biomarker associated or induced by an infection; and optionally (d) measuring a presence, an absence, or quantity of neutralizing antibody induced by the infection.
  • the method identifies the infection caused by the pathogen based on the quantity or activity of the biomarker.
  • the method identifies the infection caused by the pathogen based on the quantity or activity of the biomarker in a subject, who does not have neutralizing antibody against the pathogen.
  • the method confirms the infection caused by the pathogen based on the quantity or activity of the biomarker. In some embodiments, the method confirms the infection caused by the pathogen based on the quantity or activity of the biomarker in a subject, who does not have neutralizing antibody against the pathogen. In some embodiments, the method comprise prognosing an immunity or measuring susceptibility to infection based on detecting presence, absence, level, or activity of the biomarkers described herein. In some embodiments, the method comprise prognosing an immunity or measuring susceptibility to infection based on detecting presence, absence, quantity, or activity of the biomarkers in combination with detecting presence, absence, or quantity of the neutralizing antibodies described herein to confirm an infection caused by any one of the pathogen described herein.
  • the presence, absence, level, or activity of the biomarker is detected.
  • the method detects presence absence, level, or activity of at least one, two, three, four, five, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more biomarkers of the biomarker panel in a biological sample.
  • the method detects biomarker comprising a non-peptide coding nucleic acid sequence.
  • the method detects biomarker comprising a peptide-coding nucleic acid sequence such as mRNA or cDNA.
  • the method detects biomarker comprising peptide or protein.
  • the method detects presence, absence, or level, or activity of the biomarker by utilizing a nucleic acid sequence by subjecting a biological sample to a nucleic acid-based detection assay.
  • the nucleic acid-based detection assay comprises quantitative polymerase chain reaction (qPCR), gel electrophoresis (including for e.g., Northern or Southemblot), immunochemistry, in situ hybridization such as fluorescent in situ hybridization (FISH), cytochemistry, or sequencing.
  • the sequencing technique comprises next generation sequencing.
  • the method involves a hybridization assay such as fluorogenic qPCR (e.g., TaqManTM, SYBR green, SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin), which involves a nucleic acid amplification reaction with a specific primer pair, and hybridization of the amplified nucleic acid probes comprising a detectable moiety or molecule that is specific to a target nucleic acid sequence.
  • a number of amplification cycles for detecting a target nucleic acid in a qPCR assay is about 5 to about 30 cycles.
  • the number of amplification cycles for detecting a target nucleic acid is at least about 5 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is at most about 30 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 cycles.
  • the probe may be a hydrolysable probe comprising a fluorophore and quencher that is hydrolyzed by DNA polymerase when hybridized to a target nucleic acid.
  • the presence of a target nucleic acid is determined when the number of amplification cycles to reach a threshold value is less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 cycles.
  • hybridization may occur at standard hybridization temperatures, e.g., between about 35 °C and about 65 °C in a standard PCR buffer.
  • An additional exemplary nucleic acid-based detection assay comprises the use of nucleic acid probes conjugated or otherwise immobilized on a bead, multi -well plate, or other substrate, wherein the nucleic acid probes are configured to hybridize with a target nucleic acid sequence.
  • the nucleic acid probe is specific to one or more genetic variants disclosed herein is used.
  • the nucleic acid probe specific to a SNP or SNV comprises a nucleic acid probe sequence sufficiently complementary to a risk or protective allele of interest, such that hybridization is specific to the risk or protective allele.
  • the nucleic acid probe specific to an indel comprises a nucleic acid probe sequence sufficiently complementary to an insertion of a nucleobase within a polynucleotide sequence flanking the insertion, such that hybridization is specific to the indel.
  • the nucleic acid probe specific to an indel comprises a probe sequence sufficiently complementary to a polynucleotide sequence flanking a deletion of a nucleobase within the polynucleotide sequence, such that hybridization is specific to the indel.
  • the nucleic acid probe specific to a biomarker comprises a nucleic acid probe sequence sufficiently complementary to the polynucleotide sequence of the biomarker.
  • the biomarker comprises a transcribed polynucleotide sequence (e.g., RNA, cDNA).
  • the nucleic acid probe is, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of atleast about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length and sufficient to specifically hybridize under standard hybridization conditions to the target nucleic acid sequence.
  • the target nucleic acid sequence is immobilized on a solid surface and contacted with a probe, forexample by running the isolated target nucleic acid sequence on an agarose gel and transferring the target nucleic acid sequence from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface, for example, in an Affymetrix gene chip array, and the probe(s) are contacted with the target nucleic acid sequence.
  • the method detects presence, absence, or level, or activity of the biomarker by utilizing a protein-based assay.
  • the method detects the biomarker by an antibody -based assay, where an antibody specific to the biomarker is utilized.
  • antibody-based detection method can utilize an antibody that binds to any region of the biomarker.
  • Another exemplary method of detecting the biomarker comprises performing an enzyme-linked immunosorbent assay (ELISA).
  • the ELISA assay is a sandwich ELISA or a direct ELISA.
  • Another exemplary method of detecting the biomarker comprises a single molecule array, e.g., Simoa.
  • exemplary methods of detecting biomarkers can include immunohistochemistry, lateral flow assay, gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, or various immunological methods such as fluid or gel precipitation reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), immunofluorescent assays, and Western blotting.
  • antibodies, or antibody fragments is used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins.
  • the antibody or protein is immobilized on a solid supportfor Western blots and immunofluorescencetechniques.
  • Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody.
  • Exemplary supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • Methods disclosed herein further comprise: (a) capturing an image of a detection zone of the testing device; and (b) analyzing data from the image using one or more computer programs.
  • the one or more computer programs is run on a computing device described here.
  • capturing is performed with an imaging device described herein.
  • capturing and analyzing are performed by a single personal electronic device, such as a smartphone, tablet, or laptop computer.
  • capturing and analyzing are performed at the point of need (e.g., same time and place as performing the assay with the testing device).
  • capturing comprises taking a still photograph of a liquid phase, such as a liquid composition disclosed herein. In some embodiments, capturing comprises taking a still photograph of the solid surface of the assay assembly described herein. In some embodiments, capturing comprises taking a video of the solid surface of the assay assembly.
  • analyzing comprises detecting binding between the analyte (antibody against an antigenic peptide from the pathogen) and peptide -conjugate. In some embodiments, analyzing comprises subtracting a background signal, thereby increasing the signal to noise ratio. In some embodiments, analyzing is performed by a data analytics module of an application or computer program of the computing device. In some embodiments, the analyzing by the data analytics module comprises performing machine learning.
  • Embodiment 1 A method of identifying adaptive immunity to a coronavirus in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) introducing the biological sample with (i) a detectable peptide derived from a spike glycoprotein of a coronavirus; and (ii) one or more capture molecules derived from an angiotensin-converting enzyme 2 (ACE2) receptor; and (c) detecting a binding complex between the detectable peptide and the one or more capture molecules .
  • ACE2 angiotensin-converting enzyme 2
  • Embodiment 2 The method of embodiment 1, wherein steps (a)-(c) are performed at the point of need.
  • Embodiment 3 The method of any one of embodiments 1-2, wherein steps (a)-(c) are performed at the point of care.
  • Embodiment 4 The method of any one of embodiments 1-3, wherein the detectable peptide, the one or more capture molecules, or both of the detectable peptide and the one or more capture molecules is coupled to a surface, which surface optionally comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • a surface optionally comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 5 The method of any one of embodiments 1-5, wherein the surface is a passivated surface.
  • Embodiment 6 The method of embodiment 5 , wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (aery lie acid) (PAA), polyacrylamide, poly (N -isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethyleneglycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyrrolidone)
  • PPA poly (aery lie acid)
  • PIPAM
  • Embodiment 8 The method of any one of embodiments 4-6, wherein the surfaceis or comprises an interior surface of a capillary.
  • Embodiment 9 The method of any one of embodiments 4-7, wherein the surfaceis or comprises an interior surface of a capillary in a capillary flow cell device.
  • Embodiment 10 The method of any one of embodiments 4-5, wherein the surface comprises a porous membrane, optionally, comprising cellulose.
  • Embodiment 11 The method of any one of embodiments 4-5, wherein the surface comprises a waveguide.
  • Embodiment 12 The method of embodiment 11, wherein the waveguide is a planar waveguide.
  • Embodiment 13 The method of any one of embodiments 1-12, wherein the one ormore capture molecules comprises two or more capture molecules.
  • Embodiment 14 The method of any one of embodiments 4-13, whereinthe one ormore capture molecules is coupled to the surface by a covalent bond, a linker, or a combination thereof.
  • Embodiment 15 The method of embodiment 14, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 16 Themethod of anyone of embodiments 1-15, whereinthe one ormore capture molecules is a fusion polypeptide.
  • Embodiment 17 The method of embodiment 16, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Embodiment 18 The method of any one of embodiments 1-17, whereinthe one ormore capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 19 The method of any one of embodiments 1-18, wherein the detectable peptide or the one or more capture molecules or a combination thereof comprises a nanoparticle, a fluorescent metal, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 20 The method of any one of embodiments 1-19, wherein the detectable peptide is or comprises a fluorescence resonance energy transfer (FRET) donor or acceptor.
  • FRET fluorescence resonance energy transfer
  • Embodiment 21 The method of any one of embodiments 1-20, wherein the one ormore capture molecules comprises a detectable label coupled thereto.
  • Embodiment22 The methodof embodiment21 , wherein the detectable label comprises a FRET donor or acceptor.
  • Embodiment 23 The method of any one of embodiments 20-21, wherein the FRET is lanthanide-based FRET (LRET).
  • FRET lanthanide-based FRET
  • Embodiment 24 The method of embodiment 20 or 22, wherein the FRET donor comprises a luminescent lanthanide.
  • Embodiment 25 The method of embodiment 24, wherein the luminescent lanthanide is terbium or europium.
  • Embodiment 26 The method of embodiment 24, wherein the FRET acceptor comprises a fluorophore, such as an organic fluorophore.
  • Embodiment 27 The methodof any one of embodiments 1-26, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, andplastic.
  • Embodiment28 The method of any one of embodiments 1-27, wherein the nanoparticle is magnetic.
  • Embodiment 29 The methodof any one of embodiments 1-28, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 30 The method of any one of embodiments 1-29, further comprises calculating a percent neutralization of binding between the detectable peptide and the one or more capture molecules.
  • Embodiment 31 The method of embodiment 30, wherein the percent neutralization is calculated by a formula comprising: [(S T - S N )/(S 0 - S N )* 100], wherein
  • S T is the signal detected in (c)
  • S N is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein
  • S 0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein.
  • Embodiment 32 The method of any one of embodiments 1-30, wherein a percent neutralization of about 62% correlates to about 400 reciprocal titers when measured using a cell-based pseudovirion neutralization assay (PNA).
  • PNA cell-based pseudovirion neutralization assay
  • Embodiment 33 The method of any one of embodiments 1-29, further comprising comparing a number of the binding complexes to a number of binding complexes in a control sample with a known amount of a neutralizing antibody in it having a known IC 50 .
  • Embodiment 34 The method of embodiment 33, wherein if the number of the binding complexes is low relative to the number of binding complexes in the control sample, then identifying the subject as being immune to an infection by the coronavirus; and if the number of the binding complexes is the same as or high relative to the number of binding complexes in the control sample, then identifying the subject as not being immune to an infection by the coronavirus.
  • Embodiment 35 The method of any one of embodiments 30-33, further comprising comparing the percent neutralization for the biological sample to the percent neutralization calculated for a control sample.
  • Embodiment 36 The method of embodiment 35, wherein the control sample is a negative sera or blood sample spiked with a known amount of a neutralizing antibody in it having a known IC 50 , or a positive sera or blood sample obtained from a control subject.
  • Embodiments 7 The method of embodiment 35, wherein the control subject is a subject that (a) recovered from an infection by the coronavirus, (b) is vaccinated against the coronavirus, (c) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (d) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus.
  • Embodiment 38 The method of any one of embodiments 35-37, wherein if the percent neutralization for the biological sample is low relative to the percent neutralization calculated for the control sample, then identifying the subject as being immune to an infection by the coronavirus; and if the number of the binding complexes is the same as or high relative to the number of binding complexes in the control sample, then identifying the subject as not being immune to an infection by the coronavirus.
  • Embodiment 39 The method of any one of embodiments 35 -38, further comprising comparing the percent neutralization of the biological sample to the percent neutralization measured with the cell-based PNA, thereby correlating the cell-based PNA with the percent neutralization of the biological sample.
  • Embodiment 40 The method of any one of embodiments 35 -39, further comprising detecting a percent inhibition in the biological sample comprises between about 1-100%, 10- 90%, 20-80%, 30-70%, 40-60%, or about 50% inhibition in the biological sample.
  • Embodiment 41 The method of any one of embodiments 35 -39, further comprising detecting a percent inhibition in the biological sample comprising between about 30-40%, 40%-50%, 60-70%, 70-80%.
  • Embodiment42 Themethod of anyone of embodiments 1-41, wherein the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 43 The method of any one of embodiments 1-42, wherein the detectable peptide derived from the spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS- CoV).
  • SARS- CoV Severe acute respiratory syndrome-associated coronavirus
  • Embodiment 44 The method of embodiment 43, wherein the SARS-CoV is SARS- CoV-2 or a variant or a derivative thereof.
  • Embodiment 45 The method of any one of embodiments 43-44, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
  • Embodiment 46 The method of any one of embodiments 1-45, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 47 The method of any one of embodiments 1-46, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment 48 The method of any one of embodiments 1-47, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2 [0348]
  • Embodiment 49 The method of any one of embodiments 1-48, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment 50 The method of any one of embodiments 1-49, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • Embodiment 51 The method of any one of embodiments 1-50, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 52 The method of any one of embodiments 1-51, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible using FRET.
  • Embodiment 53 The method of embodiment 52, wherein the complex between the peptide-conjugate and the one or more capture molecules emits a first fluorescent signal and a second fluorescent signal, which ratio of the first fluorescent signal and the second fluorescent signal is detectable using FRET.
  • Embodiment 54 The method of any one of embodiments 1-53, wherein the binding complexes are optically connected to a planar waveguide optical sensor.
  • Embodiment 55 The method of any one of embodiments 4-54, wherein the surface is housed in a cartridge that is disposable.
  • Embodiment 56 The method of embodiment 55, wherein the cartridge is made from a polymer material comprising metal, agarose, acrylic, or plastic.
  • Embodiment 57 The method of any one of embodiments 1-56, further comprising providing a web -based portal on the personal electronic device.
  • Embodiment 58 The method of embodiment 57, wherein method further comprises providing an application on the web-based portal.
  • Embodiment 59 The method of embodiment 58, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
  • Embodiment 60 The method of any one of embodiments 58-59, wherein method further comprises providing a data analytics module at the application.
  • Embodiment 61 The method of embodiment 60, further comprising analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
  • Embodiment 62 The method of embodiment 61, wherein the positive or the negative result is relative to a threshold number of the binding complexes or a threshold percent neutralization calculated for a control that is indicative of immunity against the coronavirus.
  • Embodiment 63 The method of embodiment 62, wherein the threshold percent neutralization is equal to or about 60%.
  • Embodiment 64 The method of any one of embodiments 60-63, further comprising analyzing the result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules on the surface.
  • Embodiment 65 The method of any one of embodiments 60-64, further comprising analyzingthe result, by the data analytics module, to determine a ratio of the binding complexes to unbound detectable peptide or the one or more capture molecules, wherein a high ratio of the binding complexes to unbound detectable peptide or the one or more capture molecules indicates a low level of immunity, and a low ratio of the binding complexes to unbound detectable peptide or the one or more capture molecules indicates a high level of immunity.
  • Embodiment 66 The method of any one of embodiments 60-65, further comprising normalizing, by the data analytics module, the result by subtracting background noise.
  • Embodiment 67 The method of any one of embodiments 60-66, further comprising identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
  • Embodiment 68 The method of embodiment 67, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
  • Embodiment 69 The method of any one of embodiments 60-68, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
  • Embodiment 70 The method of any one of embodiments 60-69, further comprising providing a data store that is a cloud-based data store or a web-based data store, or a local data store.
  • Embodiment 71 The method of any one of embodiments 60-71, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
  • Embodiment 72 The method of embodiment 71 , wherein the external datais data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 73 The method of any one of embodiments 71-72, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
  • Embodiment 74 The method of any one of embodiments 71-73, further comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 75 The method of any one of embodiments 1-74, wherein the biological sample comprises one or more antibodies specific to the detectable peptide.
  • Embodiment 76 The method of any one of embodiments 1-74, wherein the biological sample does not consist of one or more antibodies specific to the detectable peptide.
  • Embodiment 77 The method of any one of embodiments 75-76, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
  • Emb odiment 78 The method of any one of embodiments 1-77, wherein the subj ect was, or is, exposed to the coronavirus.
  • Embodiment 79 The method of any one of embodiments 1-77, wherein exposure of the subject to the coronavirus is unknown.
  • Embodiment 80 The method of any one of embodiments 1-79, wherein the method is performed on a plurality of biological samples obtained from a plurality of subjects.
  • Embodiment 81 The method of embodiments 80, further comprising identifying a level of the adaptive immunity of the plurality of subjects to the coronavirus.
  • Embodiment 82 The method of any one of embodiments 80-81, further comprising monitoring a spread of infection of the plurality of subjects by the coronavirus.
  • Embodiment 83 The method of any one of embodiments 1-82, wherein the subject was administered a vaccine against the coronavirus.
  • Embodiment 84 The method of embodiment 83, further comprising determining that the vaccine is effective to substantially immunize the subject against the coronavirus, based, at least in part on the binding complex detected in (c).
  • Embodiment 85 The method of any one of embodiments 1-84, wherein the biological sample comprises blood, urine, saliva, or feces.
  • Embodiment 86 The method of embodiment 85, wherein the bloodis capillary blood.
  • Embodiment 87 The method of embodiment 86, wherein the capillary blood is obtained from the subject by a prick of the subject’ s finger.
  • Embodiment 88 The method of any one of embodiments 85-87, wherein method further comprises separating serum from the blood in the biological sample.
  • Embodiment 89 The method of any one of embodiments 1-88, wherein the detecting in (c) comprises capturing an image of the surface with an imaging device to detect the binding complex between the detectable peptide and the one or more capture molecules.
  • Embodiment 90 The method of embodiments 89, wherein the imaging device is the personal electronic device.
  • Embodiment 91 The method of embodiment 90, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
  • Embodiment 92 The method of any one of embodiments 1-91, further comprising calculating a percent neutralization on a scale of 0-100% based on the binding complex detected in (c), wherein:
  • Embodiment 93 The method of any one of embodiments 1-92, wherein the method is performed without a wash step to wash away unbound detectable peptide, one or more capture molecules, or any combination thereof.
  • Embodiment 94 The method of any one of embodiments 1-93, wherein the method is performed in solution.
  • Embodiment 95 The method of any one of embodiments 1-94, wherein the introducing in (a) is performed in a well or container.
  • Embodiment 96 The method of any one of embodiments 1-95, further comprising repeating the method on a plurality of biological samples obtained from the subject, wherein at least two biological samples of the plurality of biological samples are obtained at different time points.
  • Embodiment 97 The method of any one of embodiments 1-96, wherein the subject has an underlying immune-comprising condition comprising a pregnancy, an immune-mediated disease, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof.
  • Embodiment 98 The method of any one of embodiments 1-97, wherein the subject is pregnant or breastfeeding.
  • Embodiment 99 The method of any one of embodiments 1-98, wherein the subject is receiving an immunosuppressant therapy.
  • Embodiment 100 The method of embodiment 99, wherein the immunosuppressant therapy comprises a biologic drug (e.g. , adalimumab (Humira®) and infliximab (Remicade®)), calcineurin inhibitors (e.g., tacrolimus (Envarsus XR® or Protopic) and cyclosporine (Gengraf®, Neoral® or Sandimmune®)), corticosteroids (e.g., prednisone), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolate mofetil (CellCept®)), janus kinase inhibitors (e.g., tofacitinib (Xeljanz®)), mechanistic target of rapamycin (mTOR) inhibitors (e.g., sirolimus (Rapamune®)), or monoclonal antibodies (e.g., basilixim
  • Embodiment 101 The method of any one of embodiments 1-100, wherein the method is performed with an accuracy comprising greater than or equal to or about 50%, 60%, 70%, 80%, 90%, or 99%.
  • Embodiment 102 The method of any one of embodiments 1-101, wherein the method is performed with a specificity comprisinggreaterthan or equal to about 50%, 60%, 70%, 80%, 90%, or 99%.
  • Embodiment 103 The method of any one of embodiments 1-102, wherein the method is performed with a sensitivity comprisinggreaterthan or equal to about 50%, 60%, 70%, 80%, 90%, or 99%.
  • Embodiment 104 The method of any one of embodiments 1 -102, wherein the method is performed with a percent coefficient of variation (CV) of less than or equal to about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
  • Embodiment 105 The method of any one of embodiments 101-104, wherein the accuracy is measured by:
  • Embodiment 106 The method of any one of embodiments 1-105, further comprising administering to the subject a vaccine against the coronavirus, provided the percent neutralization calculated for the subjectbased on the detecting in (c) is lower than 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%..
  • Embodiment 107 The method of embodiment 106, wherein the vaccine is a full dose or booster dose of the vaccine.
  • Embodiment 108 The method of any one of embodiments 1-107, selectingthe subject for treatment with a vaccine against the coronavirus, provided the percent neutralization calculated for the subject based on the detecting in (c) is lower than 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
  • Embodiment 110 The method of any one of embodiments 1-109, further comprising identifying the subject as being sufficiently immune to an infection by the coronavirus, provided the percent neutralization calculated for the subject based on the detecting in (c) is higher than 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
  • Embodiment 11 1. The method of any one of embodiments 1-110, further comprising:
  • Embodiment 112. The method of any one of embodiments 1-113, wherein the subject is a pediatric subject. KITS
  • kits for obtaining a biological sample or detecting an analyte in the biological sample may comprise a biological sample collection device, such as, for example, a transdermal puncture device (e.g, finger prick lance) or other sample collection device (e.g., a swab).
  • the kits may comprise a dried blood spot (DBS) card described herein for storing the biological sample.
  • the kits comprise an assay assembly described herein, such as a lateral flow assay or an ELISA that is capable of detecting neutralizing antibodies against a pathogen of interest in the biological sample.
  • kits described herein comprise instructions.
  • the instructions provide information about howto obtain the biological sample.
  • the instructions comprise information about how to discard the biological sample.
  • the kits comprise information about how to download a mobile application for a personal electronic device that captures and analyzes the results of the testing device.
  • the kits comprise a survey having one or more clinical questions pertaining to a symptom of an infection by a pathogen of interest. In some embodiments, the survey is on the back of the DBS card of the kit.
  • Embodiment 1 A system comprising: (a) one or more capture molecules derived from an angiotensin-converting enzyme 2 (ACE2) receptor; and (b) apeptide-conjugate comprising: (i) a peptide derived from a spike glycoprotein of a coronavirus; and (ii) a detectable moiety.
  • ACE2 angiotensin-converting enzyme 2
  • Embodiment 2 The system of embodiment 1 further comprises (a) a surface; and (b) an imaging device configured to capture an image of a complex between the peptide -conjugate and the one or more capture molecules on the surface when the complex is coupled to the surface.
  • Embodiment 3 The system of any one of embodiments 1-2 further comprises an imaging device configured to capture an image of a complex between the peptide -conjugate and the one or more capture molecules.
  • Embodiment 4 The system of any one of embodiments 1-3 comprises a container comprising (a) and (b), wherein the container is portable.
  • the system of any one of embodiments 1-4 is a point of need system.
  • the system of any one of embodiments 1-5 is a point of care system.
  • Embodiment 7 The system of any one of embodiments 1-6, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 8 The system of any one of embodiments 1-7, wherein the surface is a passivated surface.
  • Embodiment 9 The system of any one of embodiments 1 -8, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (aery lie acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly glutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyrrolidone)
  • PPA poly (aery lie acid)
  • Embodiment 10 The system of any one of embodiments 1-9, wherein the complex is coupled to the surface.
  • Embodiment 11 The system of any one of embodiments 1-10, wherein, the complex is coupled to the surface by a covalent bond, a linker, or a combination thereof.
  • Embodiment 12 The system of any one of embodiments 1-11, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 13 The system of any one of embodiments 1-12, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 14 The system of any one of embodiments 1-13, wherein the one or more capture molecules is coupled to the surface.
  • Embodiment 15 The system of any one of embodiments 1-14, whereinthe one or more capture molecules is coupled to the surface by a covalent bond, a linker, or a combination thereof.
  • Embodiment 16 The system of any one of embodiments 1-15, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 17 The system of any one of embodiments 1-16, whereinthe one or more capture molecules is a fusion polypeptide.
  • Embodiment 18 The system of any one of embodiments 1- 17, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Fc fragment crystallizable region
  • Embodiment 19 The system of any one of embodiments 1-18, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 20 The system of any one of embodiments 1- 19, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment21 The system of any one of embodiments 1-20, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, andplastic.
  • Embodiment 22 The system of any one of embodiments 1-21, whereinthe nanoparticle is magnetic.
  • Embodiment 23 The system of any one of embodiments 1-22, the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment24 The system of any one of embodiments 1-23, wherein, the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 25 The system of any one of embodiments 1-24, wherein, the peptide derived from a spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome -associated coronavirus
  • Embodiment 26 The system of any one of embodiments 1-25, wherein the SARS-CoV is SARS-CoV-2.
  • Embodiment 27 The system of any one of embodiments 1-26, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
  • Embodiment 28 The system of any one of embodiments 1-27, wherein the at least a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 29 The system of any one of embodiments 1-28, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • Embodiment 30 The system of any one of embodiments 1-29, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
  • Embodiment 31 The system of any one of embodiments 1-30, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment 32 The system of any one of embodiments 1-31, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 4
  • Embodiment 33 The system of any one of embodiments 1-32, wherein the complex between the peptide-conjugate and the one or more capture molecules on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 34 The system of any one of embodiments 1-33, where the system further comprises a housing at least partially enclosing the surface.
  • Embodiment 35 The system of any one of embodiments 1-34, wherein the system further comprises a sample receptor configured to receive a biological sample from a subject.
  • Embodiment 36 The system of any one of embodiments 1-35, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface.
  • Embodiment 37 The system of any one of embodiments 1-36, wherein the biological sample comprises one or more antibodies specific to the peptide.
  • Embodiment 38 The system of any one of embodiments 1-37, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
  • the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
  • Embodiment 10. the subject was, oris, exposed to the coronavirus.
  • Embodiment 39 The system of any one of embodiments 1-38, wherein exposure of the subject to the coronavirus is unknown.
  • Embodiment 40 The system of any one of embodiments 1-39, wherein the subject was administered a vaccine against the coronavirus.
  • Embodiment 41 The system of any one of embodiments 1-40, wherein the biological sample comprises blood, urine, saliva, or feces.
  • Embodiment 42 The system of any one of embodiments 1-41, wherein the blood is capillary blood.
  • Embodiment 43 The system of any one of embodiments 1-42, wherein the system further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
  • Embodiment 44 The system of any one of embodiments 1-43, wherein the sample receptor comprises a filter to separate serum from the blood.
  • Embodiment 45 The system of any one of embodiments 1-44, wherein the system further comprises a data store for storing data from the image that is captured by the imaging device.
  • Embodiment 46 The system of any one of embodiments 1-45, wherein the data store is a cloud-based or a web-based data store, or a local data store.
  • Embodiment 47 The system of any one of embodiments 1-46, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
  • Embodiment 48 The system of any one of embodiments 1-47, wherein the external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 49 The system of any one of embodiments 1-48, wherein the external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
  • Embodiment 50 The system of any one of embodiments 1-49, wherein the system further comprises an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 51 The system of any one of embodiments 1-50, wherein the imaging device is a personal electronic device.
  • Embodiment 52 The system of any one of embodiments 1-51, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
  • the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
  • Embodiment 53 The system of any one of embodiments 1-52, wherein the personal electronic device comprises a web-based portal.
  • Embodiment 54 The system of any one of embodiments 1-53, wherein the web-based portal utilizes an application.
  • Embodiment 55 The system of any one of embodiments 1-54, wherein the application is configured to receive data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, and external data from one or more external device.
  • Embodiment 56 The system of any one of embodiments 1-55, wherein the application comprises a data analytics module configured to analyze the result by: (a) determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity; (b) determining an absolute number of complexes between the peptide-conjugate and the ACE2 receptor on the surface; or (c) determining a level of binding between the peptide-conjugate and the ACE2, wherein a high level of binding indicates a low level of immunity, and a low lev el of binding indicates a high level of immunity.
  • Embodiment 57 The system of any one of embodiments 1-56, wherein the positive or the negative result is relative to a threshold number of complexes between the peptide- conjugate and the ACE2 receptor on the surface.
  • Embodiment 58 The system of any one of embodiments 1-57, wherein the threshold number is predetermined relative to an index a control.
  • Embodiment 59 The system of any one of embodiments 1-58, wherein the data analytics module is further configuredto normalize the resultby subtractingbackground noise.
  • Embodiment 60 The system of any one of embodiments 1-59, wherein the data analytics module is further configuredto identify a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
  • Embodiment 61 The system of any one of embodiments 1-60, wherein the data analytics module utilizes geofencing from coordinates of the personal electronic device to identify the geographical location.
  • Embodiment 62 The system of any one of embodiments 1-61, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
  • Embodiment 63 The system of any one of embodiments 1-62, wherein system further comprises one or more capture molecules specific to one or more antibodies against the coronavirus.
  • Embodiment 64 The system of any one of embodiments 1-63, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
  • Embodiment 65 The system of any one of embodiments 1-64, wherein system further comprises a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
  • Embodiment 66 The system of any one of embodiments 1-65, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment is monoclonal, antibody or antigen-binding fragment is polyclonal.
  • Embodiment67 Aspects disclosed herein comprise system comprising: (a) one or more capture molecules derived from a spike glycoprotein of a coronavirus; (b) a peptide-conjugate comprising: (i) a peptide derived from angiotensin -converting enzyme 2 (ACE2) receptor, and (ii) a detectable moiety.
  • ACE2 angiotensin -converting enzyme 2
  • Embodiment 68 The system of embodiment 67, wherein the system further comprises (a) a surface; and (b) an imaging device configured to capture an image of a complex between the peptide-conjugate and the one or more capture molecules on the surface when the complex is coupled to the surface.
  • Embodiment 69 The system of any one of embodiments 67-68, wherein the system further comprises an imaging device configured to capture an image of a complex between the peptide-conjugate and the one or more capture molecules.
  • Embodiment 70 The system of any one of embodiments 67-69, wherein the system further comprises a container comprising (a) and (b), wherein the container is portable.
  • Embodiment 71 The system of any one of embodiments 67-70, wherein the system is a point of need system.
  • Embodiment 72 The system of any one of embodiments 67-71, wherein the point of need is a point of care system.
  • Embodiment 73 The system of any one of embodiments 67-72, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 74 The system of any one of embodiments 67-73, wherein the surface is a passivated surface.
  • Embodiment 75 The system of any one of embodiments 67-74, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (aery lie acid) (PAA), polyacrylamide, poly(N -isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly glutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyrrolidone)
  • PPA poly (aery lie acid
  • Embodiment 76 The system of any one of embodiments 67-75, wherein the complex is coupled to the surface.
  • Embodiment 77 The system of any one of embodiments 67-76, wherein the complex is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 78 The system of any one of embodiments 67-77, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 79 The system of any one of embodiments 67-78, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 80 The system of any one of embodiments 67-79, wherein the one or more capture molecules is coupled to the surface.
  • Embodiment 81 The system of any one of embodiments 67-80, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 82 The system of any one of embodiments 67 -81, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 83 The system of any one of embodiments 67-82, wherein the one or more capture molecules is a fusion polypeptide.
  • Embodiment 84 The system of any one of embodiments 67-83, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Fc fragment crystallizable region
  • Embodiment 85 The system of any one of embodiments 67-84, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 86 The system of any one of embodiments 67-85, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 87 The system of any one of embodiments 67-86, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
  • Embodiment 88 The system of any one of embodiments 67-87, wherein the nanoparticle is magnetic.
  • Embodiment 89 The system of any one of embodiments 67-88, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 90 The system of any one of embodiments 67-89, wherein the peptide derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1
  • Embodiment 91 The system of any one of embodiments 67-90, wherein the one or more capture molecules derived from a spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome -associated coronavirus
  • Embodiment 92 The system of any one of embodiments 67-91, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment 93 The system of any one of embodiments 67-92, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
  • Embodiment 94 The system of any one of embodiments 67-93, wherein the at least a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 95 The system of any one of embodiments 67-94, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment 96 The system of any one of embodiments 67-95, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
  • Embodiment 97 The system of any one of embodiments 67-96, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment 98 The system of any one of embodiments 67-97, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • Embodiment 99 The system of any one of embodiments 67-98, wherein the complex between the peptide-conjugate and the one or more capture molecules on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 100 The system of any one of embodiments 67-99, wherein system further comprises one or more capture molecules specific to one or more antibodies against the coronavirus.
  • Embodiment 101 The system of any one of embodiments 67-100, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
  • Embodiment 102 The system of any one of embodiments 67-101, wherein system further comprises a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
  • Embodiment 103 The system of any one of embodiments 67-102, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen -binding fragment.
  • Embodiment 104 The system of anyone of embodiments 67-103, wherein the antibody or antigen-binding fragment is monoclonal, antibody or antigen-binding fragment is polyclonal.
  • Embodiment 105 The system of any one of embodiments 67-104, wherein system further comprises a housing at least partially enclosing the surface.
  • Embodiment 106 The system of any one of embodiments 67-105, wherein system further comprises a sample receptor configured to receive a biological sample from a subject.
  • Embodiment 107 The system of any one of embodiments 67-106, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface.
  • Embodiment 108 The system of any one of embodiments 67-107, wherein the biological sample comprises one or more antibodies specific to the peptide.
  • Embodiment 109 The system of any one of embodiments 67-108, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
  • Embodiment 110 The system of any one of embodiments 67-109, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
  • Embodiment 111 The system of any one of embodiments 67 -110, wherein the subject was, or is, exposed to the coronavirus.
  • Embodiment 112. The system of any one of embodiments 67-111, wherein exposure of the subject to the coronavirus is unknown.
  • Embodiment 113 The system of any one of embodiments 67-112, wherein the subject was administered a vaccine against the coronavirus.
  • Embodiment 114 The system of any one of embodiments 67-113, wherein the biological sample comprises blood, urine, saliva, or feces.
  • Embodiment 115 The system of any one of embodiments 67-114, wherein the blood is capillary blood.
  • Embodiment 116 The system of any one of embodiments 67-115, wherein system further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
  • Embodiment 117 The system of any one of embodiments 67-116, wherein the sample receptor comprises a filter to separate serum from the blood.
  • Embodiment 118 The system of any one of embodiments 67-117, wherein system further comprises a data store for storing data from the image that is captured by the imaging device.
  • Embodiment 119 The system of any one of embodiments 67-118, wherein the data store is a cloud-based or a web-based data store, or a local data store.
  • Embodiment 120 The system of any one of embodiments 67-119, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
  • Embodiment 121 The system of any one of embodiments 67-120, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 122 The system of any one of embodiments 67-121, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
  • Embodiment 123 The system of any one of embodiments 67-122, wherein system further comprises an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 124 The system of any one of embodiments 67-123, wherein the imaging device is a personal electronic device.
  • Embodiment 125 The system of any one of embodiments 67-124, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
  • Embodiment 126 The system of any one of embodiments 67-125, wherein the personal electronic device comprises a web -based portal.
  • Embodiment 127 The system of any one of embodiments 67-126, wherein the webbased portal utilizes an application.
  • Embodiment 128 The system of any one of embodiments 67-127, wherein the application is configured to receive data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, and external data from one or more external device.
  • Embodiment 129 The system of any one of embodiments 67-128, wherein the application comprises a data analytics module configured to analyze the result by: (a) determining whether the resultis a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity; (b) determining an absolute number of complexes between the peptide-conjugate and the ACE2 receptor on the surface; or (c) determining a level of binding between the peptide -conjugate and the ACE2, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
  • Embodiment 130 The system of any one of embodiments 67-129, wherein the data analytics module is further configured to analyze the result by determining whether the result is a positive result or a negative result, wherein a positive result indicates a presence of an acute infection by the coronavirus, and a negative results indicates an absence of the acute infection by the coronavirus.
  • Embodiment 131 The system of any one of embodiments 67-130, wherein the positive or the negative result is relative to a threshold number of complexes between the peptide- conjugate and the ACE2 receptor on the surface.
  • Embodiment 132 The system of any one of embodiments 67-131, wherein the positive or the negative result is relative to a threshold number of complexes between the one or more capture molecules specific to the one or more antibodies against the coronavirus.
  • Embodiment 133 The system of any one of embodiments 67-132, wherein the threshold number is predetermined relative to an index a control.
  • Embodiment 134 The system of any one of embodiments 67-133, wherein the data analytics module is further configured to normalize the resultby subtractingbackground noise.
  • Embodiment 135. The system of any one of embodiments 67-134, wherein the data analytics module is further configuredto identify a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
  • Embodiment 136 The system of any one of embodiments 67-135, wherein the data analytics module is further configuredto identify a geographical location comprised of subjects for which a positive result was determined to identify regions of infection or re-infection, or to recommend further testing.
  • Embodiment 137 The system of any one of embodiments 67-136, wherein the data analytics module utilizes geofencing from coordinates of the personal electronic device to identify the geographical location.
  • Embodiment 138 The system of any one of embodiments 67-137, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
  • Embodiment 139 Aspects disclosed herein provide a method of identifying adaptive immunity to a coronavirus in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) producing a mixture by introducing the biological sample with a detectable peptide derived from a spike glycoprotein of a coronavirus; (c) bringing the mixture into contact with one or more capture molecules derived from an angiotensin -converting enzyme 2 (ACE2) receptor; (d) detecting a number of binding complexes between the detectable peptide and the one or more capture molecules; (e) if the number of the binding complexes is low relative to an index or a control, then identifyingthe subject as being immune to an infection by the coronavirus; and (f) if the number of the binding complexes is high relative to an index ora control, then identifyingthe subject as notbeingimmuneto an infection by the coronavirus.
  • ACE2 angiotensin -converting enzyme 2
  • Embodiment 140 The method of embodiment 139, wherein steps (a)-(f) are performed at the point of need.
  • Embodiment 141 The method of any one of embodiments 139-140, wherein steps (a)- (f) are performed at the point of care.
  • Embodiment 142 The method of any one of embodiments 139-141, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 143 The method of any one of embodiments 139- 142, wherein the surface is a passivated surface.
  • Embodiment 144 The method of any one of embodiments 139-143, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(m ethyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyridine)
  • PVP poly(vinyl pyrrol
  • Embodiment 145 The method of any one of embodiments 139-144, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 146 The method of any one of embodiments 139-145, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 147 The method of any one of embodiments 139-146, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 148 The method of any one of embodiments 139-147, wherein the one or more capture molecules is a fusion polypeptide.
  • Embodiment 149 The method of any one of embodiments 139-148, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Fc fragment crystallizable region
  • Embodiment 150 The method of any one of embodiments 139-149, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 151 The method of any one of embodiments 139-150, wherein the detectable peptide comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 152 The method of any one of embodiments 139-151, wherein The method of any one of embodiments 139-192, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
  • Embodiment 153 The method of any one of embodiments 139-152, wherein the nanoparticle is magnetic.
  • Embodiment 154 The method of any one of embodiments 139-153, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 155 The method of any one of embodiments 139-154, wherein the detectable peptide derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 156 The method of any one of embodiments 139-155, wherein the one or more capture molecules derived from the spike glycoprotein of a coronavirus comprises atleast a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome -associated coronavirus
  • Embodiment 157 The method of any one of embodiments 139-156, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment 158 The method of any one of embodiments 139-157, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
  • Embodiment 159 Themethodof any oneof embodiments 139-158, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 160 Themethodof any oneof embodiments 139-159, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment 161 Themethodof any oneof embodiments 139-160, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
  • Embodiment 162 Themethodof any oneof embodiments 139-161, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment 163 Themethodof any oneof embodiments 139-162, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115 [0576] Embodiment 164. The method of any one of embodiments 139-163, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 165 The method of any one of embodiments 139-164, wherein the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 166 The method of any one of embodiments 139-165, wherein the detectable peptide derived from the spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome -associated coronavirus
  • Embodiment 167 The method of any one of embodiments 139-166, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment 168 The method of any one of embodiments 139-167, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
  • Embodiment 169 Themethodof any oneof embodiments 139-168, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 170 Themethodof any oneof embodiments 139-169, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment 171 Themethodof any oneof embodiments 139-170, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
  • Embodiment 172 Themethodof any oneof embodiments 139-171, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment 173 Themethodof any oneof embodiments 139-172, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115 [0586] Embodiment 174. The method of any one of embodiments 139-173, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 175. The method of any one of embodiments 139-174, wherein method further comprises providing a web -based portal on the personal electronic device.
  • Embodiment 176 The method of any one of embodiments 139-175, wherein method further comprises providing an application on the web-based portal.
  • Embodiment 177 The method of any one of embodiments 139-176, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
  • Embodiment 178 The method of any one of embodiments 139-177, wherein method further comprises providing a data analytics module at the application.
  • Embodiment 179 The method of any one of embodiments 139-178, wherein method further comprises analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
  • Embodiment 180 The method of any one of embodiments 139-179, wherein the positive or the negative result is relative to a threshold number of complexes between the detectable peptide and the one or more capture molecules on the surface.
  • Embodiment 18 The method of any one of embodiments 139-180, wherein the threshold number is predetermined relative to an index a control.
  • Embodiment 182 The method of any one of embodiments 139-181, wherein method further comprises analyzingthe result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules on the surface.
  • Embodiment 183 The method of any one of embodiments 139-182, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of bindingbetween the peptide-conjugate andthe ACE2, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
  • Embodiment 184 The method of any one of embodiments 139-183, wherein method further comprises normalizing, by the data analytics module, the result by subtracting background noise.
  • Embodiment 185 The method of any one of embodiments 139-184, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
  • Embodiment 186 The method of any one of embodiments 139-185, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
  • Embodiment 187 The method of any one of embodiments 139-186, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
  • Embodiment 188 The method of any one of embodiments 139-187, wherein method further comprises providing a data store that is a cloud-based data store or a web-based data store, or a local data store.
  • Embodiment 189 The method of any one of embodiments 139-188, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
  • Embodiment 190 The method of any one of embodiments 139-189, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 191 The method of any one of embodiments 139-190, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
  • Embodiment 192 The method of any one of embodiments 139-191, wherein comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • aspects disclosed herein provide a method of identifying adaptive immunity to a coronavirus in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) producing a mixture by introducing the biological sample with a detectable peptide derived from an angiotensin -converting enzyme 2 (ACE2) receptor; (c) bringing the mixture into contact with one or more capture molecules derived from a spike glycoprotein of a coronavirus; (d) detecting a number of binding complexes between the detectable peptide and the one or more capture molecules; (e) if the number of the binding complexes is low relative to an index or a control, then identifyingthe subject as being immune to an infection by the coronavirus; and (f) if the number of the binding complexes is high relativeto an index ora control, then identifyingthe subject as notbeingimmuneto an infection by the coronavirus.
  • ACE2 angiotensin -converting enzyme 2
  • Embodiment 194 The method of embodiment 193, wherein steps (a)-(f) are performed at the point of need.
  • Embodiment 195 The method of any one of embodiments 193-194, wherein steps (a)- (f) are performed at the point of care.
  • Embodiment 196 The method of any oneof embodiments 193-195, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 197 The method of any oneof embodiments 193-196, wherein the surface is a passivated surface.
  • Embodiment 198 The method of any one of embodiments 193- 197, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(m ethyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyridine)
  • PVP poly(vinyl pyr
  • Embodiment 199 The method of any one of embodiments 193-198, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 200 The method of any one of embodiments 193-199, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 201 The method of any one of embodiments 193-200, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 202 The method of any one of embodiments 193-201, wherein the one or more capture molecules is a fusion polypeptide.
  • Embodiment 203 The method of any one of embodiments 193-202, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Fc fragment crystallizable region
  • Embodiment 204 The method of any one of embodiments 193-203, wherein the one or more capture molecules is bound by an antibody th at is coupled to the surface.
  • Embodiment 205 The method of any one of embodiments 193-204, wherein the detectable peptide comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 206 The method of any one of embodiments 193-205, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
  • Embodiment 207 The method of any one of embodiments 193-206, wherein the nanoparticle is magnetic.
  • Embodiment 208 The method of any one of embodiments 193-207, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 209 The method of any one of embodiments 193-208, wherein the detectable peptide derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 210 The method of any one of embodiments 193-209, wherein the one or more capture molecules derived from the spike glycoprotein of a coronavirus comprises atleast a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome-associated coronavirus
  • Embodiment 211 The method of any one of embodiments 193-210, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment 212 The method of any one of embodiments 193-211, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
  • Embodiment21 Themethodof any oneof embodiments 193-212, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment214 Themethodof any oneof embodiments 193-213, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment215. Themethodof any oneof embodiments 193-214, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2 [0628] Embodiment216.
  • Themethodof any oneof embodiments 193-215, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment217 Themethodof any oneof embodiments 193-216, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • Embodiment 218 The method of any one of embodiments 193-217, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 219. The method of any one of embodiments 193-218, wherein the biological sample comprises one or more antibodies specific to the peptide.
  • Embodiment 220 The method of any one of embodiments 193-219, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
  • Embodiment 22 The method of any one of embodiments 193-220, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
  • Embodiment222 Themethodof any one of embodiments 193-221, wherein the subject was, or is, exposed to the coronavirus.
  • Embodiment 22 The method of any one of embodiments 193-222, wherein exposure of the subject to the coronavirus is unknown.
  • Embodiment224 Themethodof any one of embodiments 193-223, wherein the subject is a plurality of subjects.
  • Embodiment 225 The method of any one of embodiments 193-224, wherein method further comprises identifying adaptive immunity of the plurality of subjects to the coronavirus.
  • Embodiment 226 The method of any one of embodiments 193-225, wherein method further comprises monitoring a spread of infection of the plurality of subjects by the coronavirus.
  • Embodiment227 Themethodof any one of embodiments 193-226, wherein the subject was administered a vaccine against the coronavirus.
  • Embodiment 228 The method of any one of embodiments 193-227, wherein method further comprises determining that the vaccine is effective to substantially immunize the subject against the coronavirus, provided the number of the binding complexes is low relative to an index or a control.
  • Embodiment 229. The method of any one of embodiments 193-228, wherein the biological sample comprises blood, urine, saliva, or feces.
  • Embodiment 230 The method of any one of embodiments 193-229, wherein the blood is capillary blood.
  • Embodiment 23 The method of any one of embodiments 193-230, wherein the capillary blood is obtained from the subject by a prick of the subject’ s finger.
  • Embodiment 232 The method of any one of embodiments 193-231, wherein method further comprises separating serum from the blood in the biological sample.
  • Embodiment 233 The method of any one of embodiments 193-232, wherein detecting in (d) comprises capturing an image of the surface with an imaging device to detect a number of binding complexes between the detectable peptide and the one or more capture molecules.
  • Embodiment 23 The method of any one of embodiments 193-233, wherein the imaging device is a personal electronic device.
  • Embodiment 235 The method of any one of embodiments 193-234, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
  • Embodiment 236 The method of any one of embodiments 193-235, wherein method further comprises providing a web -based portal on the personal electronic device.
  • Embodiment 237 The method of any one of embodiments 193-236, wherein method further comprises providing an application on the web-based portal.
  • Embodiment 238 The method of any one of embodiments 193-237, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
  • Embodiment 239. The method of any one of embodiments 193-238, wherein method further comprises providing a data analytics module at the application.
  • Embodiment 240 The method of any one of embodiments 193-239, wherein method further comprises analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
  • Embodiment 241. The method of any one of embodiments 193-240, wherein the positive or the negative result is relative to a threshold number of complexes between the detectable peptide and the one or more capture molecules on the surface.
  • Embodiment 242 The method of any one of embodiments 193-241, wherein the threshold number is predetermined relative to an index a control.
  • Embodiment 243 The method of any one of embodiments 193-242, wherein method further comprises analyzingthe result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules on the surface.
  • Embodiment 244 The method of any one of embodiments 193-243, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of bindingbetween the peptide-conjugate andthe ACE2, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
  • Embodiment 245. The method of any one of embodiments 193-244, wherein method further comprises normalizing, by the data analytics module, the result by subtracting background noise.
  • Embodiment 246 The method of any one of embodiments 193-245, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
  • Embodiment 247 The method of any one of embodiments 193-246, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
  • Embodiment 248 The method of any one of embodiments 193-247, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
  • Embodiment 249. The method of any one of embodiments 193-248, wherein method comprises further comprise providing a data store that is a cloud-based data store or a webbased data store, or a local data store.
  • Embodiment 250 The method of any one of embodiments 193-249, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
  • Embodiment 251. The method of any one of embodiments 193-250, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 252 The method of any one of embodiments 193-251, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
  • Embodiment 253 The method of any one of embodiments 193-252, wherein comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • aspects disclosed herein provide a method comprising: (a) obtaining a biological sample from the subject; (b) determiningwhetherthe subjecthas an acute infection by a pathogen by: (i) introducing the biological sample to a mixture comprising one or more capture molecules specific to an antibody against the pathogen; (ii) detecting a number of binding complexes between the one or more capture molecules specific to the antibody against the pathogen, wherein if the number of the binding complexes is high relative to an index or a control, then identifying the subject as having the acute infection by the coronavirus; or (c) determiningwhetherthe subjecthas a sufficient adaptive immunity against the pathogen by: (i) introducing the biological sample with a second mixture comprising detectable peptide; (ii) bringing the second mixture into contact with one or more capture molecules specific to the detectable peptide; (iii) detecting a number of binding complexes between the detectable peptide and the one or more capture molecules specific to the peptide, wherein if the number of the
  • Embodiment 255 The method of embodiment 254, wherein if the number of the binding complexes is high relative to an index or a control, then identifying the subject as not being immune to an infection by the coronavirus.
  • Embodiment 256 The method of any one of embodiments 254-255, wherein the first mixture and the second mixture is a single mixture.
  • Embodiment 257 The method of any one of embodiments 254-256, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 258 The method of any one of embodiments 254-257, wherein the one or more capture molecules comprises a first antibody, a second antibody or a third antibody, wherein the first antibody is specific to an immunoglobulin G antibody against SARS-CoV-2, the second antibody is specific to an immunoglobulin M antibody against SARS-CoV-2, and the third antibody is specific to an immunoglobulin A of the SARS-CoV-2.
  • Embodiment 259. The method of any one of embodiments 254-258, wherein steps (a)- (c) are performed at the point of need.
  • Embodiment 260 The method of any one of embodiments 254-259, wherein steps (a)- (c) are performed at the point of care.
  • Embodiment 26 1. The method of any one of embodiments 254-260, wherein the one or more capture molecules specific to the antibody against the pathogen are coupled to a surface. [0674] Embodiment 262. The method of any one of embodiments 254-261, wherein the one or more capture molecules specific to the labeled peptide are coupled to the surface.
  • Embodiment263 The method of any oneof embodiments 254-262, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 264 The method of any oneof embodiments 254-263, wherein the surface is a passivated surface.
  • Embodiment 265. The method of any one of embodiments 254-264, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyrrolidone)
  • PAA poly(acrylic acid)
  • Embodiment 266 The method of any one of embodiments 254-265, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 267 The method of any one of embodiments 254-266, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 268 The method of any one of embodiments 254-267, wherein the one or more capture molecules is a fusion polypeptide.
  • Embodiment 269. The method of any one of embodiments 254-268, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Embodiment 270. The method of any one of embodiments 254-269, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 27 The method of any one of embodiments 254-270, wherein the detectable peptide comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 272 The method of any one of embodiments 254-271, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
  • Embodiment 27 The method of any one of embodiments 254-272, wherein the nanoparticle is magnetic.
  • Embodiment 27 The method of any one of embodiments 254-273, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 275 The method of any one of embodiments 254-274, wherein the detectable peptide is derived from the ACE2 receptor and comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 276 The method of any one of embodiments 254-275, wherein the one or more capture molecules specific to the detectable peptideis derived from the spike glycoprotein of a coronavirus and comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome-associated coronavirus
  • Embodiment 278 The method of any one of embodiments 254-277, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
  • Embodiment279. Themethodof any oneof embodiments 254-278, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment280 Themethodof any oneof embodiments 254-279, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment282 Themethodof any oneof embodiments 254-281, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment283 Themethodof any oneof embodiments 254-282, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • Embodiment 284 The method of any one of embodiments 254-283, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 285. The method of any one of embodiments 254-284, wherein the one or more capture molecules specific to the detectable peptide is derived from the ACE2 receptor and comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 286 The method of any one of embodiments 254-285, wherein the detectable peptide is derived from the spike glycoprotein of a coronavirus and comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome-associated coronavirus
  • Embodiment287 Themethodof any one ofembodiments 254-286, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment 288 The method of any one of embodiments 254-287, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
  • Embodiment289. Themethodof any oneof embodiments 254-288, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment290 Themethodof any oneof embodiments 254-289, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment292 Themethodof any oneof embodiments 254-291, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment293. Themethodof any oneof embodiments 254-292, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • Embodiment 294 The method of any one of embodiments 254-293, wherein the complex between the detectable peptide and the one or more capture molecules specific to the detectable peptide is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 295. The method of any one of embodiments 254-294, wherein the biological sample comprises one or more antibodies specific to the peptide.
  • Embodiment 296 The method of any one of embodiments 254-295, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
  • Embodiment 297 The method of any one of embodiments 254-296, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
  • Embodiment 298 The method of any one of embodiments 254-297, wherein pathogen is a coronavirus.
  • Embodiment 299. Themethodof any one ofembodiments 254 -298, wherein the subject was, or is, exposed to the coronavirus.
  • Embodiment 300 The method of any one of embodiments 254-299, wherein exposure of the subject to the coronavirus is unknown.
  • Embodiment 301 The method of any one of embodiments 254-300, wherein the subject is a plurality of subjects.
  • Embodiment 302. The method of any one of embodiments 254-301, wherein method further comprises identifying adaptive immunity of the plurality of subjects to the coronavirus.
  • Embodiment 303 The method of any one of embodiments 254-302, wherein method further comprises monitoring a spread of infection of the plurality of subjects by the coronavirus.
  • Embodiment 304 The method of anyone of embodiments 254-303, wherein the subject was administered a vaccine against the coronavirus.
  • Embodiment 305 The method of any one of embodiments 254-304, wherein method further comprises determining that the vaccine is effective to substantially immunize the subject against the coronavirus, provided the number of the binding complexes between the detectable peptide and the one or more capture molecules specific to the detectable peptide is low relative to an index or a control.
  • Embodiment 306 The method of any one of embodiments 254-305, wherein the biological sample comprises blood, urine, saliva, or feces.
  • Embodiment 307 The method of any one of embodiments 254-306, wherein the blood is capillary blood.
  • Embodiment 308 The method of any one of embodiments 254-307, wherein the capillary blood is obtained from the subject by a prick of the subject’ s finger.
  • Embodiment 309 The method of any one of embodiments 254-308, wherein method further comprises separating serum from the blood in the biological sample.
  • Embodiment 310 The method of any one of embodiments 254-309, wherein detecting in (d) comprises capturing an image of the surface with an imaging device to detect a number of binding complexes between the detectable peptide and the one or more capture molecules.
  • Embodiment 311 The method of any one of embodiments 254-310, wherein the imaging device is a personal electronic device.
  • Embodiment 312 The method of any one of embodiments 254-311, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
  • Embodiment 31 The method of any one of embodiments 254-312, wherein method further comprises providing a web -based portal on the personal electronic device.
  • Embodiment 31 The method of any one of embodiments 254-313, wherein method further comprises providing an application on the web-based portal.
  • Embodiment 315 The method of any one of embodiments 254-314, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
  • Embodiment 316 The method of any one of embodiments 254-315, wherein method further comprises providing a data analytics module at the application.
  • Embodiment 317 The method of any one of embodiments 254-316 wherein method further comprises analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
  • Embodiment 318 The method of any one of embodiments 254-317, wherein the positive or the negative result (e.g., immune or not immune) is relative to a threshold number of complexes between the detectable peptide and the one or more capture molecules.
  • the positive or the negative result e.g., immune or not immune
  • Embodiment 319 The method of any one of embodiments 254-318, wherein the threshold number is predetermined relative to an index a control.
  • Embodiment 320 The method of any one of embodiments 254-319, wherein the positive or the negative result (e.g., acute infection present or not present) is relative to a threshold number of complexes between the one or more capture molecules specific to the antibody against the pathogen and the antibody against the pathogen.
  • the positive or the negative result e.g., acute infection present or not present
  • Embodiment 321 The method of any one of embodiments 254-320, wherein method further comprises analyzingthe result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules specific to the detectable peptide, or the antibody against the pathogen and the one or more capture molecules specific to the antibody.
  • Embodiment 322 The method of any one of embodiments 254-321, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of binding between the detectable peptide and the one or more capture molecules specific to the detectable peptide, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
  • Embodiment 32 The method of any one of embodiments 254-322, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of binding between the one or more capture molecules specific to the antibody against the pathogen and the antibody against the pathogen, wherein a high level of binding indicates a presence of an acute infection by the pathogen.
  • Embodiment 324 The method of any one of embodiments 254-323, wherein method further comprises normalizing, by the data analytics module, the result by subtracting background noise.
  • Embodiment 325 The method of any one of embodiments 254-324, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result for immunity was determined to detect a presence of herd immunity to the pathogen, or recommend further testing.
  • Embodiment 326 The method of any one of embodiments 254-325, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result for an acute infection was determined to detect a presence of infectivity or re-infectivity by the pathogen, or recommend further testing.
  • Embodiment 327 The method of any one of embodiments 254-326, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
  • Embodiment 328 The method of any one of embodiments 254-327, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
  • Embodiment 329 The method of any one of embodiments 254-328, wherein method further comprises providing a data store that is a cloud-based data store or a web-based data store, or a local data store.
  • Embodiment 330 The method of any one of embodiments 254-329, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
  • Embodiment 331 The method of any one of embodiments 254-330, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 332 The method of any one of embodiments 254-331, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
  • Embodiment 333 The method of any one of embodiments 254-332, wherein comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
  • Embodiment 334 Embodiment 334.
  • a device comprising: (a) a liquid composition comprising a peptide-conjugate comprising: (i) a peptide derived from a spike gly coprotein of a coronavirus; and (ii) a detectable moiety; and (b) a surface submerged in the liquid composition, the surface comprising one or more capture molecules coupled to the surface, the one or more capture molecules derived from an angiotensin -converting enzyme 2 (ACE2) receptor.
  • ACE2 angiotensin -converting enzyme 2
  • Embodiment 335 The device of embodiment 334, wherein the surface is a surface of a container, wherein the container contains (a) and (b).
  • Embodiment 336 The device of any one of embodiments 334-335, wherein the device is portable.
  • Embodiment 337 The device of any one of embodiments 334-336, wherein the device is a point of need device.
  • Embodiment 338 The device of any one of embodiments 334-337, wherein the point of need is a point of care.
  • Embodiment 339 The device of any one of embodiments 334-338, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 340 The device of any one of embodiments 334-339, wherein the surface is a passivated surface.
  • Embodiment 34 The device of any one of embodiments 334-340, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(m ethyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly (vinyl alcohol)
  • PVP poly(vinyl pyridine)
  • PVP poly(vinyl pyrroli
  • Embodiment 342 The device of any one of embodiments 334-341, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 34 The device of any one of embodiments 334-342, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 344 The device of any one of embodiments 334-343, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 345 The device of any one of embodiments 334-344, wherein the one or more capture molecules is a fusion polypeptide.
  • Embodiment 346 The device of any one of embodiments 334-345, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Fc fragment crystallizable region
  • Embodiment 347 The device of any one of embodiments 334-346, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 348 The device of any one of embodiments 334-347, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 349 The device of any one of embodiments 334-348, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
  • Embodiment 350 The device of any one of embodiments 334-349, wherein the nanoparticle is magnetic.
  • Embodiment 35 The device of any one of embodiments 334-350, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 352 The device of any one of embodiments 334-3514, wherein the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
  • Embodiment 353 The device of any one of embodiments 334-352, wherein the peptide derived from the spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome -associated coronavirus
  • Embodiment 354 The device of any one of embodiments 334-353, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment 355. The device of any one of embodiments 334-354, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
  • Embodiment 356 The device of any one of embodiments 334-355, wherein the at least a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 357 The device of any one of embodiments 334-356, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • Embodiment 358 The device of any one of embodiments 334-357, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
  • Embodiment 359. The device of any one of embodiments 334-358, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
  • Embodiment 360 The device of any one of embodiments 334-359, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
  • Embodiment 36 The device of any one of embodiments 334-360, wherein the complex between the peptide-conjugate and the one or more capture molecules receptor on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
  • Embodiment 362 The device of any one of embodiments 334-361, wherein the one or more capture molecules is specific to one or more antibodies against the coronavirus.
  • Embodiment 36 The device of any one of embodiments 334-362, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
  • Embodiment 36 The device of any one of embodiments 334-363, wherein device further comprise a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
  • Embodiment 365 The device of any one of embodiments 334-364, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen -binding fragment.
  • Embodiment 366 The device of any one of embodiments 334-365, wherein the antibody or antigen -binding fragment is monoclonal, antibody or antigen -binding fragment is polyclonal.
  • Embodiment 367 The device of any one of embodiments 334-366, wherein device further comprises a housing at least partially enclosing the surface.
  • Embodiment 368 The device of any one of embodiments 334-367, wherein device further comprises a sample receptor configured to receive a biological sample from a subject.
  • Embodiment 369 The device of any one of embodiments 334-368, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface.
  • Embodiment 370 The device of any one of embodiments 334-369, wherein the biological sample comprises one or more antibodies specific to the peptide.
  • Embodiment 37 The device of any one of embodiments 334-370, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
  • Embodiment 372 The device of any one of embodiments 334-371, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
  • Embodiment 373 The device of any one of embodiments 334-372, wherein the subject was, or is, exposed to the coronavirus.
  • Embodiment 374 The device of any one of embodiments 334-373, wherein exposure of the subject to the coronavirus is unknown.
  • Embodiment 375 The device of any one of embodiments 334-374, wherein the subject was administered a vaccine against the coronavirus.
  • Embodiment 376 The device of any one of embodiments 334-375, wherein the biological sample comprises blood, urine, saliva, or feces.
  • Embodiment 377 The device of any one of embodiments 334-376, wherein the blood is capillary blood.
  • Embodiment 378 The device of any one of embodiments 334-377, wherein device further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
  • Embodiment 379 The device of any one of embodiments 334-378, wherein the sample receptor comprises a filter to separate serum from the blood.
  • Embodiment 380 The device of any one of embodiments 334-379, wherein the device is a single integrated device.
  • Embodiment 381. Aspects disclosed herein provide a device comprising: (a) a liquid composition comprising a peptide-conjugate comprising: (i) a peptide derived from an angiotensin-converting enzyme 2 (ACE2) receptor; and (ii) a detectable moiety; and (b) a surface submerged in the liquid composition, the surface comprising one or more capture molecules coupled to the surface, the one or more capture molecules derived from a spike glycoprotein of a coronavirus.
  • ACE2 angiotensin-converting enzyme 2
  • Embodiment 382. The device of embodiment 381, wherein the surface is a surface of a container, wherein the container contains (a) and (b).
  • Embodiment 383. The device of any one of embodiments 381-382, wherein the device is portable.
  • Embodiment 384 The device of any one of embodiments 381-383, wherein the device is a point of need device.
  • Embodiment 385 The device of any one of embodiments 381-384, wherein the point of need is a point of care.
  • Embodiment 386 The device of any one of embodiments 381-385, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
  • Embodiment 387 The device of any one of embodiments 381-386, wherein the surface is a passivated surface.
  • Embodiment 388 The device of any one of embodiments 381-387, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
  • PEG polyethylene glycol
  • PVA poly(vinyl alcohol)
  • PVP poly(vinyl pyridine)
  • PVP poly(vinyl pyrrolidone)
  • Embodiment 389 The device of any one of embodiments 381-388, wherein the one or more capture molecules comprises two or more capture molecules.
  • Embodiment 390 The device of any one of embodiments 381-389, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
  • Embodiment 391 The device of any one of embodiments 381-390, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 392 The device of any one of embodiments 381-391, wherein the one or more capture molecules is a fusion polypeptide.
  • Embodiment 393 The device of any one of embodiments 381-392, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
  • Embodiment 394 The device of any one of embodiments 381-393, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
  • Embodiment 395 The device of any one of embodiments 381-394, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
  • Embodiment 396 The device of any one of embodiments 381-395, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
  • Embodiment 397 The device of any one of embodiments 381-396, wherein the nanoparticle is magnetic.
  • Embodiment 398 The device of any one of embodiments 381-397, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
  • Embodiment 399 The device of any one of embodiments 381-398, wherein the peptide derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1
  • Embodiment 400 The device of any one of embodiments 381-399, wherein the one or more capture molecules derived from the spike glycoprotein of a coronavirus comprises atleast a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
  • SARS-CoV Severe acute respiratory syndrome -associated coronavirus
  • Embodiment 401 The device of any one of embodiments 381-400, wherein the SARS- CoV is SARS-CoV-2.
  • Embodiment402. The device of any one of embodiments 381-401 , wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
  • Embodiment 403. The device of any one of embodiments 381-402, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
  • Embodiment 404 The device of any one of embodiments 381-403, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
  • RBD receptor binding domain
  • Embodiment 405. The device of any one of embodiments 381-404, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
  • Embodiment 406 The device of any one of embodiments 381-405, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3

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Abstract

Provided are devices, systems, methods and kits for determining whether a subject is immune to an infection by a disease-causing pathogen by measuring neutralizing antibodies against the disease-causing pathogen in a biological sample from the subject. The devices, systems, methods, and kits described herein are useful for confirming whether a vaccine against the disease-causing pathogen has elicited enough neutralizing antibodies to prevent a later infection, or lessen severity of disease caused by, the disease-causing pathogen. Such devices, systems, methods, and kits are also useful for detecting an infection in the subject.

Description

METHODS AND SYSTEMS FOR ASSESSING ADAPTIVE
IMMUNITY TO CORONAVIRUS
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 63/240,265, filed September 2, 2021, which application is incorporated hereinby reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 58552-706_601_SEQ.xml, created August 23, 2022, which is 650 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
BACKGROUND
[0003] Viral infections on a cellular level are mediated by viruses binding to receptors expressed on the surface of a target cell. For example, the spike glycoprotein of a coronavirus binds to the angiotensin-converting enzyme 2 (ACE2) receptor, and binding between the receptor-binding domain (RBD) of the spike protein and ACE2 precedes entry of the coronavirus into the cell. Individuals who are exposed to a new virus (e.g., coronavirus) may develop neutralizing antibodies againstthe virus to block viral infection. This adaptive immune response significantly reduces incidences of a second infection by the same virus.
[0004] The coronavirus disease of 2019 (COVID-19) is an ongoingpandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There is an unprecedented need for effective and safe vaccines against SARS-CoV-2. Even those who are infected with SARS- CoV-2 and recover face severe side-effects, and even death, post recovery. Post-COVID syndrome, which includes cardiac, neuro, and respiratory complications, can persist indefinitely. Onethird of recovered COVID-19 hospitalized patients are readmitted, and 1 in 8 die within 5 months. In an effort to fast track the regulatory approval process for new vaccines against SARS-CoV-2, regulatory agencies and vaccine developers may look to surrogate markers of protection against future infections by SARS-CoV-2 until proof of protection is available. This means that new vaccines entering the market, although proven to be safe, may not be effective. [0005] Infectious disease experts believe there is a strong correlation between protection from a future infection by SARS-CoV-2 and the presence of neutralizing antibody titers. Yet, out of the dozens of vaccines under clinical investigation today, a vast maj ority do not test for presence of neutralizing antibodies against SARS-CoV-2 as a primary clinical endpoint. There exists an urgent need fora confirmatory diagnostic test that measures neutralizing antibody titers to determine whether a legally commercialized vaccine is effective to confer immunity against SARS-CoV-2 (e.g., post-allowance or licensure of the vaccine). With nearly 320 million people in the United States alone in need of vaccination, the neutralizing antibody test needed must be scalable and cost-efficient.
[0006] Reliance on surrogate markers of protection may result in a larger portion of subjects that will not respond to the new legally commercialized vaccine as compared with a comparable vaccine developed within the traditional 15-20 year timeframe. There is a need for a scalable test that measures neutralizing antibodies in a population of individuals after administration of the new SARS-CoV-2 vaccine (e.g., allowed or approved for human or other animal use) and a system that enables non-responders in that population to know whether the new vaccine was effective to induce neutralizing antibodies conferring protection, or not. For example, there is minimal trial data for important sub -populations, such as those of the nonwhite race, and those currently having one or more risk factors related to COVID -19. There is also no data on larger subgroups who are excluded from clinical trials, such as those who are pregnant, breastfeeding, or have priorly been infected by COVID-19. Stratifying non- responders from responders to a new vaccine in this context will prevent the unintentional spread of COVID-19 by vaccinated individuals with a “false” sense of immunity. This is particularly critical for protecting our vulnerable populations, including individuals ages 50 and older who are significantly more likely to die from a disease caused by SARS-CoV-2 as compared to younger individuals. For example, published data show that new vaccines entering the market elicit around 40% fewer neutralizing antibodies in subjects ages 65 and older. Clinical trials show the efficacy of the new vaccines in subjects ages 65 and older could be as low as 65%.
[0007] Additionally, the vaccine administration methods taking place pose further problems. With many millions of people waiting several weeks to receive a second COVID-19 vaccine dose in some countries, it is possible that SARS-CoV-2 could evolve vaccine resistance. In addition to the timing of the administered doses being an issue, the durability of the vaccine elicited immune response in patients is not yet known. Further, as millions of people are being vaccinated, health officials are struggling to collect critically important information — such as race, ethnicity and occupation — of every person vaccinated. The data being collected is so scattered that there’ s little in sight into which h ealth care workers, or first responders, have been among the people getting the initial vaccines, as intended — or how many doses instead have gone to people who should be much further down the list.
[0008] In addition, the fragile thermostability of vaccines means that any delay during distribution poses a risk that the vaccines will have reduced potency upon arrival to its destination. As one example, delays in distribution due to unpredictable and extreme weather conditions across the United States are a source of major concern. Addressing this challenge for vaccines against SARS-CoV-2 is made even more difficult with the limited number of facilities around the world manufacturing the vaccines requiring longer delivery routes.
[0009] However, this challenge is not limited to delays in distribution. For a clinic to receive a vaccine, they must have ultra-low cold storage sufficient to hold the vaccine at low enough temperatures. The vaccine must be kept at low temperatures in order to preserve its potency. For example, even opening freezers repeatedly can harm vaccines stored in the freezer and inadvertently lower their potency. Once a clinic receives a vaccine shipment, health workers thaw out the vials in a refrigerator as they prepare to give injections to patients. But once a vaccine is thawed, it is only viable for a few days and cannot be re-frozen. Thus, there exists a need for a test that can determine whether a vaccine administered to a subject is effective to meet this unprecedented need.
[0010] Shelter-in-place mandates all over the world designed to reduce the spread of COVID- 19 are having severe and long-lasting negative socioeconomic and economic consequences. In theory, reintegration into society of individuals with enough neutralizing antibody titers conferring immunity to SARS-CoV-2 is possible, provided there is a reliable and scalable test to measure whether a subject has sufficient neutralizing antibody titers against SARS-CoV-2 to confer protection. However, existing SARS-CoV-2 tests are not scalable. Thus, there exists a need for a test that can measure neutralizing antibodies that is scalable to meet this unprecedented need.
SUMMARY
[0011] In certain aspects, disclosed herein is a method of assessing adaptive immunity against a coronavirus in subject, the method comprising: (a) bringing a biological sample of the subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from the coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample when the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is a signal from the binding complex, SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein; and (c) comparing the percent inhibition of the binding measured in (b) to a percent inhibition of binding of the first peptide or protein to the second peptide or protein of in a control sample or index, wherein the control or the index is derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus. In some embodiments, the biological sample has an average volume of less than 1 milliliter when obtained from the subject. In some embodiments, the biological sample is not a liquid when it is provided in (a). In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the one or more control subjects received a vaccine against the coronavirus within one month of comparing in (d). In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. In some embodiments, the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus. In some embodiments, the SARS virus is SARS-CoV-2 or a variant thereof. In some embodiments, the subject is a human subject that has an age comprising more than or equal to about 50 years of age. In some embodiments, the method further comprises eluting the at least the portion of the biological sample from a dried blood spot card. In some embodiments, the subject is unvaccinated against the coronavirus.
[0012] In certain aspects, disclosed herein is a method comprising: identifying a subject as having adaptive immunity against a coronavirus relative to a control subject that is vaccinated against the coronavirus, wherein the identifying comprises: (a) bringing a biological sample obtained from the subject into contact with (1) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (2) a second peptide or protein comprising a spike protein or a portion thereof derived from the corona virus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring a percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample; and (c) comparingthe percentinhibition of the bindingto a percentinhibition of binding of the first peptide or protein to the second peptide or protein in a control or an index, wherein the control or the index is derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus. In some embodiments, the biological sample has an average volume of less than 1 milliliter when obtained from the subject. In some embodiments, the biological sample comprises capillary blood . In some emb odiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the control subject received a vaccine against the coronavirus within one month of comparing in (c). In some embodiments, the method further comprises: (d) identifying the adaptive immunity against the coronavirus of the subject as being lower than the adaptive immunity of the control or the index; and (e) administering a vaccine against the coronavirus to the subject. In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. In some embodiments, the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus. In some embodiments, the SARS virus is SARS-CoV-2, or a variant thereof. In some embodiments, the human subject has an age comprising greater than or equal to about 50 years of age. In some embodiments, the first peptide or protein or the second peptide or protein is immobilized on a substrate In some embodiments, the method further comprises detectingthe binding complex using a lateral flow assay. In some embodiments, detectingthe binding complex in comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device. In some embodiments, the method further comprises eluting the biological sample from a dried blood spot card. In some embodiments, the subject is unvaccinated against the coronavirus.
[0013] In certain aspects, disclosed herein is a method of analyzing a biological sample, the method comprising: bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an accuracy of at least about 80%. In some embodiments, the subject is vaccinated against the coronavirus. In some embodiments, the subject is unvaccinated against the coronavirus. In some embodiments, the coronavirus comprises SARS-CoV-2 or a variant thereof. In some embodiments, the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide. In some embodiments, the second protein or peptide comprises the S1 subunit of the spike protein. In some embodiments, the biological sample has an average volume of less than 1 milliliter when obtained. In some embodiments, the biological sample comprises capillary blood. In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the method further comprises administering a vaccine against the coronavirus to the subject based on measuring in (b). In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. In some embodiments, the subject is an age comprising greater than or equal to about 50 years of age. In some embodiments, the first peptide or protein or the second peptide or protein is immobilized on a substrate In some embodiments, the method further comprises detecting the binding complex using a lateral flow assay. In some embodiments, detecting the binding complex in (b) comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device. In some embodiments, the method further comprises eluting the at least the portion of the biological sample from a dried blood spot card. In some embodiments, the accuracy is measured by: (a) introducing a reference sample with (i) the first peptide or protein; and (ii) the second peptide or protein, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC50; and (b) detecting a binding complex between the first peptide or protein and the second peptide or protein; and (c) determining an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50. [0014] In certain aspects, disclosed herein is a method of analyzing a biological sample, the method comprising: bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an sensitivity of at least about 80%. In some embodiments, the subject is vaccinated against the coronavirus. In some embodiments, the subject is unvaccinated against the coronavirus. In some embodiments, the coronavirus comprises SARS-CoV-2 or a variant thereof. In some embodiments, the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof. In some embodiments, the second protein or peptide comprises the S1 subunit of the spike protein. In some embodiments, the biological sample has an average volume of less than 1 milliliter when obtained. In some embodiments, the biological sample comprises capillary blood. In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the method further comprises administering a vaccine against the coronavirus to the subject based on measuring in (b). In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. In some embodiments, the subject is an age comprising greater than or equal to about 50 years of age. In some embodiments, the method further comprises eluting the at least the portion of the biological sample from a dried blood spot card. In some embodiments, the first peptide or protein or the second peptide or protein is immobilized on a substrate In some embodiments, the method further comprises detecting the binding complex using a lateral flow assay. In some embodiments, detecting the binding complex in (b) comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device. [0015] In certain aspects, disclosed herein is a method of analyzing a biological sample, the method comprising: bringing at least a portion of a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with percent coefficient of variation of less than 10%. In some embodiments, the subject is vaccinated against the coronavirus. In some embodiments, the subject is unvaccinated against the coronavirus. In some embodiments, the coronavirus comprises SARS-CoV-2 or a variant thereof . In some embodiments, the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof. In some embodiments, the second protein or peptide comprises the S1 subunit of the spike protein. In some embodiments, the biological sample has an average volume of less than 1 milliliter when obtained. In some embodiments, the biological sample comprises capillary blood. In some embodiments, the method does not consist of utilizing an immortalized cell or immortalized cell culture. In some embodiments, the method further comprises administering a vaccine against the coronavirus to the subject based on measuring in (b). In some embodiments, the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. In some embodiments, the subject is an age comprising greater than or equal to about 50 years of age. In some embodiments, the method further comprises eluting the biological sample from a dried blood spot card. In some embodiments, the percent inhibition is predictive of a clinical endpoint when the subject received a pre-approved or licensed vaccine against the coronavirus. In some embodiments, the pre-approved or licensed vaccine comprises Ad26.COV.S, mRNA-1273 or BNT162b2, or any combination thereof. In some embodiments, the percent inhibition is predictive of individual protection against another infection by the coronavirus. In some embodiments, the method further comprises administering to the subject a vaccine booster against the coronavirus or a second dose of a vaccine against the coronavirus, when the percent inhibition is below low relative to a control of the index derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus. In some embodiments, the subject has an underlying immune-compromising condition. In some embodiments, the underlying immune-comprising condition comprises a pregnancy, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof. In some embodiments, the subject is breastfeeding. In some embodiments, further comprising detecting one or more signals from one or more detectable moieties coupled to the binding complex. In some embodiments, the method further comprises detecting the binding complex using a lateral flow assay. In some embodiments, the one or detectable moieties comprises a nanoparticle, a fluorescent metal, a fluorescent dye, an enzymatic label, or a colorimetric label, or any combination thereof. In some embodiments, the method further comprises determining that the biological sample comprises about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell -based pseudovirion neutralization assay (PNA), when the percent neutralization measured is about 62%. In some embodiments, the method further comprises determining that the subject is adequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of about 62%. In some embodiments, the method further comprises determining that the biological sample comprises less than about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell -based pseudovirion neutralization assay (PNA), when the percent neutralization measured is less than about 62%. In some embodiments, the method further comprises determining that the subject is inadequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of less than about 62%. In some embodiments, the method is performed without a wash step to wash away the first peptide or protein or the second peptide or protein that are unbound following the bringing in (a). In some embodiments, the bringing the biological sample into contact with the first peptide or protein and the second peptide or protein is performed in a container. In some embodiments, the container is optically clear. In some embodiments, the container is a disposable cartridge. In some embodiments, the method further comprises repeating the method on a plurality of biological samples obtained from the subject, wherein at least two biological samples of the plurality of biological samples are obtained at different time points. In some embodiments, the subject is receiving an immunosuppressant therapy. In some embodiments, the method further comprises: (a) identifying the subject as needing a dose of a vaccine against the coronavirus, provided the percent neutralization is lower than 62%; and (b) suspending a treatment regimen comprising the immunosuppressant therapy for a period of time during which period of time, the subject is administered the dose of the vaccine. In some embodiments, the subject is a pregnant subject. INCORPORATION BY REFERENCE
[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The novel features of the inventive concept(s) are set forth with particularity in the appended claims. Abetterunderstandingofthe features and advantages of the present inventive concept(s) will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concept(s) are utilized, and the accompanying drawings of which:
[0018] FIG. 1 shows an exemplary assay measuring binding between an immobilized human angiotensin-converting enzyme 2 (ACE2) receptor and a peptide-conjugate derived from a spike glycoprotein of a SARS-CoV-2 in a biological sample obtained from a patient that has notbeen exposed to SARS-CoV-2.
[0019] FIG. 2 shows an exemplary assay measuring binding between an immobilized human ACE2 receptor and a peptide-conjugate derived from the spike glycoprotein of SARS-CoV-2 in a biological sample obtained from a patient exposed to SARS-CoV-2.
[0020] FIG. 3 shows an exemplary lateral flow assay to measure binding between human ACE2 receptor and a peptide-conjugate derived from the spike glycoprotein of SARS-CoV-2 in a biological sample obtained from a patient that has notbeen exposed to SARS-CoV-2.
[0021] FIG. 4 shows an exemplary lateral flow assay to measure binding between human ACE2 receptor and a peptide-conjugate derived from the spike glycoprotein of SARS-CoV-2 in a biological sample obtainedfrom a patient that was exposed to SARS-CoV-2.
[0022] FIG. 5 shows an exemplary system according to some embodiments.
[0023] FIG. 6 shows a computing device; in this case, a device with one or more processors, memory, storage, and a network interface, in accordance with some embodiments.
[0024] FIG. 7 shows an exemplary assay in FIG. 2 in an image-free system; results from the exemplary assay is visible and can be interpreted by the naked eye. [0025] FIG. 8A-8D shows an exemplary assay assembly to measure inhibition of binding between the receptor binding domain (RBD) of SARS-CoV-2 and its cognate receptor, ACE2. FIG. 8A illustrates assay with a first RBD (RBD 1 ) with a patient sample that does not contain neutralizing antibodies that functionally inhibitRBDl -ACE2 binding. FIG. 8B shows the same assay from FIG. 8A with a patient sample that contains neutralizing antibodies that functionally inhibit RBD 1-ACE2 binding. FIG. 8C shows a neutralization assay that measures a presence of neutralizing antibodies against at least two different RBD peptides (RBD1 and RBD2) in the absence of neutralizing antibodies (FIG. 8C) and in the presence of neutralizing antibodies (FIG. 8D).
[0026] FIG. 9A-9D illustrate that the assay assembly from FIG. 8A-8D may be performed in solution (e.g., not on a solid surface) using fluorescence resonance energy transfer (FRET). FIG. 9 A shows electron transfer between the donor fluoroph ore and the acceptor fluorophore, indicating an absence of neutralizing antibodies that block binding between RBD and ACE2. FIG. 9B shows no electron transfer between the donor fluorophore and the acceptor fluorophore, indicating a presence of neutralizing antibodies that b lockbinding between RBD. FIG. 9C shows a neutralization assay in solution in which electron transfer occurs between two different donor fluorophore and the acceptor fluorophore pairs, indicating an absence of neutralizing antibodies thatblockbindingbetween firstRBD, RBD 1, and second RBD, RBD2. FIG. 9D shows a neutralization assay in solution in which no electron transfer occurs between two different donor fluorophore and the acceptor fluorophore pairs, indicating a presence of neutralizing antib odies that block binding b etween RBD 1 and RBD2.
[0027] FIG. 10A-10E illustrate homogenous solution with reaction components, where the homogenous solution is applied to a surface with surface-bound capture molecules. FIG. 10A illustrates a homogenous solution comprising the reaction components of FIG. 8A being applied to surface-bound capture molecules of FIG. 8A. The capture molecule captures an epitope tag conjugated to RBD, which forms a complex with ACE2 conjugated to the detective moiety. FIG. 10B illustrates a homogenous solution comprising reaction components being applied to surface-bound capture molecules of FIG. 8A. The capture molecule captures an epitope tag conjugated to ACE2, which forms a complex with RBD conjugated to the detective moiety. FIG. 10C illustrates a homogenous solution comprising the reaction components of FIG. 8B being applied to surface-bound capture molecules of FIG. 8B. FIG. 10D illustrates a homogenous solution comprisingthe reaction components of FIG. 8C being applied to surfacebound capture molecules of FIG. 8C. FIG. 10E illustrates a homogenous solution comprising the reaction components of FIG. 8D being applied to surface-bound capture molecules of FIG. 8D
[0028] FIS. 11A-11B illustrate one of the many advantages of utilizing the neutralizing antibody test disclosed herein to reduce risk posed by individuals who may not know if they are adequately protected by their vaccine. FIG. 11 A illustrates two vaccines having different levels of efficacy in a population of individuals, and the risks and benefits associated therewith. FIG. 11B illustrates that the methods provided herein, in some embodiments, can reduce or remove the risk associated with usage of a commercialized vaccine.
[0029] FIG. 12A-12C illustrate a biological sample medium (BSM) capable of storing one or more biological samples in both a closed (FIG. 12A) and open (FIG. 12B) configuration. FIG 12C illustrates a survey printed on the BSM.
[0030] FIG 13 illustrates a system accordingto some embodiments disclosed herein.
[0031] FIG. 14 illustrates a workflow of the automated systems disclosed herein accordingto some embodiments.
[0032] FIG. 15 shows results neutralizing antibody inhibition of SARS-CoV-2 S1 bindingto ACE2 from a time-resolved fluorescence resonance energy transfer (TR-FRET) assay run on 384 well plate (left) and a 1536 well plate (right).
[0033] FIG. 16 shows that, unexpectedly, neutralization levels against SARS-CoV-2 can vary widely in subjects who are vaccinated against SARS-CoV-2 using. FIG. 16 shows the percent of neutralizing antibody response in 7 vaccinated individuals.
[0034] FIG. 17 shows the repeatability of IC50 measurements using systems described herein. [0035] FIGS. 18A-18C shows results from three-time separate TR-FRET assays (also referenced as Runs 1 , 2 and 3) using different reagent lots and performed on difference days. The linear trends observed when run 1 was plotted against run 2 (FIG. 18A) and run 1 against run 3 (FIG. 18B) indicate that no batch effects were observed. FIG. 18C is a plot of two additional TR-FRET assays on different sera than FIGS. 18A-18B, which also show a linear trend.
[0036] FIG. 19 shows that the neutralizing antibody test disclosed herein correlates with the cell-based Pseudovirion Neutralization Assay (PNA) neutralization assay up to a titer of IC50 of about 1,000. DETAILED DESCRIPTION
[0037] The inventors of the instant disclosure discovered an urgent and unmet need for a confirmatory diagnostic test that determines whether an allowed or approved vaccine for the treatmentof a disease caused by SARS-CoV-2 is effective to confer adaptive immunity against SARS-CoV-2 outside the clinical trial context. The inventors also discovered that existingtests for antibodies against SARS-CoV-2 fail to measure the functional inhibition (e.g., blocking) of binding between the spike protein and its cognate receptor (ACE2), which mediates infection by SARS-CoV-2. This means that existing tests are not able to determine whether a subject has sufficient adaptive immunity against SARS-CoV-2 induced by the vaccine following vaccine administration.
[0038] To address this urgent need, the inventors of the instant disclosure developed a neutralizing antibody test that measures the functional inhibition between the spike protein and ACE2, which can be used to determine whether a vaccine is effective to confer an adaptive immune response sufficient to protect a subject from a future infection. The neutralizing antibody tests described herein can also be used to measure the durability of a vaccine over time following a prior administration of the vaccine, which indicates whether a booster of the vaccine may be needed. The neutralizing antibody tests described herein can be used to screen a population of individuals. The neutralizing antibody tests described herein can be used to identify individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. The neutralizing antibody tests described herein can be used to minimize or reduce the risk associated with vaccine that has been commercialized for usage in a population (e.g., by identifying individuals that do not respond to the vaccine). The neutralizing antibody tests described herein can provide a surrogate marker of protection from a vaccine. The neutralizing antibody tests described herein can be used to classify vaccines by % efficacy in a population. With over 300 million people in the United States alone in need of vaccination, the neutralizing antibody tests described herein are both scalable and cost- efficient.
[0039] Patients recovered from COVID-19 cannot be assumed to be at low risk for rebound or re-infection, because many patients recovered from COVID-19 in several recent studies were found to never produce neutralizing antibodies against the spike protein. This is true even if the patient is given a vaccine against SARS-CoV-2. Thus, there exists a need for at testing device and systems for measuring sufficient neutralizing antibodies against SARS-CoV-2 in a patient sample conferring adaptive immunity in the patient. With over 300 million individuals in need of vaccination in the United States alone, these testing devices and systems must be scalable, cost-effective, and accessible at the point of need or point of care.
[0040] Existing SARS-CoV-2 tests marketed to aid in the diagnosis of COVID-19 at best measure exposure to SARS-CoV-2. Further, existing SARS-CoV-2 tests cannot predict severity of CO VID-19 in a subjectupon reinfection by SARS-CoV-2. For example, existing tests that detect a presence of antibodies against SARS-CoV-2 in a patient sample cannot discriminate neutralizing antibodies against SARS-CoV-2 and non-neutralizing antibodies. A “neutralizing antibody to SARS-CoV-2” asused herein, refers to an antibody that, when bound to the spike protein of SARS-CoV-2, prevents binding between the spike protein and its cognate receptor, ACE2. Further, existing SARS-CoV-2 tests that detect a presence of antibodies against SARS-CoV-2 lack specificity because they cannot assess the amount of cross reactivity between the antibodies in a patient sample and SARS-CoV-2 versus more benign (e.g., seasonal) coronaviruses. Although SARS-CoV-2 tests thatdetectbindingbetween an antibody in a patient sample and the spike protein exist, they do not necessarily indicate when a sufficient adaptive immune response to SARS-CoV-2 has occurred. Even direct antibody binding to the receptor binding domain of the spike protein does not always inhibit viral entry into a cell initiating an infection.
[0041] The gold standard test for assessing clinical immunity to a virus utilizes a cell-based assay. The cell-based assay quantitatively measures neutralizing antibody titers in patient sera by adding patient serum and a solution containing the virus to viral -susceptible cells, and analyzing the cells to determine if the virus can no longer infect the cells. In this cell-based assay, a reduction in viral induced cytotoxicity is a measure of neutralization activity in the sera. The strength of neutralization is reported in two ways: (i) the IC50 (e.g., half the cells are killed) or (ii) highest dilution at which neutralization activity disappears. This cell -based assay has limited clinical utility, because it is difficult to scale up, is cost and time intensive, and require technical training to perform. The inability to scale these cell-based assays means clinical utility for larger populations of individuals is limited. The time, cost, and technical expertise required means clinical utility of cell-based assay at the point of need is limited. Accordingly, there is an urgent need for a scalable assay that can also be used at the point of need to measure clinical immunity.
[0042] By contrast, the testing devices and systems described herein directly detect the functional inhibition of binding between the receptor binding domain of the spike protein and its cognate receptor (e.g., ACE2) by neutralizing antibodies in a patient sample. In this manner, the devices and systems described herein have increased specificity to neutralizing antibodies to SARS-CoV-2, because they can (i) discriminate neutralizing antibodies from nonneutralizing antibodies, and (ii) assess cross reactivity between antibodies in the patient sample and benign coronaviruses. The testing devices and systems described herein are designed to indicate when a sufficient adaptive immune response to SARS-CoV-2 has occurred, by detecting the functional inhibition of binding between ACE2 and the spike protein when in a presence of antibodies in a patient sample. Testing devices and systems described herein are portable, do not require technical expertise to use or perform, and are far less costly than the gold standard test (e.g., cell-based assay) for assessing clinical immunity. For at least these reasons, the devices and systems described herein are an ideal test at the point of need.
[0043] The testing devices and systems described herein are easily scalable, at least because the testing device (i) has an assay assembly that is simple (e.g., lateral flow, solution, or solid phase capture), (ii) does not require complex imaging devices, like f or e.g., BioTek® Cytation plate reader, and (iii) does not require use of a cell (or cell line). In some embodiments, the testing device is manufactured before the discovery of a new strain of virus. Upon discovery of the new strain, the assay assembly of the testing device is modified by replacing one component of the assay assembly (e.g., the recombinant RBD peptide unique to the new strain of SARS- CoV). In some embodiments, the new strain may comprise one or more mutations to SEQ ID NO: 5. In some embodiments, the new strain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1. In some embodiments, the assay assembly is configured for centralized and automated processing, enabling high throughput performance with significantly lower reagent amounts. Methods of utilizing the testing devices and systems described herein, in some cases, leverage robotics or other means of automation that can perform upwards of 60,000 tests per day using less than one-quarter of the reagents required for existing tests.
[0044] The high throughput systems and methods for analyzing a biological sample described herein may analyze more than one biological sample (e.g., hundreds or thousands of biological samples) in a single day. In some embodiments, the high throughput performance may include between about 5,000 to about 20,000 unique processed biological samples every 8 hours. In some embodiments, the high throughputperforman ce may be achievedby utilizing a biological sample medium (BSM) 1201, as shown in FIG. 12A-12B, which contains a biological sample obtained from a subject (e.g., a finger prick) and, in some embodiments, integrates the biological sample into an automated or semi-automated system for sample processing and assayingthe biological sample. In some embodiments, the BSMmay be a dried blood spot card (DBS). The automated or semi-automated systems described herein, automate sample processing, sample elution, sample preparation for assaying, and/or assaying the biological sample (e.g., without a need for an operator).
[0045] In some embodiments, as shown in FIG. 13, the automated or semi-automated systems 1300 described herein comprise a biological sample processor 1302 and an assay assembly block 1309. In some embodiments, the biological sample processor 1302 of the automated or semi-automated systems 1300 provided in FIG. 13 are configured to receive the biological sample, scan the biological sample (e.g., barcode on BSM) to register information regarding the origin of the biological sample (e.g., patient identifying information), separate a segment of the biological sample (e.g., a hole punch segment of the BSM containing the biological sample), place the segment of the biological sample into a vessel (e.g., a well), image the vessel to ensure that the segment of the biological sample is correctly placed into the vessel , extract the biological sample from the BSM, and/or purify the biological sample. In some embodiments, the assay assembly block 1309 of the automated or semi-automated systems 1300 provided in FIG. 13 are configured to dilute the optionally purified sample into serial dilutions (e.g., dilutions of 1 :20, 1 : 160, 1 :320, 1 :640 sample to solution), and/or conduct the assay (e.g., FRET, ELISA) disclosed herein on the serial dilutions, to yield the assay results 1312
[0046] In addition to directly detecting the functional inhibition of binding between the receptor binding domain of the spike protein and its cognate receptor (e.g., ACE2) by neutralizing antibodies in a patient sample, the testing devices described herein measure a presence or quantity of all antibodies(e.g., neutralizing or otherwise) againstthe pathogen (e.g, SARS-CoV-2) that are unique to a subject. In some embodiments, the testing devices differentiate between antibody classes, such as immunoglobulin G (IgG) and immunoglobulin M (IgM). In some embodiments, a presence or a quantity of IgG, IgM, or a combination thereof, in a patient sample is indicative of an acute infection. Thus, the testing device described herein can determine whether a subject is suffering from an acute infection by the pathogen (e.g, SARS-CoV-2), and whether the subject has developed a sufficient adaptive immunity to the pathogen such that they are immune from a future infection. The testing devices and systems described here are capable of adapting to rapidly evolving viral outbreaks in a manner that is considerably faster than existing technologies. In some embodiments, the testing devices test for more than one strain of a virus. For example, the testing devices and systems described herein are capable of identifying whether an individual is immune to human 2002-2003 and 2003-2004 SARS-CoV, with S proteins corresponding to GenBank accession no. AY274119 and GenBank accessionno. AY525636, respectively. The testing devices and systems, in some embodiments, utilize recombinant RBD peptides that each have a different epitope region unique to the strain of SARS-CoV. Proper epitope presentation is accomplishedby engineering the recombinant peptides to exhibit the same tertiary and quaternary structures as the corresponding pathogenic virus.
[0047] Provided herein are testing devices and systems for measuring adaptive immunity to a pathogen in a subject. In some embodiments, the testing devices are point of need or point of care devices. In some embodiments, the testing devices are configured to perform an assay on a biological sample of the subject to detect sufficient antibody titers to confer adaptive immunity to a pathogen of interest in the subject.
[0048] The biological sample includes cell, tissue, or bodily fluid obtained from the subject. Non-limiting examples of biological samples include aqueous humour, vitreous humour, bile, breast milk, cerebrospinal fluid, endolymph, perilymph, female ejaculate, amniotic fluid, gastric juice, menses, mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, urine, feces, whole blood, blood serum, blood plasma, sputum, cerebrospinal fluid, synovial fluid, lymphatic fluid, nasal swab, or cheek swab. In some embodiments, the biological sample comprises blood, urine, saliva, or feces. In some embodiments, the blood is capillary blood. In some embodiments, the capillary blood is obtained by a finger prick. In some embodiments, the capillary blood sample comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 drops of blood. In some embodiments, the capillary blood sample comprises a volume of blood less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μL. In a non-limiting example, the subject pricks a finger, obtains a drop of blood from the finger prick, and places the drop of blood on a blood card to be stored. The subject may do this 3-4 times for the same blood card. The subject mails the blood card to the laboratory for processing.
[0049] In some embodiments, the assay is a competition assay comprising one or more capture molecules, optionally coupled to a surface, and a detectable peptide-conjugate. In some embodiments, a signal from the detectable peptide-conjugate is detected by capturing an image of a detection zone of the device by an imaging device. In some embodiments, the imaging device is a smartphone. In some embodiments, the assay is a lateral flow assay (LFA). In some embodiments, the pathogen of interest is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
[0050] In some embodiments, the detectable peptide-conjugate comprises a peptide derived from a spike glycoprotein of a coronavirus, and the one or more capture molecules is derived from an angiotensin-converting enzyme 2 (ACE2) receptor, or a surrogate thereof (e.g., heparin or a fragment thereof). In some cases, the detectable peptide-conjugate comprises a peptide derived from the ACE2 receptor, and the one or more capture molecules is derived from the spike glycoprotein of a coronavirus. In some embodiments, the coronavirus is a severe acute respiratory syndrome coronavirus (SARS-CoV). In some embodiments, the SARS-CoV is SARS-CoV-2. In some embodiments, the ACE2 receptor is derived from a human ACE2 receptor. In some embodiments, more than one detectable peptide-conjugate is utilized to measure neutralizing antibodies against more than one strain of SARS-CoV.
[0051] Systems described herein comprise the testing device and the imaging device. In some embodiments, systems further comprise a computing device with an application (e.g., web or mobile) comprising a data analytics module for receiving an analyzing data from the imaging device to provide a result. In some embodiments, the imaging device is the computing device (e.g., smartphone). In some embodiments, the imaging device is notthe computing device. In some embodiments, the result is a positive result indicating that a subject is immune to an infection by the pathogen. In some embodiments, the result is a negative result indicating that the subject is not immune to an infection by the pathogen. In some embodiments, the application further comprises a communication module configured to display via a graphical user interface (GUI) one or more results to a user. In some embodiments, systems comprise a data store configured to store and retrieve data from the imaging device, the external device, or both.
[0052] In some embodiments, systems comprise an external device, such as a wearable tracking device (e.g., Aurora®, Fitbit ®, Apple Watch). In some embodiments, the data analytics module receives and analyzes external data from the external device. In some embodiments, external data comprise body temperature, heart rate, heart rate variability, sleep quality, or sleep quantify of the subject. In some embodiments, data analytics module is configured to analyze the external data in combination with the data received from the imaging device to identify the subject as being infected with a specific pathogen.
[0053] In some embodiments, systems described herein comprise multiple users with multiple computing devices. Within a geographical location of interest, systems described herein are designed to determine whether a population of users has become immune to an infection by the pathogen (e.g., SARS-CoV-2). In some instances, global position system (GPS) tracking of the personal computing device, the imaging devices, or both, can be used to produce a geofence surrounding the geographical location of interest.
TESTING DEVICES AND SYSTEMS
[0054] Disclosed herein, in some embodiments, are testing devices that can be deployed at the point of need to determine whether a patient is immune to an infection by the pathogen. In some embodiments, devices comprise an assay assembly capable of assaying a biological sample obtained from the patient. In some embodiments, the testing devices comprise one or more components, such as a housing, a sample receiver, a sample processor, a sample purifier, or a detection zone. In some embodiments, the testing systems described herein comprise one or more testing devices described herein and a biological sample medium (BSM) that stores the biological sample. In some embodiments, the one or more components of the testing devices or systems described herein elutes the biological sample from the BSM prior to assaying the sample using the assay assembly ofthe testing device or system.
Assay Assembly
[0055] Disclosed herein, in some embodiments are devices comprising an assay assembly that is capable of detecting a target analyte. In some embodiments, the target analyte is an antibody specific to a pathogen of interest. In some embodiments, the antibody specific to the pathogen of interest functionally blocks binding between the pathogen to its cognate receptor (e.g., a “neutralizing antibody”). In some embodiments, the neutralizing antibody is elicited in a subject by a vaccine or an infection by the pathogen. In some embodiments, the testing devices comprise two assay assemblies, the first assay assembly capable of detecting an antibody specific to a pathogen of interest, and the second assay assembly capable of detecting a neutralizing antibody that functionally blocks binding between the pathogen and its cognate (host) receptor. In some embodiments, the target analyte is a biomarker. It is contemplated that any combination of assay assemblies described herein may be used in combination, depending on the desired result. In some embodiments, the combination is housed in a single testing device. In some embodiments, the combination is housed in more than one testing device.
[0056] In some embodiments, the pathogen comprises a virus, a bacteria, a parasite, a fungus, or a combination thereof. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a severe acute respiratory syndrome coronavirus (SARS- CoV). In some embodiments, the SARS-CoV is SARS-CoV-2. In some embodiments, the coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV). In some embodiments, the coronavirus is an alpha coronavirus (e.g., 229E, NL63). In some embodiments, the coronavirus is a beta coronavirus (e.g., OC43, HKU1). In some embodiments, the virus is an influenza virus.
[0057] In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequences, or portions thereof, provided in SEQ ID NOS: 1-116. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO : 12, or a portion thereof. In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, 47-48, 71-72, 95-96, or 115, or a portion thereof. In some embodiments, the coronavirus comprises a viral genome comprising a sequence that is greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7-10, 13-20, 43, 67, or 91 , or a portion thereof.
[0058] In some embodiments, the pathogen ofinterest is a variant of the pathogen, for example, a pathogen that has evolved through mutations in the genes encoding the pathogen. In the case of a virus, the variant virus may have a mutation in an amino acid sequence of a viral protein, such as an envelope protein, nucleocapsid protein, membrane protein, hemaggluitinin, neuraminidase, or spike protein. In some embodiments, the virus (e.g., coronavirus) comprises a mutation in one or more of the sequences provided in SEQ ID NOS: 1-116. In some embodiments, the mutation does not affect, or minimally affects, host receptor binding by the spike protein of the coronavirus. In some embodiments, the mutation is an insertion, a deletion, or a substitution at an amino acid (or a plurality of amino acids) of one or more sequences provided in SEQ ID NOS: 1-116 for coronaviruses (e.g., SARS, SARS-CoV, and NL63). In some embodiments, the Non-limiting examples of mutations in an amino acid sequence of the spike protein of SARS-CoV-2 (SEQ ID NO: 5) are provided in Long et al., Molecular Architecture of Early Dissemination andMassive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area, Am. Soc. M. Bio., 11 :6 (November/December 2020) e02707-20, which is hereby incorporated by reference in its entirety. In some embodiments, the variant of SARS-CoV-2 comprises a spike protein having an amino acid sequence comprising a substitution of an aspartic acid (Asp) to a glycine (Gly) at amino acid position 614 (Asp614Gly) with reference to SEQ ID NO: 5 (SARS-CoV-2). In some embodiments, the variant of SARS- CoV-2 comprises a spike protein having an amino acid sequence comprising one or more substitution selected from Ala442Val, Ala448Val, Ala553Pro or Vai, Gly682Arg, Ser758Pro, and Cys812Phe, with reference to SEQ ID NO: 5 (SARS-CoV-2).
[0059] In some embodiments, the antibody specific to coronavirus is specific to the receptor binding domain of the spike protein of the coronavirus, such that the antibody blocks binding of the spike protein to its cognate receptor (e.g., ACE2). In some embodiments, the analyte is a complex comprising the spike protein bound to the antibody at the receptor binding region of the spike protein. In some embodiments, the antibody specific to the pathogen of interest belongs to an immunoglobulin class comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, or immunoglobulin D.
[0060] In some embodiments, the target analyte (or second target analyte) is a biomarker. In some embodiments, the assay assembly is configured to detect at least one, two, three, four, five, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1,000 or more biomarkers in the biological sample. In some embodiments, the biomarker comprises a non -peptide coding nucleic acid sequence. In some embodiments, the biomarker comprise a peptide coding nucleic acid sequence such as mRNA or cDNA. In some embodiments, the biomarker comprises a peptide or a protein.
[0061] In some embodiments, the biomarker is derived from the pathogen of interest (e.g., coronavirus). In some embodiments, the assay assembly is adapted to detect a region of the pathogen of interest. In some embodiments, the biomarker comprises orfla, orflab, spike protein (S protein), 3a, 3b, envelope protein (E protein), matrix protein (M protein), p6, 7a, 7b, 8b, 9b, nucleocapsid protein (N protein), orf 14, nspl (leader protein), nsp2, nsp3, nsp4, nsp5 (3C-like proteinase), nsp6, nsp7, nsp8, nsp9, nspl 0 (growth -factor-like protein), nspl2 (RNA- dependent RNA polymerase, or RdRp), nspl 3 (RNA 5 '-triphosphatase), nspl 4 (3'-to-5' exonuclease), nsp15 (endoRNAse), and nspl 6 (2'-O-ribose methyltransferase), a portion thereof, or combinations thereof.
[0062] In some embodiments, the biomarker is a proinflammatory marker. In some embodiments, the proinflammatory marker comprises 4-1BBL, acylation stimulating protein, adipokine, albinterferon, APRIL, Arh, BAFF, Bcl-6, CCL1, CCL1/TCA3, CCL11, CCL12/MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L/CD178, Cerberus (protein), chemokines, CLCF1, CNTF, colonystimulating factor, common b chain (CD 131), common g chain (CD 132), CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL2/MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCR3, CXCR4, CXCR5, EDA-A1, Epo, erythropoietin, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Flt-3L, FMS-like tyrosine kinase 3 ligand, Foxp3, GATA-3, GcMAF, G-CSF, GITRL, GM-CSF, granulocyte colony -stimulating factor, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5/IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFN-α, IFN-β, IFN-γ, IFNω/IFNW1, IL-1, IL-10, IL-10 family, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17A–F, IL-18, IL-18BP, IL-19, IL-1A, IL-1B, IL-1F10, IL-1F3/IL-1RA, IL- 1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1-like, IL-1RA, IL-1RL2, IL-1α, IL-1β, IL-2, IL-20, IL-21, IL-22, IL-23, IL-24, IL-28A, IL-28B, IL-29, IL-3, IL-31, IL-33, IL-35, IL-4, IL-5, IL- 6, IL-6-like, IL-7, IL-8/CXCL8, IL-9, inflammasome, interferome, interferon, interferon beta- 1a, interferon beta-1b, interferon gamma, interferon type I, interferon type II, interferon type III, interferons, interleukin, interleukin 1 receptor antagonist, Interleukin 8, IRF4, Leptin, leukemia inhibitory factor (LIF), leukocyte-promoting factor, LIGHT, LTA/TNFB, LT-β, lymphokine, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, macrophage colony- stimulating factor, macrophage inflammatory protein, macrophage-activating factor, M-CSF, MHC class III, miscellaneous hematopoietins, monokine, MSP, myokine, myonectin, nicotinamide phosphoribosyltransferase, oncostatin M (OSM), oprelvekin, OX40L, platelet factor 4, promegapoietin, RANKL, SCF, STAT3, STAT4, STAT6, stromal cell-derived factor 1, TALL-1, TBX21, TGF-α, TGF-β, TGF-β1, TGF-β2, TGF-β3, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF4, TNFSF8, TNF-α, TNF-β, Tpo, TRAIL, TRANCE, TWEAK, vascular endothelial growth inhibitor, XCL1, or XCL2. In some embodiments, the biomarkers includes GM-CSF, IL-1ɑ, IL-5, IL-7, IL-12/23 p40, IL-15, IL- 16, IL-17A, TNF-β, VEGF, INF-y, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p70, IL-13, TNF-α, Eotaxin, MIP-1β, Eotaxin-3, TARC, IP-10, MIP-1ɑ, IL-8, MCP-1, MDC, MCP-4, IFN-g, or VEGF-A, or a combination thereof. [0063] In some embodiments, the assay assembly comprises one or more capture molecules. Non-limiting examples of capture molecules include a nucleic acid molecule, peptide, protein, or fragments thereof. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. In some embodiments, the nucleic acid molecule or peptide is an aptamer that binds to an analyte described herein. In some embodiments, the peptide or protein is derived from the receptor binding domain (RBD) or the spike protein from a coronavirus. In some embodiments, the peptide or protein is derived from the ACE2 receptor surfaces for biomolecular detection (e.g., human ACE2). In some embodiments, the peptide or protein is an antibody or antigen-binding fragment that directly or indirectly binds the RBD of the spike protein or ACE2. In some embodiments, the capture molecule may include heparin, or a fragment thereof. [0064] In some embodiments, the one or more capture molecules is coupled to a solid surface. In some embodiments, the one or more capture molecules is not coupled to a solid surface. In some embodiments, the solid surface is made of silicon, glass (silicon dioxide), nitrocellulose, gold, silver, polystyrene, graphene, or crystal. In some embodiments, the solid surface is ahead or a plate. In some embodiments, the plate comprises reaction wells, such as in a 96-well plate, a 384-well plate, or a 1536-well plate. In some embodiments, the bead is sub stantially spherical in shape.
[0065] In some embodiments, the surface is passivated. In some embodiments, the surface comprises a polymer coating comprising a polymer selected from polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly glutamic acid (PGA), poly -lysine, polyglucoside, streptavidin, and dextran.
[0066] In some embodiments, the one or more capture molecules is coupled to the surface directly or indirectly. Capture molecules coupled indirectly to the surface may, for example, be coupled to the surface by a linker. In some embodiments, the linker is a chemical linker, a peptide link, a polymer linker, or a combination thereof. In some embodiments, an epitope tag is conjugated to the one or more capture molecules. Non -limiting examples of an epitope tag include MBP, His-tag, V5 tag, c-my, HA, S-tag, strep-tag, strep-MAB, VSV-G, GST, GFP, mCherry, CFP, BFP, and DYKDDDDK (SEQ ID NO: 116). In some embodiments, the one or more capture molecules is bound by a primary capture antibody that is bound to the surface covalently or non-covalently. In some embodiments, the one or more capture molecules is conjugated to an epitope tag and the epitope tag is coupled to a primary capture antibody that is bound to the surface. Capture molecules coupled directly to the surface may, for example, be covalently or non-covalently bound to the surface.
[0067] The one or more capture molecules described herein may be immobilized to the surface directly or indirectly using a suitable surface modification chemistry depending on the intended use. Non-limiting examples of suitable surface modification chemistries are provided in Sonawane el al., Surface Modification Chemistries ofMaterials used in Diagnostic Platforms with Biomolecules, Journal of Chemistry (2016), which is hereby incorporated by references in its entirety. [0068] In some embodiments, the one or more capture molecules is derived from an angiotensin-converting enzyme 2 (ACE2) receptor or a fragment thereof, or a surrogate of ACE2 (e.g., heparin) or a fragment thereof . In some embodiments, the fragment of ACE2 comprises an extracellular portion of ACE2 that binds to the spike protein. In some embodiments, the extracellular portion of ACE2 comprises the peptidase domain. In some embodiments, the fragment of ACE2 is a fragment of the extracellular portion of ACE2 that is sufficient for binding to the spike protein of a coronavirus, which are provided in Li et al., Structure of SARS coronavirus spike receptor -binding domain complexed with receptor. Science 309, 1864-1868 (2005); and Yan et al., Structural basis for the recognition ofSARS- CoV-2 by full-length human ACE2. Science. 2020 Mar 27; 367(6485): 1444-1448, which are incorporated by reference herein in their entireties. In some embodiments, ACE2 is human ACE2 (Entrez ID 59272). In some embodiments, the one or more capture molecules comprises a portion of the ACE2 polypeptide provided in SEQ ID NO: 1. In some embodiments, the one or more capture molecules comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the human ACE2 polypeptide (SEQ ID NO: 1). In some embodiments, the one or more capture molecules comprisesan amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence from amino acid 19 to amino acid 615 of SEQ ID NO: 1. In some embodiments, the one or more capture molecules is a peptide or protein comprising an amino acid sequence encoding a variant of the human ACE2 polypeptide (SEQ ID NO: 1), or a fragment thereof . In some embodiments, the one or more capture molecules comprises an amino acid sequence comprising a Serl9Pro or a Asp329Gly substitution with reference to SEQ ID NO: 1 Variants of ACE2 are provided in Hussain et al., Structural variants in human ACE2 may influence its binding with SARS-CoV-2 spike protein, J. Med. Virol. 2020; 92:1580- 1586, which is incorporated by reference herein in its entirety. In some embodiments, the one or more capture molecules comprises at least 2, 3 , 4, 5, 6, 7, 8, 9, or 10 capture molecules.
[0069] The one or more capture molecules, in some embodiments, is a surrogate for ACE2. A non-limiting example of a surrogate for ACE2 is heparin. In some embodiments, the one or more capture molecules comprises heparin, or a fragment thereof that is sufficient for binding to the spike protein. Heparin may competitively bind to the spike protein of a SARS-CoV with an affinity that is similar to that of ACE2. In some embodiments, the affinity of binding between heparin and the spike protein is less than about 1E-7, 1E-8, 1E-9, or 1E-10 Kd. In some cases, the binding affinity is from about 1E-9 to about 1E-10 Kd. In some embodiments, a surrogate of ACE2 (e.g., heparin) provided herein has a binding affinity to a spike protein of less than about 1E-7, 1E-8, 1E-9, 1E-10, or lE-11Kd In some embodiments, the affinity of binding between heparin (or other surrogate of ACE2) and the spike protein is 1.20E-8. In some embodiments, the affinity of binding between heparin (or other surrogate of ACE2) and the spike protein is 8.30E-10. In some embodiments, the one or more capture molecules is a surrogate f or ACE2 with a binding affinity or avidity for the spike protein of less than about 1E-7, 1E-8, 1E-9, 1E-10, or 1E-11 Kd. Suitable methods of identifying surrogates for ACE2 to comparable binding affinities to the spike protein are provided at least in Guan-Yu et al., The discovery of potential natural products for targeting SARS-CoV-2 spike protein by virtual screening, bioRxiv 2020.06.25.170639. Suitable methods of calculating the affinity of binding are provided in Vangone A, Bonvin A. PRODIGY : a contact-based predictor ofb in ding affinity in protein-protein complexes. Bio-Protocol. (2017) 7:2124.
[0070] In some embodiments, the one or more capture molecules is derived from a spike glycoprotein of a coronavirus. In some embodiments, the coronavirus is a severe acute respiratory syndrome coronavirus (SARS-CoV). In some embodiments, the SARS-CoV is SARS-CoV-2. In some embodiments, the coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV). In some embodiments, the coronavirus is an alpha coronavirus (e.g., 229E, NL63). In some embodiments, the coronavirus is a beta coronavirus (e.g., OC43, HKU1). In some embodiments, the one or more capture molecules comprises a portion of the spike protein. In some embodiments, the portion comprises subunit 1 of the spike protein. In some embodiments, the portion comprises the receptor binding domain (RBD) of subunit 1 of the spike protein. In some embodiments, the one or more capture molecules comprises an amino acid sequence atleast70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the spike protein, the subunit 1 of the spike protein, or the RBD of the subunit 1 of the spike protein, or a combination thereof. In some embodiments, the one ormore capture molecules comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 capture molecules.
[0071] In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4
[0072] In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1. In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6
[0073] In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the one or more capture molecules comprises a peptide or protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24, 47-48, 71-72, 95-96, or 115
[0074] In some embodiments, the one or more capture molecules comprises a peptide or a protein having an amino acid sequence atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 50 amino acids of SEQ ID NO: 1-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. In some embodiments, the one or more capture molecules comprises a peptide or a protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 100 amino acids of SEQ ID NO: 1-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. In some embodiments, the one or more capture molecules comprises a peptide or a protein having an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least 150 amino acids of SEQ ID NO: 1-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0075] In some embodiments, the one or more capture molecules comprises an amino acid sequence comprising asparagine at amino acid positions 90, 322, 546, and 343 of the amino acid sequence of the spike protein, such as SEQ ID NO: 2, 5, or 11. In some embodiments, a substitution of an amino acid residue corresponding to an amino acid position selected from 344, 360, 472, 479, 480, and 487 in SEQ ID NO: 2, 5, or 11. In some embodiments, the spike protein has an amino acid sequence comprising one ormore of 344K,360F, 472L, 479N, 480D, and 487T with reference to SEQ ID NO: 2, 5, or 11 In some embodiments, the spike protein has an amino acid sequence comprising one or more of 344R, 360S, 472P, 479 N, 480G, and 487S with reference to SEQ ID NO: 2, 5, or 11 In some embodiments, the spike protein has an amino acid sequence comprising 344R, 360S, 472L, 479K, 480D, 487S with reference to SEQ ID NO: 2, 5, or 11
[0076] In some embodiments, the spike protein is derived from a mammal. In some embodiments, the spike protein is derived from a human. In some embodiments, the spike protein is derived from a non -human mammal, such as a palm civet or a bat.
[0077] In some embodiments, the one or more capture molecules is an antibody, or antibody fragment, specificto one or more antibodies against the pathogen described herein (e.g., IgM, IgG, IgA, IgE, IgD). In some embodiments, the one ormore capture molecules is a monoclonal antibody or a polyclonal antibody. In some embodiments, the one or more capture molecules comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 capture molecules.
[0078] In some embodiments, the one ormore capture molecules is a fusion protein comprising a peptide directly or indirectly bound to the surface. In some embodiments, the peptide comprises a fragment crystallizable (Fc) region of a monoclonal antibody. In some embodiments, the Fc region is derived from an antibody belongingto an immunoglobulin class comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, or immunoglobulin D.
[0079] Referring to FIG. 1, a biological sample obtained from a subject that has not been exposed to a coronavirus is assayed using the assay assembly described herein. In some embodiments, a labeled capture molecule (“CoV-2 S-Au”) comprises a peptide-conjugate comprising a detection agent (e.g., gold nanoparticle) and a peptide derived from the spike protein of SARS-CoV-2. A fluid formulation comprising the peptide-conjugate is contacted with a biological sample from the subject. In this example, the target analyte is an activity of one or more antibodies comprising blocking the binding between the spike protein of SARS- CoV-2 and ACE2. In this example the patient has not been exposed to the coronavirus, so a presence of the analyte is not expected to be detected.
[0080] In some embodiments, an immobilized capture molecule is coupled to a solid surface. In some embodiments, the immobilized capture molecule is human ACE2 receptor, surrogate thereof (e.g., heparin), or a fragment thereof. The liquid formulation is applied to the solid surface. A high number of complexes between the immobilized human ACE2 and the detectable peptide-conjugate is detected, correlating with an absence or a low amount of analyte in the sample. In some embodiments, an image of the surface is captured with an imaging device. In some embodiments, the image is a video or still image. In some embodiments, the imaging device comprises a reflectance reader. In some embodiments, the imaging device is a personal electronic device, such as a smartphone. When imaged from above the surface, a low signal indicates a high degree of binding between human ACE2 and the peptide-conjugate (therefore, a low amount of the analyte). When imaged from below the surface, a high signal indicates a high degree of binding between the human ACE2 and the peptide-conjugate.
[0081] In contrast, a biological sample obtained from a subject that was exposed to a coronavirus assayed usingthe assay assembly described herein, is provided in FIG. 2. A low number of complexes between the immobilized human ACE2 and the detectable peptide- conjugate is detected, which correlates with a presence absence or a high amount of analyte in the sample.
[0082] In some embodiments, the surface of the image is not captured. Referring to FIG. 7, a biological sample obtained fromthe subject exposed to a coronavirus is assayed usingthe assay assembly described here. A low number of complexes between the immobilized human ACE2 and the detectable peptide-conjugate is detectable by the human eye in the detection zone of the device.
[0083] In some embodiments, the assay assembly is adapted for nucleic acid molecule detection, such as to detect a nucleic acid molecule encoding a biomarker or an antibody described herein. In a non-limiting example, the assay assembly is adapted to detect RNA (or cDNA) encoding a region of the pathogen of interest or a marker of an adaptive immune response, such as a cytokine.
[0084] In some embodiments, the assay assembly performs reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, the assay assembly is a singleplex (e.g., individual assays) or multiplexed (e.g., detection of more than one biomarker in a single reaction). The assay assembly comprises a primer pair and a probe set to amplify and detect the biomarker. In some embodiments, the assay assembly comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 primer pairs. In some embodiments, the assay assembly comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 probes. A “probe” is a nucleic acid molecule comprising greater than or equal to about 30 contiguous nucleobases adapted to hybridize to the nucleic acid sequence of the target region of the biomarker. In some embodiments, the RT -PCRis qualitative PCR (qPCR), and the probe comprises a detectable moiety. In some embodiments, the detectable moiety comprises TaqMan™, SYBR green, SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin, or a combination thereof. In some embodiments, theprobemay be a hydroly sableprobe comprising a fluorophore and quencher that is hydrolyzedby DNA polymerase when hybridizedto a target nucleic acid, such as for a TaqMan™ assay assembly.
[0085] In some embodiments, the assay assembly is an array comprising probes conjugated or otherwise immobilized on a surface described herein (e.g., a bead, multi-well plate), wherein the probes are configured to hybridize with the nucleic acid sequence of the target region. In some embodiments, the target region of the biomarker comprises a mutation, such as a single nucleotide variation or an indel. In some embodiments, the surface comprises an Affymetrix gene chip array, and the like.
[0086] In some embodiments, the assays provided herein have an accuracy of at least 80%, for example, atleast 85%, atleast 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, atleast 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater. In some embodiments, the assays provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the assays provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, atleast 90%, atleast 91%, atleast 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the assays provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%. [0087] In some embodiments, the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the ref erence neutralizing antib ody has a known IC50; and (b) detecting a binding complex b etween the detectable peptide and the one or more capture molecules; and (c) determine an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50.
[0088] In some embodiments, accuracy is measured by comparingthe positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population. For example, in a population of human subjects vaccinated against a coronavirus, SARS-CoV-2, accuracy wouldbe measured by comparingthe true positives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) and true negatives (e.g., subjects that were exposed to the SARS-CoV-2 and did become infected).
Fluorescence Resonance Energy Transfer (FRET) Assay
[0089] In some embodiments, the assay assembly is a solution -based assay and does not consist of a solid surface. In some embodiments, the solution-based assay comprises labeling any one of the capture molecules described herein by conjugating the capture molecule to a detectable moiety of any one of the moietiesdescribed herein. In some embodiments, the signal is detected when the capture molecule forms a complex between the capture molecule and the target analyte. In some embodiments, the signal is not detected when the capture molecule forms a complex between the capture molecule and the target analyte. Exemplary solution-based assay comprising the labeled capture molecules includes Forster resonance energy transfer (FRET), fluorescence polarization (FP), radioligand binding assay, bioluminescent binding assay, and immunoprecipitation (e. g. ELISA and western blotting). In some embodiments, the solutionbased assay comprises unlabeled capture molecules, where the detection of the complex formed between the capture molecule and the target analyte is detected by a separate mechanism. Exemplary solution-based assay comprising unlabeled capture molecules includes surface plasmon resonance (SPR), plasmon -waveguide resonance, whispering gallery microresonator (WGM), resonant waveguide grating (RWG), mass spectrometry, nuclear magnetic resonance, X-ray crystallography, thermal denaturation assays (TDA), and isothermal titration calorimetry (ITC).
[0090] In some embodiments, the assay assembly is a (FRET) system. In some embodiments, the FRET system comprises coupling the FRET donor and FRET acceptor to any one of the capture molecules described herein. In some embodiments, the FRET donor and the FRET acceptor are fluorescent or luminescent molecules. In some embodiments, the luminescent molecule is a luminescent metal, such as lanthanide (e.g., terbium or europium). In some embodiments, the fluorescent molecule comprises an organic fluorescent dye, a fluorescent protein, semiconductor quantum dots, metal chelates, various noble metal and other nanoparticles, intrinsically fluorescent amino acids, biological cofactors, and polymers . In some embodiments, the FRET is lanthanide -based FRET (LRET). In some embodiments, a first peptide or protein derived from an antigenic component of a pathogen disclosed herein (e.g. coronavirus) is coupled to a first detectable moiety, such as the FRET donor or FRET acceptor. In some embodiments, the second peptide or protein derived from a cognate receptor of the pathogen (e.g., ACE2 receptor) is coupled to a second detectable moiety, such as a FRET acceptor or FRET donor. In some embodiments, the FRET donor and FRET acceptor emit detectable signals that differ from one another, such that when the FRET donor and the FRET acceptor are in close proximity, they emit a combined signal that differs from either of them alone. In some instances performing the FRET assay comprises mixing a biological sample with the first peptide or protein (coupled to the FRET donor) and the second peptide or protein (coupled to the FRET acceptor) under conditions sufficient for the first peptide or protein and the second peptide or protein to form a binding complex in absence of neutralizing antibodies in the biological sample, and measuring a ratio of the signal emitted from the binding complex to the signal emitted from either of the FRET acceptor or the FRET donor alone. The oppositive configuration of this FRET assay is al so possible, wherein the first peptide or protein is coupled to the FRET acceptor and the second peptide or protein is coupled to the FRET donor. When there is a presence of neutralizing antibodies in the biological sample, there will be fewer binding complexes formed between the first peptide or protein and the second peptide or protein, so the ratio of the signal for either of the FRET acceptor of the FRET donor will be larger in relation to the signal emitted from the binding complex.
[0091] In some embodiments, the antigenic component of the pathogen is a portion of the spike protein in a coronavirus, such as SARS-CoV-2, and the cognate receptor is ACE2, such as human ACE2. As a non -limiting example, FIG. 9A-9D illustrates how FRET is utilized in solution to identify a presence or a quantity of neutralizing antibodies that functionally block binding between the RBD of the spike protein derived from SARS-CoV and the cognate receptor, ACE2. FRET relies on the excitation energy of a donor fluorescent or luminescent molecule to a nearby acceptor fluorescent or luminescent molecule. In some embodiments, the firstpeptide or protein (e.g., RBD peptide, or portion thereof) is coupled to a donor fluorophore, and the second peptide or protein (e.g., ACE2, or portion thereof) is coupled to an acceptor fluorophore. In some embodiments, the donor or the acceptor fluorophore may be any suitable fluorescent protein. In some embodiments, the fluorescent proteins comprise a color selected from, blue, cyan, green, and yellow.
[0092] Referring to FIG. 9A, when a biological sample that is obtained from an individual that does not have neutralizing antibodies against the RBD, excitation of the acceptor fluorophore is observed. Whereas, no excitation of the acceptor fluorophore is observed if the biological sample contains neutralizing antibodies against RBD, as shown in FIG. 9B.
[0093] Operatingin a similar manner to FIG. 8A-8B, FIG. 9C-9D provides an assay assembly in solution (e.g., not involving a solid surface) using FRET.
[0094] The FRET assay may be performed in a vessel, such as a container or a test tube. In some embodiments, the FRET assay is performed in an assay plate, such as, a 96 -well plate, a 384-well plate, or a 1536-well plate. In some embodiments, the FRET assay is performed in a high-throughput method to decrease the amount of reagents and the cost of performing the assay. In some embodiments, the FRET assay utilizes small volumes of reagents, for example, less than 100 nL, less than 90 nL, less than 80 nL, less than 70 nL, less than 60 nL, or less than 50 nL of reagents.
[0095] In some embodiments, the biological sample (e.g., blood from a finger prick) is stored in or on a biological sample medium (BSM), such as a dried blood spot card (DBS), described herein. The biological sample is removed from the BSM and placed into the vessel (e.g., a well of a 96-well plate) to be eluted from the BSM. Next, the eluted biological sample is assayed using the FRET system deposited on a solid surface described herein.
[0096] In some embodiments, the FRET assay confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’ s biological sample. In some instances, the FRET assay prognoses a subject’s immunity to any one of the pathogen described herein. In some embodiments, the FRET assay prognoses a subject’s immunity to coronavirus. In some embodiments, the FRET assay prognoses a subject’s immunity to SARS-CoV-2. In some embodiments, the FRET assay measuresthe subject’s susceptibility to an infection by any one of the pathogens described herein. In some embodiments, the FRET assay measures the subject’s susceptibility to an infection by coronavirus. In some embodiments, the FRET assay measures the subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the FRET assay identifies individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the FRET assay minimizes or reduces the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the FRET assay identifies neutralizing antibodies as a surrogate marker of protection from a vaccine (e.g., in place of traditional, randomized, placebo-controlled trials). In some embodiments, the FRET assay classifies vaccines by % efficacy in a population.
[0097] In some embodiments, the FRET assays provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the FRET assays provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, atleast 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the FRET assays provided herein have a specificity of atleast 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the FRET assays provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%. In some embodiments, the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the ref erence neutralizing antib ody has a known IC50; and (b) detecting a binding complex b etween the detectable peptide and the one or more capture molecules; and (c) determine an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50. In some embodiments, accuracy is measured by comparing the positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population. For example, in a population of human subjects vaccinated against a coronavirus, SARS-CoV-2, accuracy would be measured by comparingthe true positives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) andtrue negatives (e.g., subjects th at were exposed to the SARS-CoV-2 and did become infected).
[0098] In some embodiments, the FRET assay is performedin solution, such that neither of the first peptide or protein or the second peptide or protein are coupled to a solid phase. In such cases, the FRET assay may be performed in a container. In some embodiments, the container is optically clear. In some embodiments, the container is a cartridge. In some embodiments, the cartridge is disposable, such as a single-use disposable cartridge. In some embodiments, the cartridge is made of a material comprising a plastic, Polyethylene Terephthalate (PET or PETE), High-Density Polyethylene (HDPE), Polyvinyl Chloride(PVC or Vinyl), Low -Density Polyethylene (LDPE), Polypropylene (PP), or Polystyrene (PS or Styrofoam), or any combination thereof.
[0099] In some embodiments, the FRET assay is performed under conditions sufficient for either the first peptide or protein or the second peptide or protein are coupled to a surface of a solid-phase. For example, if the first peptide or protein is coupled to the surface, it will capture (or bind) to the second peptide or protein in solution under otherwise sufficient conditions for their binding when there is an absence of neutralizing antibodies in the solution. Whereas the firstpeptide or protein coupledto the surface will capture fewer ofthe secondpeptideor protein in the solution in the presence of neutralizing antibodies in the solution. The opposite configuration is also possible, where the second peptide or protein is coupled to the surface, which captures the first peptide or protein in solution. In some embodiments, the first peptide or protein or the second peptide or protein are coupledto a surface of a solid -phase directly or indirectly by way of a linker or primary capture molecule (e.g., antibody, antigen -binding fragment) specific to the first peptide or protein or the second peptide or protein. In some embodiments, the surface is an interior surface of a capillary, such as a capillary of a capillary flow cell or other micro fluidic device. In some embodiments the interior of the flow cell is passivated. In some embodiments, the passivated surface comprises a polymer layer coupled thereto comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (acrylic acid) (PAA), polyacrylamide, poly(N -isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethyleneglycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran. Such passivation will reduce nonspecific binding to the surface, and increase signal to noise. In some embodiments, the surface is a waveguide, such as a planar waveguide. In some embodiments the surface compri ses a particle, such as ahead or nanoparticle. In some embodiments, the particle is made of a material comprising metal, agarose, acrylic, or plastic, or any combination thereof. In some embodiments, the particle is magnetic. [0100] In some embodiments, whether the FRET assay is performed in solution or solid-phase, the FRET assay does not comprise a wash step to wash away the first peptide or protein or the second peptide or protein that are unbound. In some embodiments, the signal to background noise is resolved using a software tool kit described herein such as FRET-Image Background- subtracted Ratiometric Analysis (IBRA). In some embodiments, signal to background noise is resolved using FRET -IBRA using the methodologies disclosed in Munglani, Gautam et al. “Fast and flexible processing of large FRET image stacks usingthe FRET -IBRA toolkit.” PLoS computational biology vol. 18,4 e1009242. 4 Apr. 2022, doi:10. 1371/journal.pcbi.1009242, which is incorporated by reference in its entirety.
[0101] In some embodiments, the signal from the binding complex (between the first peptide or protein and the second peptide or protein) and the signals from the first peptide or protein and the second peptide or protein that are unbound are detected with an optical sensor in optical communication therewith. In some embodiments, the optical sensor is a planar waveguide optical sensor. In some embodiments, the optical sensor comprises a photomultiplier tube (PMT) detector, a charge-coupled device (CCD) detector, or complementary metal-oxide semiconductor (CMOS) detector.
[0102] In some embodiments, methods further comprise calculating the percent neutralization in the biological sample from the ratio of the signals detected from the FRET assay. In some embodiments, the percentneutralization is calculated with a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is the signal corresponding to the binding complex, SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization ofbindingbetween the first peptide or protein and the second peptide or protein. In some embodiments, percent neutralization of about 62% correlates to about 400 reciprocal titers when measured using a cell-based pseudovirion neutralization assay (PNA). In some embodiments, a percent neutralization of 0% correlates with 0 antibody titers using the cell-based PNA. a percent neutralization of 100% correlates with above 800 antibody titers usingthe cell -based PNA, or any combination thereof. A non-limiting example of a PNA is provided in Example 23.
Lateral Flow Assay
[0103] In some embodiments, the assay assembly is a lateral flow assay (LFA). In some embodiments, the surface is a membrane comprising porous paper, a polymer structure, a sintered polymer, or a combination thereof. In some embodiments, the LFA assembly has one or more zones situated laterally, including a detectable zone. The detectable zone comprise at least a control region and a test region.
[0104] In some embodiments, the LFA assembly further comprises a sample receptor (e.g., a sample pad) configured to receive a biological sample. In some embodiments, the sample receptor comprises a filter designed to separate a component of the biological sample to be tested. For instance, if a biological sample were blood, the sample component would be blood serum. In some embodiments, the LFA assembly further comprises an absorbent pad.
[0105] In some instances, the target analyte moves without the assistance of external forces, e.g., by capillary action. In some instances, the target analyte moves with assistance of external forces, e.g., by facilitation of capillary action by movement of the lateral flow assembly (e.g, shaking, turning, centrifuging, applying an electrical field or magnetic field, applying a pump, applying a vacuum, or rocking).
[0106] Any suitable lateral flow test strip detection format known to those of skill in the art is contemplated for use in an assay assembly of the present disclosure. Lateral flow test strip detection formats are well known and have been described in the literature. Lateral flow test strip assay formats are generally described by, e.g., Sharma et al., (2015) Biosensors 5:577- 601, incorporated by reference herein in its entirety. Detection of nucleic acids using lateral flow test strip sandwich assay formats is described by, e.g., U.S. Pat. No. 9, 121 ,849, “Lateral Flow Assays,” incorporated by reference herein in its entirety. Detection of nucleic acids using lateral flow test strip competitive assay formats is described by, e.g., U.S. Pat. No. 9,423,399, “Lateral Flow Assays for Tagged Analytes,” incorporated by reference herein in its entirety.
[0107] Disclosed herein, in some embodiments, are signal detection devices, such as an imaging device. In some embodiments, the imagingdevice is a personal electronic device (e.g, smartphone or tablet). In some embodiments, the imaging device comprises a fluorescence reader, a colorimeter, or a sensor.
[0108] In some embodiments, the peptide -conjugate described herein comprises detection reagent or a label. Non-limiting examples of a detection reagent include a fluorophore, a chemical, a nanoparticle, an antibody, a peptide, and a nucleic acid probe. In some embodiments, the nanoparticle comprises a material selected from agarose, plastic, acrylic, or metal. In some embodiments, the nanoparticle is a microsphere. In some embodiments, the nanoparticle is magnetic. In some embodiments, the imaging device detects color, reflectance, fluorescence, bioluminescence, chemiluminescence, light, or an electrical signal. [0109] In some embodiments, the LFA assembly is in a sandwich format, a competitive format In a sandwich assay format, the detected signal is directly proportional to the amount of the target analyte present in the sample, so that increasing amounts of the target analyte lead to increasing signal intensity. In a competitive assay format, the detected signal has an inverse relationship with the amount of analyte present, and increasing amounts of analyte lead to decreasing signal intensity.
[0110] In a lateral flow sandwich format, also referred to as a “sandwich assay,” the biological sample (test sample) is applied to a sample receptor (“sample pad”) at a distal end of the LFA test strip. The biological sample flows through the test strip, from the sample pad to a conjugate pad located adjacent to, and downstream from, the sample pad. In some embodiments, the conjugate pad comprises a labeled capture molecule, e.g., an antibody or aptamer labeled with e.g., a dye, enzyme, or nanoparticle. A complex between the capture molecule and the target analyte is formed if the target analyte is present in the test sample. This complex then flows to a first test zone or sector (e.g., a test line) comprising an immobilized second capture molecule which is specific to the target analyte, thereby trapping any labeled capture molecule -target analyte complexes. In some embodiments, the intensity or magnitude of signal, e.g., color, fluorescence, reflectance, at the first test zone or sector is used to indicate the presence or absence, quantity, or presence and quantity of target analyte in the test sample. In some embodiments, the assay assembly comprises a second test zone or sector comprising a third capture molecule that binds to excess labeled capture molecule. If the applied test sample comprises the target analyte, little or no excess labeled capture molecule will be present on the test strip following capture of the target analyte by the labeled capture molecule on the conjugate pad. Consequently, the second test zone or sector will not bind any labeled capture molecule, and little or no signal (e.g., color, fluorescence, reflectance) at the second test zone or sector is expected to be observed. The absence of signal at the second test zone or sector thus provides assurance that signal ob servedin the firsttest zone or sector is due to the pres ence of the target analyte.
[0111] In some embodiments, the sandwich assay is configured to receive a biological sample disclosed herein and retain sample components (e.g., target analyte). In some embodiments, the sandwich assay is configured to receive a flow solution that flushes unwanted cellular components (other than the analyte) of the biological sample, leavingthe target analyte behind. In some embodiments, the sandwich assay comprises a membrane that binds the target analyte to help retain the target analyte when the flow solution is applied. Non-limiting examples of a membrane that binds a target analyte includes chitosan modified nitrocellulose.
[0112] In some embodiments, the assay assembly comprises a sandwich assay. In some embodiments, the target analyte is an antibody specific to a pathogen of interest (e.g., SARS- CoV-2). In some cases, capture molecule is a peptide derived from a spike protein from a coronavirus (e.g., SARS-CoV-2). In some embodiments, immobilized capture molecule is an antibody specific to the target analyte, such as an antibody or antigen -binding fragment. In some embodiments, the capture molecule is labeled, and a signal correspondingto the presence of the target analyte is visualized. In some embodiments, a labeled secondary molecule (e.g, antibody or antibody fragment) specific to the capture molecule added, and a signal correspondingto the presence of a complex between the target analyte, the capture molecule, and the secondary molecule is visualized. In some embodiments, signal is observed at the first test zone comprising color, fluorescence, or reflectance emitted from the labeled capture molecule. In some embodiments, the labeled capture molecule is a peptide -conjugate comprising a peptide derived from a spike protein from a coronavirus, and a label comprising a nanoparticle (e.g., microsphere), an enzymatic label, or a fluorescent dye.
[0113] In some embodiments, the assay assembly detects more than one target analyte. In some embodiments, the assay assembly provides two or more capture molecules that are specific to two or more antibodies specific to a pathogen of interest, such as, for example IgG, IgM, or IgA antibodies against SARS-CoV-2. In some embodiments, the two or more capture molecules are labeled. In some embodiments, the two or more capture molecules are not labeled. In some embodiments, two or more labeled secondary molecules specific to the two or more capture molecules are added to the assay assembly, and visualized as described herein. [0114] In a lateral flow competitive format, also referred to as a “competitive assay,” the test sample is applied to a sample pad at one end of the test strip, and the target analyte binds to a labeled capture molecule to form a complex between the target analyte and the labeled capture molecule in a conjugate pad downstream of the sample application pad. In the competitive format, the first test zone comprises an immobilized capture molecule specific to second capture molecule that is labeled. In the absence of the target analyte, the immobilized capture molecule and the labeled second capture molecule form a detectable complex. In the presence of the target analyte, fewer complexes between the capture molecule and the labeled second capture molecule form due to competition for binding to the labeled second capture molecule. In some embodiments, the intensity or magnitude of signal, e.g., color, fluorescence, reflectance, at the first test zone or sector is inversely proportionate to the presence or absence, quantity, or presence and quantity of the target analyte in the test sample.
[0115] In some embodiments, the assay assembly comprises a competitive assay. Referring to FIG. 3, the labeled capture molecule comprises a peptide-conjugate comprising a detection agent (e.g., gold nanoparticle) and a peptide derived from the spike protein of a coronavirus. A fluid formulation comprising the peptide-conjugate is contacted with a biological sample from a patient and human ACE2, wherein the peptide derived from the spike protein of the coronavirus comprises a receptor binding domain (RBD) specific to the human ACE2. In this example, the target analyte is one or more antibodies against the spike protein of the coronavirus. In this example the patient has not been exposed to the coronavirus, so a presence of the analyte is not expected to be detected.
[0116] The liquid formulation comprises at least one detectable complex comprising the peptide-conjugate RBD and the human ACE2, because the biological sample doesnot consist of antibodies against the RBD of the peptide. The liquid formulation comprisingthe peptide- conjugate, the biological sample, and the human ACE2 is applied to a solid surface at least partially enclosed in a housing (“test cartridge”). The liquid formulation is applied to the distal end of the solid surface at a sample pad, and flows unidirectionally over the solid surface towards the opposite distal end of the solid surface. In some embodiments, the biological sample is blood, and the sample pad separates serum from blood, permitting only the serum to flow across the solid surface. In some embodiments, the solid surface is a nitrocellulose membrane. In some embodiments, the first test zone comprises an immobilized antibody specific to the human ACE2. In some embodiments, the second test zone comprises an immobilized antibody specific to the RBD of the spike protein. In some embodiments, a high signal is observed at the first test zone, because there is an absence of target analyte in the biological sample. In some embodiments, a high signal is observed at the second test zone (positive control), for the same reason.
[0117] In alternative embodiments, the immobilized capture molecule in the first test zone is human ACE2 receptor, and the fluid formulation does not contain human ACE2. In this example, the liquid formulation comprises the peptide-conjugate and the biological sample. No complexes form between the peptide-conjugate in the absence of the target analyte (antibodies against the RBD of the spike protein). The liquid formulation is applied to the distal end of the solid surface at a sample pad, and flows unidirectionally over the solid surface towards the opposite distal end of the solid surface. Like above, in the first and second test zones, a high signal is observed in the absence of the test analyte.
[0118] Referring to FIG. 4, the same competition assay is performed on biological sample obtained from a subject that has been exposed to the coronavirus. In some embodiments, there is a presence of the target analyte in the biological sample. Therefore, a low signal is observed at the first and second test zones, which indicates a high amount of competitive binding between the target analyte and the peptide-conjugate.
[0119] Referring to FIG. 8A-8D, the assay assembly measures analyte in the biological sample. In some embodiments, the assay assembly measures one analyte (e.g. an neutralizing antibody) in the biological sample. Referring to FIG. 8A-8B, one capture molecule is used, for example RBD. FIG. 8A exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that does not have neutralizing antibodies against RBS. The peptide-conjugate (e.g., ACE2) conjugated to a detectable moiety binds to the RBD in the absence of neutralizing antibodies. The detectable moiety is visualized usingthe naked eye, or an imaging device described herein. FIG. 8B exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that has neutralizing antibody against RBD. The peptide-conjugate (e.g., ACE2) conjugated to a detectable moiety does not bind to the RBD in the presence of neutralizing antibody in the patient sample.
[0120] In some embodiments, the assay assembly measures more than one analyte (e.g., neutralizing antibody) in the biological sample. Referring to FIG. 8C-8D, more than one capture molecule is used, for example RBD1 and RBD2. In some embodiments, two or more capture molecules are used. In some embodiments, the two or more capture molecules comprise a first capture molecule and a second capture molecule, wherein the first capture molecule comprises a first variant RBD and the second capture molecules comprises a second variant RBD. In some embodiments, at least 3, 4, 5, 6, 7, 8, 9, or 10 capture molecules are used. In some embodiments, one of the capture molecules are the same. FIG. 8C exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that does nothave neutralizing antibodies against either ofRBDl andRBD2. The peptide -conjugate (e.g., ACE2) conjugated to a detectable moiety binds to the RBD1 and RBD2 in the absence of neutralizing antibodies. The detectable moiety is visualized using the naked eye, or an imaging device described herein. FIG. 8D exemplifies an assay assembly after it is performed with a biological sample that is obtained from an individual that has neutralizing antibodies against both RBD1 and RBD2. The peptide-conjugate (e.g., ACE2) conjugated to a detectable moiety does not bind to the RBDl and RBD2 in the presence of neutralizing antibodies in the patient sample.
[0121] In a lateral flow test strip detection format, more than one target analyte is detected using the test strip through the use of additional test zones or sectors comprising, e.g., probes specific for each of the target analytes.
[0122] In some instances, the lateral flow device is a layered lateral flow device, comprising zones or sectors that are present in layers situated medially, e.g., above or below each other. In some instances, one or more zones or sectors are present in a given layer. In some instances, each zone or sector is present in an individual layer. In some instances, a layer comprises multiple zones or sectors. In some instances, the layers are laminated. In a layered lateral flow device, processes controlled by diffusion and directed by the concentration gradient are possible driving forces. For example, multilayer analytical elements for fluorometric assay or fluorometric quantitative analysis of an analyte contained in a sample liquid are described in EP0097952, “Multilayer analytical element,” incorporated by reference herein.
[0123] A lateral flow device comprises one or more functional zones or sectors. In some embodiments, the test assembly comprises 1 to 20 functional zones or sectors. In some instances, the functional zones or sectors comprise at least one sample purification zone or sector, at least one target analyte amplification zone or sector, at least one target analyte detection zone or sector, and at least one target analyte detection zone or sector.
[0124] In some embodiments, the assay assembly comprises a detection zone made up of at least the first and the second test zones (test, and control). In some embodiments, the assay assembly comprises more than one test zone. In some embodiments, the assay assembly comprises a firsttestzone and a second test zone, whereinthe firsttestzone indicatesa presence or a quantity of neutralizing antibodies that competitively bind to the peptide-conjugate; and the second test zone indicates a presence or a quantity of antibodies against the pathogen (neutralizing or otherwise). In some embodiments, the assay assembly comprises a third test zone. In some embodiments, the second test zone detects IgG antibodies against the pathogen. In some embodiments, the third test zone detects IgM antibodies against the pathogen. In some embodiments, the assay assembly comprises a plurality of test zones, wherein each test zone of the plurality measures a different analyte. In some embodiments, an image of the detection zone is captured with an imaging device. In some embodiments, the image is a video or still image. In some embodiments, the imaging device comprises a fluorescence reader, a colorimeter, or a sensor. In some embodiments, the imaging device is a personal electronic device, such as a smartphone.
[0125] In some embodiments, the assay assembly is a lab -on-chip system (e.g., Maverik®). In some embodiments, the solid surface comprises a silicon chip. In some embodiments, the imaging device comprises photonic biosensors that measure changes in refractive index caused by binding between the analyte and the peptide-conjugate to form complexes, as describedin Iqbal M., et al. (2010) Label -Free Biosensor Arrays Based on Silicon Ring Resonators and High-Speed Optical Scanning Instrumentation. IEEE J Sei Quantum Elec 16.
[0126] In some embodiments, the lateral flow assay assembly confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some embodiments, a subject may be a member of a variety of races. For example, the subject may be of the White, Black, Hispanic, or Asian race. In some embodiments, a subject may be of a variety of ages. For example, the subject may be age 0 to age 18, age 18 to age 65, or age 65 and above. In some embodiments, a subject may be a male or a female. In some embodiments, a subjectmay be pregnant, breastfeeding, or may have priorly beeninfected with a coronavirus. In some embodiments, a subject may be a high risk subject and have one or more risk factors related to a coronavirus. In some embodiments, a subject may be obese. In some instances, the lateral flow assay assembly prognoses a subject’s immunity to any one of the pathogens described herein. In some embodiments, the lateral flow assay assembly prognoses a subject’s immunity to coronavirus. In some embodiments, the lateral flow assay assembly prognoses a subject’s immunity to SARS-CoV-2. In some embodiments, the lateral flow assay assembly measures the subject’s susceptibility to an infection by any one of the pathogens described herein . In some emb odiments, the lateral flow assay assembly measures the subjects susceptibility to an infection by coronavirus. In some embodiments, the lateral flow assay assembly measures the subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the lateral flow assay assembly identifies individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the lateral flow assay assembly minimizes or reduces the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the lateral flow assay assembly identifies neutralizing antibodies as a surrogate marker of protection from a vaccine (e.g., in place of traditional, randomized, placebo-controlled trials). In some embodiments, the lateral flow assay assembly classifies vaccines by % efficacy in a population. [0127] In some embodiments, the lateral flow assays provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the lateral flow assays provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, atleast 88%, atleast 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the lateral flow assays provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the lateral flow assays provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%. In some embodiments, the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC50; and (b) detecting a binding complex between the detectable peptide and the one or more capture molecules; and (c) determine an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50. In some embodiments, accuracy is measured by comparing the positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population. For example, in a population of human subjects vaccinated against a coronavirus, SARS-CoV-2, accuracy wouldbe measured by comparingthe truepositives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) and true negatives (e.g., subjects that were exposed to the SARS-CoV-2 and did become infected).
Agglutination Assay
[0128] In some embodiments, the assay assembly comprises an assembly for agglutination assay. Agglutination assay relies on agglutination of particles or nanoparticles due to a capture molecule or peptide-conjugate forming a complex with the target analyte in the sample, thus indicating the presence of the target analyte in the sample. In some embodiments, the agglutination assay comprises separating free and bound label by chromatographically separating agglutinated and non -agglutinated antibody -coated colored particles along a membrane. In some embodiments, the agglutination assay comprises a colored latex agglutination reaction, where agglutinated and non -agglutinated particles are separated by a capillary which allows non -agglutinated latex beads through but traps the aggregated latex beads. In some embodiments, the agglutination assay comprises multivalent analytes whereby in the absence of analyte label passes through a filter, but in the presence of analyte an agglutinate is formed which is trapped. In some embodiments, the agglutination assay comprises a system, where a test mixture is formed by contacting the sample with colored particles having analyte-specific receptors on the surface of the particles. The test mixture is passed through a filter having pores which are larger than the colored particles but smaller than the particle-analyte aggregates, thus causingtrappingof the aggregates. Presence of aggregates from the mixture is determined by checking the color of the filtrate. In some embodiments, the agglutination assay comprises alateral flow assay without immobilizingantibodyforverylarge analytes. In some embodiments, the agglutination assay comprises a 2-zone system, where one zone having large pores and one zone having small pores, such that analyte passes through the large pores but becomes trapped by the zone of small pores. Such arrangement is used in conjunction with a small label (e.g. gold particle) which passes through both zones. In the presence of target analyte, a fraction of the gold particle becomes bound to the target analyte and becomes trapped at the zone with small pores. In some embodiments, the agglutination assay is a non-capillary agglutination assay.
[0129] In some instances, the agglutination assay comprises covalently or non -covalently linking a particle or nanoparticle with at least one capture molecule, at least one detectable moiety, and/or at least one peptide-conjugate. In some embodiments, the particle or nanoparticle comprises macromolecule, colloidal metal (such as gold or silver) particle, bead (e.g. latex bead), charcoal, kaolinite, or bentonite. In some embodiments, the particle or nanoparticle also function as detectable moiety. In some embodiments, the particle or nanoparticle comprises of the same material as the detectable moiety. In some embodiments, the particle, nanoparticle, or detectable moiety comprises material that is agglutinable, with other (preferably similar or identical) particle, nanoparticle, or detectable moiety. The ability of the particle, nanoparticle, and detectable moiety to agglutinate enables the formation of larger agglutinates, resulting in increased stability or the detectable signal generated from the agglutinated particle, nanoparticle, or detectable moiety.
[0130] In some embodiments, the detectable moiety comprises detectable material such as a fluorescent dye, an enzymatic label, or a colorimetric label. Detectable moiety includes enzymatic label (e.g., horseradish peroxidase (HRP), beta -galactosidase, alkaline phosphatase, etc), fluorescent dye, luminescent moiety, radioactive moiety, colorimetric label, colored latex particle or nanoparticle, and metal-conjugated moiety such as metallic nanolayer, metallic nanoparticle, or metallic nanoshell-conjugated moiety. Suitable metallic nanoparticle or metallic nanoshell moiety includes gold particle, silver particle, copper particle, platinum particle, cadmium particle, composite particle, gold hollow sphere, gold -coated silica nanoshell, and silica-coated gold shell. Metallic nanolayer suitable for detectable moiety includes nanolayer comprising cadmium, zinc, mercury, gold, silver, copper, and platinum.
[0131] In some embodiments, the detectable moiety is directly or indirectly tagged for a colorimetric assay (e.g., for detection of HRP or beta -galactosidase activity), visual inspection using light microscopy, immunofluorescence microscopy, confocal microscopy, by flow cytometry (FACS), autoradiography electron microscopy, immuno staining, or subcellular fractionation. In some embodiments, the detectable moiety is directly incorporated into the capture molecule. For example, a radioactive amino acid is inserted into the capture molecule comprising a peptide. In some embodiments, the detectable moiety is part of the peptide- conjugate. In some embodiments, the detection method comprises visually examining for signs of agglutination. In some embodiments, the detection method comprises visibly examining the agglutination fora color change oraphysical-chemical change. Physical -chemical changes can occur with oxidation reactions or other chemical reactions, which is detected both visually or via a spectrophotometer. In some embodiments, the agglutination of the particles or nanoparticles due to formation of a complex formed between: capture molecule and target analyte; capture molecule and peptide-conjugate; and/or target analyte and peptide-conjugate is visually examined. In some embodiments, the complex formed is examined using color, reflectance, fluorescence, bioluminescence, or chemiluminescence. In some embodiments, the target analyte is a monoclonal or polyclonal antibody against a protein of any one of the pathogen described herein. In some embodiments, the target comprises a neutralizing antibody. In some embodiments, the target analyte is a monoclonal or polyclonal neutralizing antibody recognizing a spike glycoprotein of a coronavirus. In some embodiments, the target analyte is a monoclonal or polyclonal neutralizing antibody recognizing a spike glycoprotein of SARS- CoV-2. In some embodiments, the target analyte is targeted and captured by the capture molecules described herein.
[0132] In some embodiments, agglutination assay assembly comprises particle or nanoparticle linked to capture molecules comprisingboth ACE2 and spike glycoprotein. If the target analyte comprisingthe neutralizing antibody recognizing spike glycoprotein is absent in a biological sample, agglutination is ob served due to the complex formed by the spike glycoprotein binding to ACE2. In such case, the observed agglutination or signal of the agglutination is indicative of the absence of the target analyte in the biological sample. Conversely, if the target analyte comprising the neutralizing antibody recognizing the spike glycoprotein is present in the biological sample, the binding of the target analyte to the spike glycoprotein prevents the complex formation between spike glycoprotein and ACE2. In this case, the absence of agglutination or signal of the agglutination is indicative of the presence of the target analyte in the biological sample. In some embodiments, the agglutination assay assembly confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some instances, the agglutination assay assembly prognoses a subject’s immunity to any one of the pathogen describedherein. In some embodiments, the agglutination assay assembly measures the subj ect’ s susceptibility to an inf ection by any one of the pathogen described herein.
ELISA
[0133] In some embodiments, the assay assembly comprises an assembly for enzyme -linked immunosorbent assay (ELISA). In certain embodiments, one or more capture molecules are attached or immobilized on a solid support disclosed herein. In some cases, the peptide- conjugate is attached or immobilized on the solid support. In some embodiments, the one or more capture molecules comprise a first peptide or protein and a second peptide or protein. In some embodiments, the first peptide or protein is derived from an antigenic component of a pathogen of interest. In some embodiments, the second peptide or protein is derived from the cognate receptor of the pathogen of interest. In some embodiments, the first peptide or protein or the second peptide or protein is attached or immobilized to the solid support. In some embodiments, the target analyte is attached or immobilized on the solid support. In some embodiments, the solid support is a bead such as colloidal particle, metallic nanoparticle, nanoshell, or latex bead; a flow path in a lateral flow immunoassay device (e.g., a porous membrane); a blot (e.g., Western blot, a slot blot, or dot blot); a flow path in an analytical or centrifugal rotor, or a tube or well (e.g., in a plate suitable for an ELISA or microarray). In some embodiments, the solid support is a plate such as a microtiter plate with multiple wells. In some embodiments, the one or more capture molecules or one or more peptide conjugates is biotinylated, or conjugated to streptavidin, avidin, or neutravidin. In some embodiments, the target analyte is attached to the surface of the well of the microtiter plate via direct binding to the well of the microtiter plate. In some embodiments, the target analyte is attached to the well via binding to one or more capture molecules that are attached to the well of the microtiter plate. In some embodiments, the target analyte is attached or immobilized to the well via binding to one or more peptide-conjugates that are attached or immobilized to the well of the microtiter plate. In some embodiments, the target analyte is attached to the well via binding to one or more peptide-conjugates that form a complex with the one or more capture molecules that are attached or immobilized to the well of the microtiter plate. Residual or nonspecific protein-binding on the solid support can then blocked with an blocking agent, such as bovine serum albumin (BSA) or heat-inactivated normal goat serum (NGS).
[0134] In some embodiments, the solid support is incubated with a biological sample suspected of containing the target analyte. The sample is applied and/or attached to the solid support undiluted or diluted. After incubation, the solid support is washed to remove unbound protein and then incubated with an optimal concentration of the one or more capture molecules conjugated to a detectable moiety or to a substrate of the detectable moiety. In some cases, one or more capture molecules conjugated to the detectable moiety comprises secondary antibody, which binds to the neutralizing antibody in the target analyte. In some embodiments, the target analyte comprises any one of the antibody or any one of the neutralizing antibody described herein.
[0135] The detectable moiety comprises an enzyme, including HRP, beta-galactosidase, alkaline phosphatase (ALP), and glucose oxidase. Sufficient time is allowed for specific binding between the one or more capture molecules and the target analyte to occur. Color is allowed to develop and the optical density of the contents of the well is determined visually or instrumentally (measured at an appropriate wave length) via the use of a spectrophotometer. In some embodiments, the ELISA assay assembly confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some instances, the ELISA assay assembly prognoses a subject’s immunity to any one of the pathogen described herein. In some embodiments, the ELISA assay assembly measures the subject’s susceptibility to an infection by any one of the pathogen described herein.
[0136] In some embodiments, the ELISA assays provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the ELISA assay s provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the ELISA assays provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, atleast 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater. In some embodiments, the ELISA provided herein have a coefficient of variance less than about 20%, 15%, 10%, 9%, 8%, 7%, 6% , 5%, 4%, 3%, 2%, or 1%. In some embodiments, the accuracy is measured by: (a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the ref erence neutralizing antib ody has a known IC50; and (b) detecting a binding complex b etween the detectable peptide and the one or more capture molecules; and (c) determine an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50. In some embodiments, accuracy is measured by comparing the positive results (e.g., protected) and negative results (e.g., not protected) from the neutralizing antibody test disclosed herein with true positive results (both true positive and true negative) in a selected population. For example, in a population of human subjects vaccinated against a coronavirus, SARS-CoV-2, accuracy would be measured by comparing the true positives (e.g., subjects that were exposed to the SARS-CoV-2 and did not become infected) andtrue negatives (e.g., subjects th at were exposed to the SARS-CoV-2 and did become infected).
Testing Device and System Components
[0137] Disclosed herein, in some embodiments, are testing devices comprising a housing. In some embodiments, a testing device is at least partially enclosed by the housing. In some embodiments, the testing device is fully enclosed by the housing. In some embodiments, the housing comprises a synthetic polymer material, such as plastic.
[0138] In some embodiments, the housing is configured to provide information to an imaging device describedherein. Information can include, butis notlimited to, information to normalize an image of the testing device and identifying information (e.g., barcode, RFID chip). In some embodiments, the identifying information comprises test parameters, test result interpretation instructions, expected values for imaging controls, and the like. [0139] Disclosed herein, in some embodiments, are testing devices comprising a sample receptor. In some embodiments, the sample receptor is a sample receiver, a sample processor, a sample purifier, or a combination thereof. In some embodiments, the sample receptor is a sample receiver configured to receive and retain a biological sample obtained from a subject. In some embodiments, the sample receptor is a sample processor configured to remove a component of the sample or separate the sample into multiple fractions (e.g., blood cell fraction and plasma or serum).
[0140] Useful separation materials may include specific binding moieties that bind to or associate with the substance. Binding is covalent or noncovalent. Any suitable binding moiety known in the art for removing a particular substance is used. For example, antibodies and fragments thereof are commonly used for protein removal from samples. In some instances, a sample purifier disclosed herein comprises a binding moiety that binds a nucleic acid, protein, cell surface marker, or microvesicle surface marker in the biological sample. In some instances, the binding moiety comprises an antibody, antigen binding antibody fragment, a ligand, a receptor, a peptide, a small molecule, or a combination thereof.
[0141] The sample receptor is a sample purifier, configured to remove an unwanted substance or non-target component of a biological sample. Depending on the source of the biological sample, unwanted substances can include, but are not limited to, proteins (e.g., antibodies, hormones, enzymes, serum albumin, lipoproteins), free amino acids and other metabolites, microvesicles, nucleic acids, lipids, electrolytes, urea, urobilin, pharmaceutical drugs, mucous, bacteria, and other microorganisms, and combinations thereof. In some embodiments, the sample purifier separates components of a biological sample disclosed herein. In some embodiments, the sample purifier disclosed herein removes one or more components of a sample that would inhibit, interfere with or otherwise be detrimental to the analyses of the target analyte. In some embodiments, the resulting modified sample is enriched for the target analyte. This is considered indirect enrichment of target analytes. Alternatively, or additionally, target analytes may be captured directly, which is considered direct enrichment of target analytes.
[0142] In some embodiments, sample purifiers disclosed herein comprise a filter. In some embodiments, sample purifiers disclosed herein comprise a membrane. Generally, the filter or membrane is capable of separating or removing cells, cell particles, cell fragments, blood components other than cell-free nucleic acids, or a combination thereof, from the biological samples disclosed herein. [0143] In some embodiments, the sample purifier facilitates separation of plasma or seium from cellular components of a blood sample. In some embodiments, the sample purifier facilitates separation of plasma or serum from cellular components of a blood sample before starting a molecular amplification reaction or a sequencing reaction. Plasma or serum separation is achieved by several different methods such as centrifugation, sedimentation or filtration. In some embodiments, the sample purifier comprises a filter matrix for receiving whole blood, the filter matrix having a pore size that is prohibitive for cells to pass through, while plasma or serum can pass through the filter matrix uninhibited. In some embodiments, the filter matrix combines a large pore size at the top with a small pore size at the bottom of the filter, which leads to very gentle treatment of the cells preventing cell degradation or lysis, during the filtration process. This is advantageous because cell degradation or lysis would result in release of nucleic acids from blood cells or maternal cells that would contaminate target cell-free nucleic acids. Non-limiting examples of such filters include Pall VividTM GR membrane, Munktell Ahlstrom filter paper, and TeraPore filters.
[0144] In some embodiments a vertical filtration system is used to facilitate separation of plasma or serum from a cellular component of a blood sample. In this instance, the filtration is driven by capillary force to separate a component or fraction from a sample (e.g., plasma from blood). By way of non-limiting example, vertical filtration may comprise gravitation assisted plasma separation. A high-efficiency superhydrophobic plasma separator is described, e.g, by Liu et al., A High Efficiency Superhydrophobic Plasma Separation, Lab Chip 2015.
[0145] In some embodiments, the sample purifier comprises a lateral filter (e.g., sample does not move in a gravitational direction or the sample moves perpendicular to a gravitational direction). The sample purifier may comprise a vertical filter (e.g., sample moves in a gravitational direction). The sample purifier may comprise vertical filter and a lateral filter. The sample purifier may be configured to receive a sample or portion thereof with a vertical filter, followed by a lateral filter. The sample purifier may be configured to receive a sample or portion thereof with a lateral filter, followed by a vertical filter. In some embodiments, a vertical filter comprises a filter matrix. In some embodiments, the filter matrix of the vertical filter comprises a pore with a pore size that is prohibitive for cells to pass through, while plasma can pass the filter matrix uninhibited. In some embodiments, the filter matrix comprises a membrane that is especially suited for this application because it combines a large pore size at the top with a small pore size at the bottom of the filter, which leads to very gentle treatment of the cells preventing cell degradation during the filtration process. [0146] In some embodiments, the filter comprises a material that moves, draws, pushes, or pulls the biological sample through the filter. In some embodiments, the material is a wicking material. Examples of appropriate materialsusedin the sample purifierto remove cells include, but are not limited to, polyvinylidene difluoride, polytetrafluoroethylene, acetylcellulose, nitrocellulose, polycarbonate, polyethylene terephthalate, polyethylene, polypropylene, glass fiber, borosilicate, vinyl chloride, or silver. In some embodiments, the separation material is a hydrophobic filter, for example a glass fiber filter, a composite filter, for example Cytosep (e.g., Ahlstrom Filtration or Pall Specialty Materials, Port Washington, NY), or a hydrophilic filter, for example cellulose (e.g., Pall Specialty Materials). In some embodiments, whole blood is fractionated into red blood cells, white blood cells and serum components for further processing according to the methods of the present disclosure using a commercially available kit (e.g., Arrayit Blood Card Serum Isolation Kit, Cat. ABCS, Arrayit Corporation, Sunnyvale, CA).
[0147] In some embodiments, the sample purifier comprises at least one filter or at least one membrane characterized by at least one pore size. In some embodiments, at least one pore size of at least one filter is about 0.05 microns to about 10 microns. In some embodiments, the pore size is about 0.05 microns to about 8 microns. In some embodiments, the pore size is about 0.05 microns to about 6 microns. In some embodiments, the pore size is about 0.05 microns to about4 microns. In some embodiments, the pore size is about 0.05 microns to about 2 microns. In some embodiments, the pore size is about 0.05 microns to about 1 micron. In some embodiments, at least one pore size of at least one filter is about 0.1 microns to about 10 microns. In some embodiments, the pore size is about 0.1 microns to about 8 microns. In some embodiments, the pore size is about 0.1 microns to about 6 microns. In some embodiments, the pore size is about 0.1 microns to about 4 microns. In some embodiments, the pore size is about 0.1 microns to about 2 microns. In some embodiments, the pore size is about 0.1 microns to about 1 micron.
[0148] In some embodiments, the sample processor is configured to separate blood cells from whole blood. In some embodiments, the sample processor is configured to isolate plasma from whole blood. In some embodiments, the sample processor is configured to isolate serum from whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 1 milliliter of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 1 milliliter of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 500 microliters (μL) of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 400 μL of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 300 μL of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 200 μL of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 150 μL of whole blood. In some embodiments, the sample processor is configured to isolate plasma or serum from less than 100 μL of whole blood.
[0149] Disclosed herein, in some embodiments, are devices comprising a detection zone. In some embodiments, the detection zone comprises a test region and a control region. In some embodiments, the imaging device captures an image of the detection zone. In some embodiments, the control region is a positive control. In some embodiments, the control region is a negative control. In some embodiments, the control region comprises a positive and a negative control.
[0150] In some embodiments, multiple test regions are provided in the detection zone. In some embodiments, the detection zone comprises a first test region comprising a competition assay assembly to measure a presence of neutralizing antibodies in a patient sample against a first SARS-CoV-2 spike protein receptor binding domain (RBD). The first test region, in some embodiments, comprises a first capture molecule described herein. In some embodiments, the first capture molecule comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the competition assay assembly further comprises a peptide- conjugate comprising a peptide derived from a cognate receptor of the spike protein (e.g., human ACE2) conjugated to a detectable moiety.
[0151] In some embodiments, the detection zone comprises a second test region comprising a second competition assay assembly to measure a presenceofneutralizingantibodiesin a patient sample against a second SARS-CoV-2 spike protein RBD that differs from the first SARS- CoV-2 spike protein RBD by at least 1 amino acid. In some embodiments, the first SARS- CoV-2 spike protein RBD and the second SARS-CoV-2 spike protein RBD differ by at least 2 amino acids. In some embodiments, the first SARS-CoV-2 spike protein RBD and the second SARS-CoV-2 spike protein RBD differ by at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
[0152] In some embodiments, the second test region comprises a second assay assembly configured to measure total antibodies (e.g., neutralizing or otherwise) against SARS-CoV-2 or another virus. In some embodiments, the second assay assembly comprises a capture molecule that binds to anti-nucleocapsid IgG or IgM, anti-spike RBD IgG or IgM, anti-Spike S1 S2 IgG or IgM, anti-spike S2 IgG or IgM, anti-spike S1 IgG or IgM, or a combination thereof. In some embodiments, the capture molecule binds to anti-nucleocapsid IgG or IgM for SARS-CoV, anti-Sl IgG or IgM for MERS, anti-H1 IgG or IgM for influenza and anti-H3 IgG or IgM for influenza.
[0153] In some embodiments, the testing device comprises multiple detection zones. In some embodiments, the testing device comprises a first detection zone and a second detection zone. In some embodiments, the first detection zone comprises the first and second testing regions described herein. In some embodiments, the first and second detection zones are partially enclosed in a single housing. In some embodiments, the first and second detection zones are not enclosed in a single housing. The testing device of the present disclosure may comprise any number of detection zones or testing regions, depending on the desired output. In some embodiments, the testing device comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 detection zones. In some embodiments, at least one of the detection zones comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 testing regions. In some embodiments, the detection zones are partially enclosed in a single housing. In some embodiments, the detection zones are not enclosed in a single housing
Biological Sample Medium
[0154] A biological sample medium (BSM) may be used to store a biological sample to be assayed using the testing devices, systems or methods described herein. In some embodiments, the BSM is a dried blood spot card (DBS). Referringto FIG. 12B, in some embodiments, the BSM 1201 may comprise one or more regions 1205 for containing the one or more biological sample(s). In some embodiments, the one or more regions may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more regions. In some embodiments, the one or more regions may comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 regions. In some embodiments, the one or more regions may comprise 2 to 5 regions (e.g., 2, 3, 4, or 5). In some embodiments, the one or more regions 1205 may comprise spatially distinct adjacent regions. In some embodiments, the biological sample(s) stored on the BSM may comprise two or more biological samples. In some embodiments, the one ormore regions may comprise atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, or more samples. In some embodiments, the one or more regions may comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 samples. In some embodiments, the one or more regions may comprise 2 to 5 samples (e.g., 2, 3, 4, or 5). [0155] Still referring to FIG. 12B, in some embodiments, the BSM may comprise a sealable member 1207 to protect the one or more regions of one or more deposited biological sample 1205 during transport of the BSM. As shown in FIG. 12A-12B, in some embodiments, the BSM may comprise subject identifying information associated with a barcode 1202, numeric 1203, text-based characters 1204, RFID 1206, or any combination thereof to identify the subject from whom the one or more biological samples originated. As shown in FIG. 12C, in some embodiments, the BSM may comprise a fillable region 1208 for a subject to provide answers to a survey related to the health of the subject, including for example symptoms related to an infection by a pathogen of interest (e.g., SARS-CoV-2).
[0156] In some embodiments, the fillable region is positions on a dried spot card (DSC). In some embodiments, the survey may include one ormore clinical questions 1210 pertaining to a symptom associated with an infection by a pathogen of interest. In some embodiments, the BSM may comprise a survey specific to coronavirus symptoms. In some embodiments, the associated symptoms may include cough, nasal congestion, chest pain, headache, diarrhea, vomiting, difficulty breathing or any combination thereof. In some embodiments, the survey may comprise a response severity scale 1211 of associated symptoms. In some embodiments, said severity scale may include terminology to indicate symptom s never present, sometimes present, often present, or present every day. In some embodiments, clinical questions may include indicating whether or not the subject has been exposed to a second subject who has confirmed contracting one or more viruses or microbes. In some embodiments, clinical questions may further comprise a unique computer recognizable code 1209 (e.g. QR code, alpha numeric character, RFID or any combination thereof) that a computer software image processing algorithm may recognize, and associate said medical differential diagnosis survey response with. In some embodiments, a computer software image processing algorithm may further provide a combined interpretation of a subject’s assay results in view of survey severity scale responses.
[0157] As referred to in FIG. 14, the systems and methods described herein, in some embodiments may perform steps on a BSM including: (a) providing a stack of uniformly oriented BSMs 1401; (b) reading the BSM barcode 1402, (c) opening the BSM to expose biological sample(s) 1403, (d) reading BSM patient info 1404, (e) identifying a region on the BSM 1405, (f) punchingthe biological sample spot 1406, (g) droppingthe punch into a well of a multi-well microtiter plate 1407, (h) confirming the position of punch sample in the well 1408, (i) repeating steps (a)-(h) until sufficient biological samples have been punched 1409, (j) stackingthe multi-well microtiter plates 1410, (k) transferring the stackedmulti -well microtiter plates to the automated centralizing assay system 1411, (l) foldingthe BSM for storage 1412 or any combination thereof. One or more of the steps performed by the system may be automated.
Automated and Centralizing Systems
[0158] In some embodiments, the systems described herein are automated high throughput systems. In some embodiments, the systems described may comprise an automated system capable of : (a) receiving and processing a biological sample in preparation for assaying, and (b) conductingthe assay . In some embodiments, one ormore of (a) or (b) is automated. In some embodiments, (a) and (b) are automated. For example, an automated system may comprise system components operated by one or more robots. In some embodiments, the system components are otherwise in mechanical, electrical, fluidic or optical communication with one another such that human intervention is not necessary to perform the disclosed functions . Such automated systems may be capable of completingthe methods disclosedherein with or without the intervention of a human user. In some embodiments, the systems utilize a BSM comprising the biological sample to be assayed with the system.
Biological Sample Processor. As shown in FIG. 13, in some embodiments, the systems disclosed herein comprise a biological sample (BS) processor 1302 and an assay assembly block 1309. In some embodiments, the BS processor comprises: (a) a BS receiver 1303; (b) a BS scanner 1304; (c) a BS separator 1305; (d) BS imager 1306; (e) BS chemical extractor 1307; and (f) a BS purifier 1308. In some embodiments, the biological sample may be contained within a BSM and further processed by the high throughput system.
[0159] In some embodiments, the BS receiver 1303 may mechanically manipulate the BS through linear translation or rotation of the BS to properly position the BS for further processing. In some embodiments, theBS receiver comprises a receptacle for a container (e.g, a vial, 96 well plate). In some embodiments, BS receiver is a crate with multiple receptacles for containers. In some embodiments, theBS receiver comprises a mechanical arm with a distal hand-like member configuredto hold the container and transferthe container to theBS scanner to be scanned.
[0160] In some embodiments, the BS processor 1302 may comprise a BS scanner 1304 in electrical communication with the BS processor 1302. In some embodiments, the BS scanner 1304 may interpret and record subject identifying information on the BS. In some embodiments, the BS scanner 1304 may scan identifying information providedin, for example, barcode 1202, numeric 1203, text-based characters 1204, RFID 1206 or any combination thereof, as shown in FIG. 12A.
[0161] In some embodiments, the BS processor 1302 may further comprise a BS separator 1305 capable of mechanically separating a segment of the BS. In the case where the BS is stored on a BSM, such as a dried blood spot card, the BS separator 1305 may comprise a cutting tool that isolates a segment of the BSM containing the BS. In some embodiments, the cutting tool may comprise a knife or a punch . In some embodiments, the punch comprises a plurality of diameters. In some embodiments, the punch outer-diameter may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more millimeters. In some embodiments, the punch outer-diameter may comprise no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 millimeters. In some embodiments, the one or more punched segments of BS may be placed into an assay chamber for further processing In some embodiments, such as when the BS is stored in a container (e.g., a vial), the BS separator may be a syringe or a needle for isolating a portion of the BS to be assayed. In some embodiments, theBS separator 1305 may aspirate a segment of the BS using a needle. In some embodiments, the needle may be 18 AWG, 19AWG, 20AWG, 21AWG, 22AWG, 23AWG, 24AWG, 25 AWG, 26AWG, 27AWG, or 28 AWG. In some embodiments, the one or more aspirated segment of BS may be placed into an assay chamber for further processing the BS. In some embodiments, the BS processor 1302 may further comprise a BS imager 1306 that may provide quality control feedback position of the BS in the assay chamber to ensure proper processing. After proper placement of one or more segments of BS in the assay chamber, the BS may then be catalogued and stored to be referenced in the future.
[0162] In some embodiments, the assay chamber may comprise a well plate. In some embodiments, the well plate may be a 96-well, 384-well, or a 1536-well plate. In some embodiments, the well plate may be a microtiter well plate. In some embodiments, the assay chamber comprises a unique identifier that may be recognized as it moves between the biological sample processor 1302 and the assay assembly block 1309 described herein. In some embodiments, after successful placement of the one or more B S segments in the assay chamber, the BS may be further processed by the biological sample processor 1302 in preparation for assaying
[0163] Biological Sample Preparation. Biological sample preparation, in some embodiments, is accomplished by a (a) BS chemical extractor 1307 and (b) a BS purifier 1308. Suitable buffers that may be used to elute the BS from the BSM includes phosphate-buffered saline (PBS). In some embodiments, the BS chemical extractor 1307 may be capable of eluting the BS to isolate the BS (e.g., from a biological sample medium). In some embodiments, the chemical extractor elutes the BS from a biological sample medium in the assay chamber. In some embodiments, the BS chemical extractor 1307 may be in fluidic communication with one or more reservoirs of elution buffers andthe assay chamber to dispense elution buffer as needed into the assay chamber. In some embodiments, the assay chamber containing the elution buffer and BS may be agitated by mechanical shaking to facilitate the elution process. In some embodiments, the eluted BS may be purified by the BS purifier 1308 priorto transferring the assay chamber to the assay assembly block 1309. In some embodiments the purification process may comprise centrifugation to isolate BS by density. In some embodiments, upon sufficient BS processing, the biological sample processor 1302 may mechanically transfer the assay chamber to the assay assembly block 1309.
[0164] Assaying the Biological Sample . Assayingthe biological sample, in some embodiments, may be accomplished by an assay assembly block 1309. In some embodiments, the assay assembly block comprises: (a) BS Diluter 1310 and (b) BS assay conductor 1311. In some embodiments, theBS diluter 1310 may be in fluidic communication with the assay chamber to prepare the serial dilutions of the BS. In some embodiments, assay chamber identification may be interpreted by the assay assembly block to indicate which assay to conduct on which BS. In some embodiments, upon completion of the assay by the BS assay conductor 1311, the assay results 1312 will be saved with respect to the identification of the assay chamber and more broadly the subject providing the biological sample assayed.
[0165] In some embodiments, the system comprises the use of a preprogramed and automated robotics to perform one or more of the steps above, including the assays described herein. In some embodiments, the automated centralizing assay system comprises the use of preprogramed and automated robotics to perform the assay comprising a lateral flow assay, agglutination assay, ELISA, or FRET assay as described herein. In some embodiment, the system is capable of performing at least or about 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or more than 100,000 tests per day. In some embodiments, the system utilizes small volumes of reagents, such as less than 100 nL, less than 90 nL, less than 80 nL, less than 70 nL, less than 60 nL, or less than 50 nL of reagents. In some embodiments, the system performs the assay (e.g., a solution-based assay) in a vessel, such as a container or a test tube. In some embodiments, the system performs the assay (e.g., a solution -based assay) in an assay plate, such as a 96-well plate, a 384-well plate, or a 1536-well plate. [0166] In some embodiments, the biological sample (e.g., blood from a finger prick) is stored in or on a BSM, such as a DSC, described herein. The biological sample is removed from the BSM and placed into the vessel (e.g., a well of a 96 -well plate) to be eluted from the BSM. Next, the eluted biological sample is assayed using an assay (e.g., FRET, ELISA) described herein deposited on a solid surface.
[0167] In some embodiments, biological samples are collected from the subjects via the use of blood or sample collection cards. The biological samplesis tested by the automated centralizing assay system. In some embodiments, the blood or sample collection card comprises multiple slots for the blood or sample collection. In such instances, the biological sample from each slot is tested separately by a different assay method. For example, a blood collection card (i.e. a blood spot card) comprises five slots that is tested by the automated centralizing assay system performing fives assays such as lateral flow assay, an ELISA, a biomarker panel, a qPCR, and a neutralizingantibody titer assay. In some embodiments, the same assay is performedmultiple times (e.g., duplicates) to ensure accuracy of the results. In some embodiments, the assay is performed at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, multiple dilutions are tested, for e.g., 1 :40, 1 :80, 1 :160, and 1 :320 dilutions are tested at the same time. In some embodiments, the dilutions tested are one or more of 1 :5, 1 : 10, 1 : 15, 1 :20, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, 1 :50, 1 :55, or 1 :60, 1 :65, 1 :70, 1 :75, 1 :80, 1 :85, 1 :90, 1 :95, 1 :100, 1 : 100, 1 : 105, 1 :110, 1 : 115, 1 :120, 1 :125, 1 : 130, 1 :135, 1 :140, 1 : 145, 1 :150, 1 :155, 1 : 160, 1 : 165, 1 :170, 1 :175, 1 : 180, 1 :185, 1 :190, 1 : 195, 1 :200, 1 :205, 1 :210, 1 :215, 1 :220, 1 :225, 1 :230, 1 :235, 1 :240, 1 :245, or 1 :250. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different dilutions are tested simultaneously.
[0168] In some embodiments, the automated centralizing assay system confirms the efficacy or effectiveness of a vaccine in a population. In some embodiments, the biological samples are collected from a population before and after the population is vaccinated. In some embodiments, additional biological samples are collected at any time frame or frequency described herein to confirm presence and sufficient quantity of neutralizing antibody (induced by the vaccine) in the population. In some embodiment, the automated centralizing assay system can prognose immunity or measure susceptibility of the population to any one of the pathogen described herein by confirming the absence or insufficient quantity of neutralizing antibody in the population. In some embodiments, the automated centralizing assay system confirms presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some instances, the automated centralizing assay system prognoses a subject’s immunity to any one of the pathogen described herein. In some embodiments, the automated centralizing assay system prognoses a subject’s immunity to coronavirus. In some embodiments, the automated centralizing assay system prognoses a subject’s immunity to SARS-CoV-2. In some embodiments, the automated centralizing assay system measures the subject’s susceptibility to an infection by any one of the pathogens described herein. In some embodiments, the automated centralizing assay system measures the subject’s susceptibility to an infection by coronavirus. In some embodiments, the automated centralizing assay system measures the subject’s susceptibility to an infection by SARS-CoV- 2. In some embodiments, the automated centralizing assay system identifies individual s in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the automated centralizing assay system minimizes or reduces the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the automated centralizing assay system identifies neutralizing antibodies as a surrogate marker of protection from a vaccine (e.g., in place of traditional, randomized, placebo-controlled trials). In some embodiments, the automated centralizing assay system classifies vaccines by % efficacy in a population.
[0169] In some embodiments, the automated centralizing assay systems provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the automated centralizing assay systems provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or greater. In some embodiments, the automated centralizing assay systems provided herein have a specificity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
COMPUTER-IMPLEMENTED SYSTEMS
[0170] Disclosed herein, in some embodiments, are systems. Referring to FIG. 5, the system 500 comprises, in some embodiments, a testing device, an imaging device 501, and a computing device, for determining whether a subject is immune to an infection by a pathogen of interest (e.g., SARS-CoV-2). The imaging device and/or the computing device are configured to receive and analyze data generated by the testing device and/or one or more external devices, to provide a result to the subject. The result is provided to the subject via a graphical user interface (GUI) by the imaging device and/or computing device using an application (web application or mobile application). Systemsfurthercompriseone ormore data store for storing and retrieving the data.
[0171] Disclosed herein, in some embodiments, are data analyzed by one or more components of the system described herein. In some embodiments, the data are structured or unstructured. In some embodiments, the data are generated by the imaging device when an image is captured of the detection zone of the testing devices described herein. In some embodiments, the data are external data generated from an external device. In some embodiments, the external device comprises a diagnostic device, a prognostic device, or a health or fitness tracking device. In some embodiments, the external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, or sleep quantity, or a combination thereof.
[0172] Provided here, in some embodiments, are imaging devices for capturing an image of the detection zone of the testing devices described herein. In some embodiments, the imaging device is a camera. In some embodiments, the imaging device is a computing device with a camera, such as a smartphone, laptop, or tablet. In some embodiments, the imaging device is not a computing device, but is in communication with the computing device via a communication network. In some embodiments, the communication network is wireless, such as wireless Internet or Bluetooth.
[0173] Referring to FIG. 5, the system 500 comprises an imaging device 501, an external device 502, a data store 505, all in communication via a communication network 503, equipped with cloud-based computing executed by the data analytics module 507 and communications module 508. In this example, the imaging device transmits data from an image captured of the detection zone of the testing device described herein, via the communication network, to the data analytics module 507 in the cloud 506 to be analyzed. The data analytics module 507 transmits the result to the communications module 508 to be packaged for display to the user. In this example, theresultis displayed on the imaging device 501, whichis a personal electronic device belongingto the user (e.g., the subject in this example). The data store 505 is a remote server in this example. Alternatively, the data store is a cloud-based data store. [0174] Also provided, in some embodiments, are computing devices comprising a computing system configured to analyze data described herein to provide a result. Referring to FIG. 6, a block diagram is shown depicting an exemplary computing device that includes a computing system 600 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and/or methodologies for static code scheduling of the present disclosure. The components in FIG. 6 are examples only and do not limit the scope ofuse or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.
[0175] Computer system 600 can include one or more processors 601, a memory 603, and a storage 608 that communicate with each other, and with other components, via a bus 640. The bus 640 can also link a display 632, one or more input devices 633 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 634, one or more storage devices 635, and various tangible storage media 636. All of these elements can interface directly or via one or more interfaces or adaptors to the bus 640. For instance, the various tangible storage media 636 can interface with the bus 640 via storage medium interface 626. Computer system 600 can have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.
[0176] Computer system 600 includes one or more processor(s) 601 (e.g., central processing units (CPUs) or general purpose graphics processing units (GPGPUs)) that carry out functions. Processor(s) 601 optionally contains a cache memory unit 602 for temporary local storage of instructions, data, or computer addresses. Processor(s) 601 are configured to assist in execution of computer readable instructions. Computer system 600 can provide functionality for the components depicted in FIG. 6 as a result of the processor(s) 601 executing non -transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 603, storage 608, storage devices 635, and/or storage medium 636. The computer-readable media can store software that implements particular embodiments, and processor(s) 601 can execute the software. Memory 603 can read the software from one or more other computer-readable media (such as mass storage device(s) 635, 636) or from one or more other sources through a suitable interface, such as network interface 620. The software can cause processor(s) 601 to carry out one or more processes or one or more steps of one or more processes described or illustrated herein. Carrying out such processesor steps can include defining data structures stored in memory 603 and modifying the data structures as directed by the software.
[0177] The memory 603 can include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 604) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phase-change random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 605), and any combinations thereof. ROM 605 can act to communicate data and instructions unidirectionally to processor(s) 601, and RAM 604 can act to communicate data and instructions bidirectionally with processor(s) 601. ROM 605 and RAM 604 can include any suitable tangible computer-readable media described below. In one example, a basic input/output system 606 (BIOS), including basic routines that help to transfer information between elements within computer system 600, such as during start-up, is stored in the memory 603
[0178] Fixed storage 608 is connected bidirectionally to processors) 601, optionally through storage control unit 607. Fixed storage 608 provides additional data storage capacity and can also include any suitable tangible computer-readable media described herein. Storage 608 is used to store operating system 609, executable(s) 610, data 611, applications 612 (application programs), and the like. Storage 608 can also include an optical disk drive, a solid-state memory device (e.g., flash -based systems), or a combination of any of the above. Information in storage 608 may, in appropriate cases, be incorporated as virtual memory in memory 603.
[0179] In one example, storage device(s) 635 is removably interfaced with computer system 600 (e.g., via an external port connector (not shown)) via a storage device interface 625. Particularly, storage device(s) 635 and an associated machine-readable medium can provide non-volatile and/or volatile storage of machine-readable instructions, data structures, program modules, and/or other data for the computer system 600. In one example, software can reside, completely or partially, within a machine -readable medium on storage device(s) 635. In another example, software can reside, completely or partially, within processor(s) 601.
[0180] Bus 640 connects a wide variety of subsystems. Herein, reference to a bus can encompass one or more digital signal lines serving a common function, where appropriate. Bus 640 is any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example and not by way of limitation, such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.
[0181] Computer system 600 can also include an input device 633. In one example, a user of computer system 600 can enter commands and/or other information into the computer system 600 via input device(s) 633. Examples of an input device(s) 633 include, but are not limited to, an alpha-num eric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, atouch screen, a multi-touch screen, ajoystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device is a Kinect, Leap Motion, or the like. Input device(s) 633 is interfaced to bus 640 via any of a variety of input interfaces 623 (e.g., input interface 623) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.
[0182] In particular embodiments, when computer system 600 is connected to network 630, computer system 600 can communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 630. Communications to and from computer system 600 is sent through network interface 620. For example, network interface 620 can receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 630, and computer system 600 can store the incoming communications in memory 603 for processing Computer system 600 can similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 603 and communicated to network 630 from network interface 620. Processor(s) 601 can access these communication packets stored in memory 603 for processing.
[0183] Examples of the network interface 620 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 630 or network segment630 include, butarenotlimited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 630, can employ a wired and/or a wireless mode of communication. In general, any network topology is used.
[0184] Information and data is displayed through a display 632. Examples of a display 632 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 632 can interface to the processor(s) 601, memory 603, and fixed storage 608, as well as other devices, such as input device(s) 633, via the bus 640. The display 632 is linked to the bus 640 via a video interface 622, and transport of data between the display 632 and the bus 640 is controlled via the graphics control 621. In some embodiments, the display is a video projector. In some embodiments, the display is a head-mounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.
[0185] In addition to a display 632, computer system 600 can include one or more other peripheral outputdevices 634including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices is connected to the bus 640 via an outputinterface 624. Examples of an outputinterface624 include,butarenotlimited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.
[0186] In addition or as an alternative, computer system 600 can provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which can operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein. Reference to software in this disclosure can encompass logic, and reference to logic can encompass software. Moreover, reference to a computer-readable medium can encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both. [0187] Those of skill in the art will appreciate that the various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein is implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality.
[0188] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein is implemented or performed with a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combinationthereof designed to perform the functions described herein. A general purpose processor is a microprocessor, but in the alternative, the processor is any conventional processor, controller, microcontroller, or state machine. A processor can also be implemented as a combination of computing devices, e.g, a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.
[0189] The steps of a method or algorithm described in connection with the embodiments disclosed herein is embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module can reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art. An exemplary storage medium is coupled to the processor such that the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium is integral to the processor. The processor and the storage medium can reside in an ASIC. The ASIC can reside in a user terminal. In the alternative, the processor and the storage medium can reside as discrete components in a user terminal.
[0190] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, smart phones, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers, invariousembodiments, include those with booklet, slate, and convertible configurations, known to those of skill in the art. In some embodiments, the smartphoneis an Apple iPhone or an androiddevice (e.g., Samsung Galaxy). [0191] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. In some embodiments, the operating system is provided by cloud computing. Those of skill in the art will also recognize that suitable mobile smartphone operating systems include, by way of non -limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®. Those of skill in the art will also recognize that suitable media streaming device operating systems include, by way of non -limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in the art will also recognize that suitable video game console operating systems include, by way of non -limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.
[0192] Disclosed herein are computing systems comprising a data processor. In some embodiments, the data processor is a mobile processor. In some embodiments, the data processor is configured to receive data from an imaging device, an external device, or a data store, or a combination thereof. In some embodiments, data processor analyzes the data to provide a result. In some embodiments, the imaging device and the data processor are housed in the same device, such as a smartphone.
[0193] In some embodiments, the data processor is configured to provide a computer program or application, comprising a data analytics module. In some embodiments, the application is a web application. In some embodiments, the application is a mobile application. In some embodiments, the data analytics module is configured to receive data from an imaging device and analyze the data to provide a result. In some embodiments, the data analytics module is configured to receive external data from an external device. In some embodiments, the external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, or sleep quantity, or a combination thereof. In some embodiments, the external device comprises a diagnostic device, a prognostic device, or a health or fitness tracking device. In some embodiments, the health tracking device is the Aurora®, Fitbit®, or Apple Watch. In some embodiments, systems comprise multiple external devices. In some embodiments, the data analytics module is configured to analyze the external data to provide a result. In some embodiments, the data analytics module is configured to analyze the external data and the data received from the imaging device to provide a result.
[0194] In some embodiments, the mobile application comprises a communication module. In some embodiments, the communication module is configured to communicate the result to the subject. In some embodiments, the communication module is configured to display the result to the subject via a graphical user interface (GUI) of an electronic device. In some embodiments, the electronic device is the imaging device described herein, the computing device described herein, or a combination thereof. In some embodiments, the electronic device is a smartphone, such as those described herein.
Computer program
[0195] Disclosed herein, in some embodiments, the data processor is configured to run a computer program. A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions is implemented as program modules, such as functions, obj ects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types. In light of the disclosure provided herein, those of skill in the art will recognize that a computer program is written in various versions of various languages.
[0196] The functionality of the computer readable instructions is combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof
[0197] In some embodiments, the computer programs described herein are configured to perform any one of the methods described herein. In some embodiments, the computer program is configured to analyze data obtained from an assay described herein (e.g., lateral flow assay, FRET assay) and provide a result based on the analysis of the data.
[0198] In some embodiments, the computer program is configured to confirm the presence, absence, or quantity of the neutralizing antibody described herein from a subject’s biological sample. In some instances, the computer program is configured to prognose a subject’s immunity to any one of the pathogen described herein. In some embodiments, the computer program is configured to prognose a subject’s immunity to coronavirus. In some embodiments, the computer program is configured to prognose a subject’s immunity to SARS-CoV-2. In some embodiments, the computer program is configured to measure a subject’s susceptibility to an infection by any one of the pathogen described herein. In some embodiments, computer program is configured to measure a subject’s susceptibility to an infection by coronavirus. In some embodiments, the computer program is configured to measure a subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the computer program is configured to identify individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the computer program is configured to identify neutralizing antibodies as a surrogate marker of protection from a vaccine. In some embodiments, the computer program is configured to classify a vaccine by % efficacy in a population. In some embodiments, the computer program is configured to minimize or reduce the risk associated with vaccine usage following market entry (e.g., by identifying individuals that do not respond to the vaccine).
Non-transitory computer readable storage medium
[0199] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device. In further embodiments, a computer readable storage medium is a tangible component of a computing device. In still further embodiments, a computer readable storage medium is optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media
Web application
[0200] Disclosed here, in some embodiments, are data processors comprising one or more web applications. In light of the disclosure provided herein, those of skill in the art will recognize that a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft®.NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, and XML database systems. In further embodiments, suitable relational database systems include, by way of non -limiting examples, Microsoft® SQL Server, my SQL™, and Oracle®. Those of skill in the art will also recognize that a web application, in various embodiments, is written in one or more versions of one or more languages. A web application is written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as HypertextMarkupLanguage (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous Javascript and XML (AJAX), Flash® Actionscript, Javascript, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tel, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®
Mobile application
[0201] Also disclosed herein, in some embodiments, are data processors comprising one or more mobile applications. In some embodiments, the mobile application is provided to a mobile digital processing device at the time it is manufactured. In some embodiments, the mobile application is provided to a mobile digital processing device via the computer network described herein. Mobile applications disclosed herein are configured to locate, encrypt, index, and/or access information. Mobile applications disclosed herein are configured to acquire, encrypt, create, manipulate, index, and peruse data.
[0202] A mobile application is created by suitable techniques using hardware, languages, and development environments known to the art. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, Java™, Javascript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.
[0203] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non -limiting examples, Airplay SDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite,. NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non -limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non -limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.
[0204] Those of skill in the art will recognize that several commercial forums are available for distribution of mobile applications including, by way of non -limiting examples, Apple® App Store, Google® Play, Chrome Web Store, BlackBerry® App World, App Store for Palm devices, App Catalog for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, and Samsung® Apps. Standalone Application
[0205] Disclosed here, in some embodiments, are data processors comprising one or more standalone applications. A standalone application is an independent computer process; not an add-on to an existing process, e.g., not a plug-in. Those of skill in the art will recognize that standalone applications are often compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB.NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications.
Software Modules
[0206] Disclosed herein, in some embodiments, the data processor comprises a computer program configured with one or more software modules. In some embodiments, the one or more software module is a data analytics module. In some embodiments, the one or more software module is a communication module.
[0207] In view of the disclosure provided herein, software modules are created by techniques known to those of skill in the art using machines, software, and languages known to the art. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, and a standalone application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location [0208] In some embodiments, the computing system described herein comprise a data analytics module. In some embodiments, the data analytics module is configured to run one or more algorithms. In some embodiments, the one or more algorithms comprise a machine learning algorithm. The machine learning algorithm is capable of supervised learning, unsupervised learning, reinforcement learning, semi-supervised learning, self-supervised learning, multiinstance learning, inductive learning, deductive inference, transduction learning, multi-task learning, active learning, online learning, transfer learning, or ensemble learning. In some embodiments, the data analytics module is configured with artificial intelligence (Al), such as a limited memory Al.
[0209] In some embodiments, the data analytics module receives data from the testing device, and optionally, external data from one or more external devices, and analyzes the data to provide a result. Referring to analysis of the data received from the testing device, the data analytics module normalizes the resultby subtracting background signal intensity of the testing device (e.g., background plasma concentration, non-specific binding to the solid surface). Referring to external data, the data analytics module identifies whether external data indicates symptoms to an acute infection by a pathogen.
[0210] In some embodiments, the result is either positive or negative. In some embodiments, the positive result indicates an acute infection and negative result indicates a lack of an acute infection. In some embodiments, the positive result indicates that the subject is immune to a future infection by a pathogen of interest. In some embodiments, the data analytics module transmits the result to a communications module for display to the subject.
[0211] In some embodiments, the communication module is configured to display one or more results to the subject via a graphical user interface (GUI).
Data Store
[0212] Disclosed herein, in some embodiments, are systems comprising one or more data stores. In some embodiments, the data store is a database suitable for the storage and retrieval of data. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, and XML databases. Further nonlimiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, and Sybase. In some embodiments, a database is internet-based. In further embodiments, a database is web -based. In still further embodiments, a database is cloud computing-based. In a particular embodiment, a database is a distributed database. In other embodiments, a database is based on one or more local computer storage devices, such as a smartphone.
Graphical User Interface
[0213] Also disclosed herein, in some embodiments, are graphical user interfaces (GUI) configured to display a resultto a user. In some embodiments, the user is the subject. In some embodiments, the GUI comprises one or more dashboards of an application (e.g., web application or mobile application). In some embodiments, the dashboard comprises relevant health information to a subject. In some embodiments, the GUI is a part of personal electronic device, such as a smartphone or tablet, belonging to the user.
Web Portal
[0214] Disclosed herein, in some embodiments, is a web portal providing a single access point for multiple users to access information about the immune status of a subject. In some embodiments, a web portal provides access to a subject, a subject’s doctor, or a healthcare worker responding to an urgent public health crisis. In some embodiments, the portal provides a single access point for a population of individuals, wherein the data is anonymized. In this example, the web portal provides access to policy makers, health care professionals, governmental organization, and non-governmental organizations responding to a public health crisis. Among other information, the portal can indicate one or more geographical locations comprising subjects that are either acutely infected by a pathogen of interest, or immune to the pathogen of interest.
METHODS
[0215] Disclosed herein, in some embodiments, are methods of measuring a target analyte disclosed herein in biological sample obtained from subject. In some embodiments, a subject may be a member of a variety of races. For example, the subject may be of the White, Black, Hispanic, or Asian race. In some embodiments, a subject may be of a variety of ages. For example, the subject may be age 0 to age 18, age 18 to age 65, or age 65 and above. In some embodiments, a subject may be a male or a female. In some embodiments, a subject may be pregnant, breastfeeding, or may have priorly been infected with a coronavirus. In some embodiments, a subject may be a high risk subject and have one or more risk factors related to a coronavirus. In some embodiments, a subject may be obese. In some embodiments, methods comprise utilizing the testing devices described herein. In some embodiments, methods further comprise analyzing data generatedby the testing device disclosed herein, and providing a result to a user of an electronic device. In some embodiments, providing the result comprises displayingthe result on a GUI of the electronic device. In some embodiments, the analyzing and displaying is performed by a single computing device (e.g., smartphone, tablet). In some embodiments, analyzing and displaying is performed at the point of need (e.g., at the time and space that the analyte is detected in the biological sample using the testing device described herein).
[0216] Provided herein are methods of processing a biological sample comprising: (a) providing a biological sample from a subject; (b) measuring a presence, an absence, or a level of a labeled complex between (i) a capture molecule and (ii) a peptide-conjugate comprising a peptide derived from a pathogen in the presence of the biological sample; (c) classifying the biological sample as having a presence or a level of the neutralizing antibody.
[0217] In some embodiments, methods further comprise determining whether a subject suffers from an acute infection by the pathogen (e.g., SARS-CoV-2). In some embodiments, methods further comprise determining whether the subject is immune to an infection by the pathogen (e.g., SARS-CoV-2). In some embodiments, methods further comprise determining whether a vaccine administered to the subject is effective to immunize the subject against the pathogen. In some embodiments, the methods further comprise determining whether the biological sample is safe for transfusion into another subject (e.g., blood, or blood plasma transfusion). In some embodiments, the methods further comprise determining whether the biological sample is safe as a convalescent plasma therapy for the prevention of an infection by the pathogen or treatment of a disease or condition associated with the pathogen. In some embodiments, the methods further comprise identifying the subject as having an acute infection by the pathogen of interest. In some embodiments, the methods further comprise identifying a subject in need of a vaccination to the pathogen of interest. The methods described herein may be performed at the point of care or point of need.
[0218] In some embodiments, the pathogen comprises a coronavirus or an influenza virus.
[0219] In some embodiments, the methods provided herein confirm presence, absence, or quantity of the neutralizing antibody described herein from a subject’ s biological sample. In some instances, the methods described herein prognose a subject’s immunity to any one of the pathogens described herein. In some embodiments, the methods described herein prognose a subject’s immunity to coronavirus. In some embodiments, the methods described herein prognose a subject’s immunity to SARS-CoV-2. In some embodiments, the methods provided herein measure the subject’s susceptibility to an infection by any one of the pathogens described herein. In some embodiments, the methods provided herein measure the subject’s susceptibility to an infection by coronavirus. In some embodiments, the methods provided herein measure the subject’s susceptibility to an infection by SARS-CoV-2. In some embodiments, the methods provided herein identify individuals in a population that respond to a vaccine and individuals in a population that do not respond to a vaccine. In some embodiments, the methods provided herein minimize or reduce the risk associated with usage of a commercialized vaccine in a population (e.g., by identifying individuals that do not respond to the vaccine). In some embodiments, the methods provided herein identify neutralizing antibodies as a surrogate marker of protection from a vaccine. In some embodiments, the methods provided herein classify vaccines by % efficacy in a population.
[0220] In some embodiments, the methods provided herein have an accuracy of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or greater. In some embodiments, the methods provided herein have a sensitivity of at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater. In some embodiments, the methods provided herein have a specificity of at least 80%, for example, atleast 85%, atleast 86%, atleast 87%, at least 88%, at least 89%, atleast 90%, atleast 91%, atleast 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
[0221] In some embodiments, the pathogen is a virus, a bacterium, a fungus, or a parasite. In some embodiments, the virus is a DNA virus or an RNA virus. In some embodiments, the virus is a single stranded virus, or a double stranded virus. In some embodiments, the virus is a plus strand or a minus strand DNA or RNA virus. In some embodiments, the virus replicates through reverse transcription of an RNA intermediate. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is a severe acute respiratory syndrome coronavirus (SARS-CoV). In some embodiments, the SARS-CoV is SARS-CoV-2. In some embodiments, the coronavirus is Middle East Respiratory Syndrome coronavirus (MERS-CoV). In some embodiments, the coronavirus is an alpha coronavirus (e.g., 229E, NL63). In some embodiments, the coronavirus is a beta coronavirus (e.g., OC43, HKU1). In some embodiments, the virus is an influenza virus.
[0222] In some embodiments, the coronavirus comprises a protein having an amino acid sequence with greater than or equal to about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more sequences, or portions thereof, provided in SEQ ID NOS: 1-116. In some embodiments, the pathogen of interest is a variant of the pathogen, for example, a pathogen that has evolved through mutations in the genes encoding the pathogen. In the case of a virus, the variant virus may have a mutation in an amino acid sequence of a viral protein, such as an envelope protein, nucleocapsid protein, membrane protein, hemagglutinin, neuraminidase, or spike protein. In some embodiments, the virus (e.g., coronavirus) comprises a mutation in one or more of the sequences provided in SEQ ID NOS: 1-116. In some embodiments, the mutation does not affect, or minimally affects, host receptor binding by the spike protein of the coronavirus. In some embodiments, the mutation is an insertion, a deletion, or a substitution at an amino acid (or a plurality of amino acids) of one or more sequences provided in SEQ ID NOS: 1-116.
[0223] In some embodiments, the human is an animal, such as a mammal. In some embodiment, the mammal is a dog, cat, monkey, non-human primate, rodent, or a farm animal. In some embodiments, the rodent is a gerbil, hamster, chinchilla, rat, mouse, or guinea pig In some embodiments, the farm animal is a horse, a pig, a cow, a sheep, an alpaca, or a lama. In some embodiments, the mammal is a human subject. In some embodiments, the subject is pediatric (e.g., age 0-18). In some embodiments, the subject is not pediatric. In some embodiments, the subject is age 50 or older. In some embodiments, the subject is an age between 50-100, 55-95, 60-90, 65-85, or 70-80. In some embodiments, the subject is female or male. In some embodiments, the subject has been exposed to the pathogen. In some embodiments, the subject has not been exposed to the pathogen. In some embodiments, the subject exhibits one or more symptoms comprising a cough, fever, tiredness, or difficulty breathing. In some embodiments, the subject has an underlying health problem comprising high blood pressure, a heart problem, diabetes, immunodeficiency, autoimmune disease. In some embodiments, the subject is immunocompromised. In some embodiments, the subject has an underlying immune-comprising condition comprising a pregnancy, an immune- mediated disease, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof. In some embodiments, the subject is breastfeeding. In some embodiments, the subject is receiving an immunosuppressant therapy for treatment of a disease or a condition. In some embodiments, the immunosuppressant therapy comprises a biologic drug (e.g., adalimumab (Humira®) and infliximab (Remicade®)), calcineurin inhibitors (e.g., tacrolimus (Envarsus XR® or Protopic) and cyclosporine (Gengraf®, Neoral® or Sandimmune®)), corticosteroids (e.g., prednisone), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolate mofetil (CellCept®)), janus kinase inhibitors (e.g., tofacitinib (Xeljanz®)), mechanistic target of rapamycin (mTOR) inhibitors (e.g., sirolimus (Rapamune®)), or monoclonal antibodies (e.g, basiliximab (Simulect®)), or any combination thereof.
[0224] Described herein are methods for testing a population using the testing device or systems described herein. In some embodiments, the population comprises subjects of any demographics, age, or geographical locations. In some embodiments, all subjects in the population are vaccinated against the pathogen. In some embodiments, some subjects in the population are vaccinated against the pathogen. In some embodiments, none of the subjects in the population are vaccinated against the pathogen. In some embodiments, the number of vaccinated subjects in the population is unknown. In some embodiments, all subjects in the population are previously infected by the pathogen. In some embodiments, some subjects in the population are previously infected by the pathogen. In some embodiments, none of the subjects in the population are previously infected by the pathogen. In some embodiments, the number of subjects previously infected by the pathogen is unknown. In some embodiments, all subjects in the population are currently infected by the pathogen. In some embodiments, some subjects in the population are currently infected by the pathogen. In some embodiments, none of the subjects in the population are currently infected by the pathogen. In some embodiments, the number of subjects currently infected by the pathogen is unknown. In some embodiments, the population is a general population. In some embodiments, the population is a population undergoing clinical trial, where all subjects of the population are vaccinated or unvaccinated. In some embodiments, the population is a population that is at higher risk of the infection. For example, the population that is at higher risk of the infection includes healthcare workers, essential workers, elderly, subjects with pre-existing condition, subjects with respiratory disease or disorder, or subjects with comorbidity or any of the health problems described herein. In some embodiments, the population comprises individuals who are vaccine responders (e.g., develop neutralizing antibodies as described herein in response to the vaccine). In some embodiments, the population comprises individuals who are vaccine nonresponders (e.g., do not develop neutralizing antibodies as described herein in response to the vaccine). In some embodiments, the population comprises individuals who have been infected and/or exposed to the natural pathogen.
[0225] In some embodiments, obtaining the biological sample from the subject or population of subjects is direct or indirect. Indirectly obtaining a biological sample from a subject may include receiving it from a laboratory or processing/ storage facility by mail, or otherwise. In some embodiments, indirectly obtaining the biological sample from the subject or the population comprises the use of blood collection means such as blood card. In some embodiments, the blood card comprises multiple slots for collecting multiple samples of blood droplets for multiple testing. For example, a biological sample medium (BSM) (e.g., a dried blood spot card) comprises multiple slots is tested by the automated centralizing assay system performing assays such as control assay to verify the quality of the biological sample comprisingthe blood, lateral flow assay, ELISA, biomarker panel, qPCR, western blotting and a neutralizing antibody titer assay. Non-limiting examples of biological samples include cell, tissue, or bodily fluid obtained from the subject. Non-limiting examples of biological samples include aqueous humour, vitreous humour, bile, breastmilk, cerebrospinal fluid, endolymph, perilymph, female ejaculate, amniotic fluid, gastric juice, menses, mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, urine, feces, whole blood, blood serum, blood plasma, sputum, cerebrospinal fluid, synovial fluid, lymphatic fluid, nasal swab, or cheek swab. In some embodiments, the biological sample comprises blood, urine, saliva, or feces. In some embodiments, the blood is capillary blood. In some embodiments, the blood is not venous blood (e.g., from a phlebotomy). In some instances, methods disclosed herein comprise obtaining a blood sample by administering a finger prick. In some embodiments, the biological sample is a swab sample (e.g., buccal swab, nasopharyngeal swab). Directly obtaining the biological sample from the subject, in some embodiments, is performed by a healthcare professional (e.g., nurse or doctor), laboratory technician, or the subject at the point of need or the point of care.
[0226] In some embodiment, the target analyte comprises macromolecules such as peptides, proteins, nucleic acids, pathogens, or pathogen particles that can be detected for presence or quantity (i.e. concentration) in a biological sample. In some embodiments, the target analyte is targeted and formed complex with an antigen or with an antibody. In some embodiments, the target analyte comprises an antibody against a pathogen. In some embodiments, the antibody against the pathogen is an antibody against an antigenic peptide derived from the pathogen. In some cases, the antibody is neutralizing antibody induced by an infection . In some instances, the antibody is neutralizing antibody induced by vaccination. As a non-limiting example, the analyte may be an antibody against a portion of the spike protein derived from a coronavirus (e.g., SARS-CoV-2). In some embodiments, the analyte is the activity of the antibody. In some embodiments, the activity is blocking binding between the spike protein of a coronavirus and its cognate receptor (e.g., ACE2). In some embodiments, the analyte is a complex comprising the spike protein bound to the antibody at the receptor binding region of the spike protein. In some embodiments, the antibody belongs to an immunoglobulin class comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, or immunoglobulin D.
[0227] In some embodiments, the detectable peptide-conjugate is the peptide-conjugate described herein comprising a detection agent and a peptide. In some embodiments, the peptide is an antigenic peptide. In some embodiments, the analyte (e.g., antibody) is specific to at least a portion of the peptide. In some embodiments, the peptide comprises at last a portion of the spike glycoprotein of a coronavirus (e.g., SARS-CoV-2) described herein. In some embodiments, the peptide is a receptor to the antigenic peptide. In some embodiments, the receptor comprises ACE2 receptor described herein.
[0228] In some embodiments, measuring comprises performing an assay on the biological sampleto detecta number of complexes formedbetweenthe analyte and the peptide -conjugate, or the peptide-conjugate and the capture molecule, or both. In some embodiments, the assay comprises an assay assembly described herein. In some embodiments, the assay is a lateral flow assay. In some embodiments, the lateral flow assay is a competition assay. In some embodiments, measuring a number of complexes formed between the analyte and the peptide- conjugate, or the peptide-conjugate and the capture molecule, or both can be performed in one step without the need for washing any portion of the assay assembly.
[0229] In some embodiments, the index or the control is derived from a subject that has not been exposed to the pathogen (negative control). In some embodiments, the index or control is from a subject that has been exposed to the pathogen (positive control). In some embodiments, the index or control is obtained from a convalescent plasma donor. In some embodiments, the index or control is synthetically derived. In a non-limiting example of a control that is synthetically derived, the control can be sample with cells engineered to express a peptide or protein that is known to induce production of neutralizing antibodies against the pathogen to a known degree. In some embodiments, the control or index is a maximum absolute dilution of a reference sample in which neutralizing activity is observed in the reference sample. The higherthe dilution, the stronger the neutralizing activity . In a non -limiting example, the dilution of 1:160 may be considered sufficient neutralizing activity and anything above 1:160 is considered to be high neutralizing activity. In another example, the dilution of 1 :40 may be considered to be low or weak neutralizing activity. In this example, neutralizing activity in the diluted reference sample is a correlate of the level of neutralizing antibodies titers in the reference sample.
[0230] In some embodiments, if the level of neutralizing antibodies detected in the biological sample using the testing devices, systems and methods described herein correlates with a dilution that is above a threshold dilution (the control), then the biological sample is classified as having a sufficient presence or level of neutralizing antibodies to confer adaptive immunity against a pathogen. In some embodiments, the biological sample is classified as having a sufficient presence or high level of neutralizing antibodies when the level of neutralizing antibodies correlates with a dilution that is above 1 :60, or above or equal to about 1 :65, 1 :70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245,1:250, 1:255, 1:260,1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, :375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465,
1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530,
1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595,
1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, or 1:640. In some embodiments, the dilution comprises four (4) dilutions equal to about 1:80, 1 :160, 1:320, and 1:640.
[0231] Conversely, if the level of neutralizing antibodies detected in the biological sample correlates with a dilution that is below a threshold dilution, then the biological sample is classified as having a low level or insufficient presence of neutralizing antibodies to confer adaptive immunity. In some embodiments, the biological sample is classified as having an insufficient presence or a low level of neutralizing antib odies when the level of the neutralizing antibodies correlates with a dilution that is below or equal to about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, or 1:60.
[0232] Methods described herein, in some embodiments, do not consist of utilizing a cell culture, such as an immortalized cell line expressing human ACE2. In some embodiments, methods do not consist of handling or administering to a cell or cell line a purified or isolated pathogen, such as a live virus of pseudovirus.
[0233] In some embodiments, the methods comprise measuring the target analyte in a biological sample from a subject or in a population of subjects multiple times. In some embodiments, the analyte is measured at least once every 12 hours, 24 hours, 36 hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, one year, 18 months, two years, three years, four years, five years, six years, seven years, eight years, nine years, decade, or a combination thereof. In some embodiments, the analyte is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In some embodiments, the analyte is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months following an event. In some embodiments, the event is an exposure to the pathogen, a first symptom of a disease or condition caused by the pathogen, a first dose of a vaccine against the pathogen, or a first dose of an anti-viral therapy to treat an acute infection by the pathogen. In some embodiments, the analyte is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years following the event. In some embodiments, the analyte is measured before the event, such as, for example before a first exposure to the pathogen to identify a subject or population of subjects who do not have sufficient immunity against the pathogen and who may be in need of a vaccine.
Convalescent Plasma Therapy
[0234] The use of convalescent plasma for therapy to prevent SARS-CoV-2 infection requires demonstration that the plasma contains sufficient titers of neutralizing antibodies to confer immunity to SARS-CoV-2. Existing methods of detecting sufficient neutralizing antibody titers for this purpose are limited at the point of need because they require use of an immortalized cell line expressing human angiotensin converting enzyme II (ACE2), e.g., 293T cells. By contrast, the testing devices described herein are portable and methods of using the testing devices described herein are cell-free (e.g., do not require use of a cell or cell line). In this manner, the testing devices are more cost effective, do not require a laboratory technician, and is scaled quickly to meet the growing demand of individuals in need of convalescent plasma therapeutic interventions.
[0235] Disclosed herein, in some embodiments are methods of screening a biological sample for use as a convalescent plasma therapy, the method comprising: (a) providing a biological sample from a donor subject; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (ii) a neutralizing antibody against the pathogen; (c) classifying the biological sample as having a presence or a level of the neutralizing antibody; and (d) identifying the biological sample as safe or not safe as a convalescent therapy based at least in part on the classifying in (c). In some embodiments, the level is high relative to an index or a control. In some embodiments, the biological sample is identified as safe for convalescent therapy provided the presence or the level of the labeled complex between the peptide-conjugate and the neutralizing antibody against the pathogen is measured. In some embodiments, the biological sample is identified as unsafe for convalescent therapy provided the presence or the level of the labeled complex between the peptide-conjugate and the capture molecule is measured.
Detecting Serological Antibodies and Neutralizing Antibodies
[0236] Disclosed herein, in some embodiments, are combined methods of identifying adaptive immunity to a pathogen in a subject and identifying the total antibodies against the pathogen that the subject is producing due to a past exposure. In some embodiments, the method comprises: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; (c) measuring a presence, an absence or a level of an antibody against the pathogen (e.g., neutralizing or otherwise); (d) classifying the biological sample as having a presence or a level of the neutralizing antibody and/or a presence or a level of a type of antibody (e.g., IgG, IgM, IgA) against the pathogen; and (d)identifyingthe subject as having sufficient adaptive immunity against the pathogen, based at least in part, on classifying in (c); and/or (e) identifying the subject as having been exposed to the pathogen prior based on classifying in (d). In some embodiments, if the number of the labeled complexes between (ii) and (iii) is high relative to an index or a control, then the subject has a sufficient adaptive immunity to an infection by the pathogen. In some embodiments, if the number of the complexes between (i) and (ii) is high relative to an index or a control, then the subject has a sufficient adaptive immunity to the infection by the pathogen. In some embodiments, the methods described herein are performed using the testing device of the present disclosure. In some embodiments, measuring a presence, an absence or a level of an antibody against the pathogen (e.g., neutralizing or otherwise) can be performed in one step without the need for washing any portion of the testing device. In some embodiments, the capture molecule or peptide-conjugate may comprise a peptide or protein that is encoded by a nucleic acid sequence, or that has an amino acid sequence, that is at least 80% identical to any one of SEQ ID NOS: 21-23, 25-42, 44-46, 49-66, 68-70, 73-90, 92-94, or 97-114.
[0237] The presence, absence, or the level of the antibody against the pathogen may be measured by detecting a complex between the antibody (e.g., IgG, IgM, IgA) and a capture molecule that is directly or indirectly labeled. In some embodiments, a level of the antibody that is high, relative to an index or a control is indicative that the subject was exposed to the pathogen. In contrast, if the level of the antibody is low relative to the index or control, the subject was not exposed to the pathogen.
[0238] In some embodiments, a presence, absence or a level of a biomarker is measured in the biological sample. In some embodiments, the biomarker is a marker of inflammation, such as a cytokine (e.g., interleukin 6). In some embodiments, the presence, absence or level of multiple biomarkers is measured in addition to the total antibodies and neutralizing antibodies againstthe pathogen. In some embodiments, thepathogenis SARS-CoV-2, or avariantthereof.
Neutralizing Antibody (Neutralization) Titer
[0239] Described, in some embodiments, are methods formeasuringneutralizing antibody titer for determining sufficient quantity of neutralizing antibody to confer adaptive immunity against any one of the pathogens described herein. In some embodiments, a biological sample of neutralizing antibody to be tested is serial -diluted and mixed with a viral suspension. The mixture is applied to known number of cells for inoculation. In some embodiments, the neutralizing antibody titer is determined by calculatingthe highest dilution of biological sample of neutralizing antibody that prevents infection of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of replicate inoculations. In some embodiments, the neutralization titer is determined by calculatingthe highest dilution of the biological sample of neutralizing antibody that prevents infection at 50% of replicate inoculation. In some cases, the method of measuring neutralization titer comprises the use of viral plaque assay, fluorescent focus assay, and endpoint dilution assay. Each of these three assays relies on serial viral dilutions added to cells to measure neutralization titer. Other exemplary measurements for determining neutralization titer include qPCR or ELISA for quantifying the amount of pathogen genome or particle necessary to infect a set number of cells in the presence of the neutralizing antibody. In some embodiments, the quantity of neutralizing antibody detected in a biological sample is sufficient in inducing adaptive immunity when the concentration of the neutralizing antibody in the biological sample prevents infection at 50% of replicate inoculation. In some embodiments, the quantity of neutralizing antibody detected in a biological sample is insufficient in inducing adaptive immunity when the concentration of the neutralizing antibody in the biological sample prevents infection below 50% of replicate inoculation.
Population Screening
[0240] Described herein, in some embodiments, are methods utilizing the systems, devices, and compositions described herein for screening a population described herein for prognosing immunity or susceptibility to infection caused by any one of the pathogen described herein. In some embodiments, the method comprises detecting presence, absence, or quantity of analyte comprising the neutralizing antibody in the population. In some embodiments, the method prognoses immunity or susceptibility to the infection based on the presence, absence, or quantity of the neutralizing antibody in the biological samples obtained from the subjects in the population. In some embodiments, the method prognoses the population to have adaptive immunity against the infection when the presence or sufficient quantity of the neutralizing antibody is detected in the biological samples. In some embodiments, the method prognoses the subjectto have susceptibility or inadequate immunity to the infection when the neutralizing antibody is absent or is detected at an insufficient quantity in biological samples. In some embodiments, the method identifies the subjects with absence or insufficient quantity of neutralizing antibody as subjects who are in need of vaccination. In some embodiments, the population is screened at any one of the time frames or frequencies described herein.
[0241] In some embodiments, the method determines if the populationis in need of vaccination against the infection based on the presence, absence, or quantity of the neutralizing antibody in the biological samples. In some embodiments, the method determines that the population does not need to be vaccinated due to presence or sufficient quantity of the neutralizing antibody detected in the biological samples. In some embodiments, the method determines that the population does not need to be vaccinated due to herd immunity when the percentage of the subjects of the population with presence or sufficient quantity neutralizing antibody is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the method determines that the population or subjects within the population need to be vaccinated due to absence or insufficient quantity of the neutralizing antibody detected in the biological samples. Confirmatory Diagnostic for a Vaccine
[0242] Described herein, in some embodiments, are confirmatory diagnostic testing devices and methods of their use to determine the efficacy or effectiveness of a vaccine against a pathogen described herein. In some embodiments, methods of determining the efficacy or effectiveness of a vaccine described herein comprise detecting presence, absence, or quantity of neutralizing antibody against the pathogen in a biological sample from a subject that was administered the vaccine.
[0243] Disclosed herein, in some embodiments are methods of determining the efficacy or effectiveness of a vaccine against a pathogen, the method comprising: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide- conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; (c) classifying the biological sample as having a presence or a level of the neutralizing antibody; and (d) identifying the vaccine as effective to induce a sufficient or insufficient adaptive immune response against the pathogen in the subject. In some embodiments, the level is high relative to an index or a control. In some embodiments, the vaccine is effective, provided the presence or the level of the labeled complex between the peptide-conjugate and the neutralizing antibody against the pathogen is measured. In some embodiments, the vaccine is not effective, provided the presence or the level of the labeled complex between the peptide-conjugate and the capture molecule is measured. In some embodiments, methods further comprise measuring a presence, absence or a level of a biomarker described herein. In some embodiments, the biomarker is interleukin 6. In some embodiments, measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide -conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen can be performed in one step without the need for washing.
[0244] In some embodiments, methods confirm that the vaccine lacks efficacy or effectiveness in the subject, provided an absence of the neutralizing antibody is detected in the biological sample obtained from the subject that was administered the vaccine. In some embodiments, methods confirm that the vaccine lacks efficacy or effectiveness, provided the level of the neutralizing antibody detected is low relative to an index or a control level. An absence or a low level of the neutralizing antibody is identified when a presence of the labeled complex between the peptide-conjugate and the capture molecule is detected. In some embodiments, methods confirm that the vaccine is effective, provided a presence of the neutralizing antibody is detected in the biological sample obtained from the subject that was administered the vaccine. In some embodiments, methods confirm that the vaccine is effective provided a level of the neutralizing antibody that is detected is high relative to an index or a control level.
[0245] In some embodiments, the subject is part of a general population, where not everyone in the general population was or is administered the vaccine. In some embodiments, the testing devices and methods described herein confirm which subjects of the general population are in need of the vaccine by detecting the absence or the low level of the neutralizing antibody in biological samples obtained from the subjects.
[0246] In some embodiments, the testing devices and methods described herein measure neutralizing antibodies against a pathogen in a biological sample from a subject prior to enrolling the subject in the clinical trial. In some embodiments, the presence or level of the neutralizing antibody detected in the biological sample excludes the subject from participating in the clinical trial for the vaccine. The absence or level of the neutralizing antibody detected in the biological sample is an inclusion criteria for the subject to enroll in the clinical trial for the vaccine. In some embodiments, the testing devices and methods described herein measure neutralizing antibodies against a pathogen in a biological sample from a subject during the clinical trial for the investigation of the efficacy of the vaccine. In some embodiments, a biological sample from the vaccinated subject is tested using the testing devices and methods described herein when an adverse event is observed during the clinical trial. In some embodiments, the adverse event is correlated with a presence, absence or level of neutralizing antibodies in the biological sample.
[0247] In some embodiments, the testing devices and methods described herein confirm efficacy or effectiveness of the vaccine in a population of subjects. In some embodiments, the population being tested for efficacy or effectiveness of the vaccine is a group receiving the vaccine (as opposed to a different group receiving the placebo) in a clinical trial. In some embodiments, the populationbeingtested for efficacy or effectiveness ofthe vaccine is agro up that is outside the clinical trial context, such as after the vaccine is allowed or approved for use in preventing a disease caused by the pathogen. In some embodiments, the testing devices and methods described herein detect presence, absence, or quantity of the neutralizing antibody against a pathogen in a biological sample from each subject in the group before and after the group is administered the vaccine. In some embodiments, the testing devices and methods described herein confirm adaptive immunity in the vaccinated population based on presence or high level of the detected neutralizing antibody in the biological samples obtained from the vaccinated subjects in the population. In some embodiments, the testing devices and methods described herein confirm a lack of sufficient adaptive immunity in the vaccinated population based on absence or low level of the detected neutralizing antibody in the biological samples obtained from the vaccinated subjects in the population. In this instance, a booster of the vaccine may be recommended for at least one subject of the group, or the entire group.
[0248] In some embodiments, neutralizing antibodies induced by the vaccine are measured multiple times after administration of the vaccine to determine whether the vaccine is effective to confer immunity against the pathogen (e.g., SARS-CoV-2). In some embodiments, the neutralizing antibodies are measured after administration of the vaccine at least once every 12 hours, 24 hours, 36 hours, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, one year, per 18 months, two years, three years, four years, five years, six years, seven years, eightyears, nine years, 10 years, or a combination thereof. In some embodiments, the neutralizing antibodies are measured atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20 ormore times. In some embodiments, the neutralizing antibodies are measured atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months following administration of the vaccine to the subject. In some embodiments, the neutralizing antibodies are measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years following administration of the vaccine to the subject.
Identifying vaccine responders and non-responders
[0249] In some embodiments, methods are provided using the systems, devices, and compositions described herein for identifying those subjects that respond to a vaccine (e.g., responders) and those subjects that do notrespond to a vaccine (e.g., non -responders). In some embodiments, the systems, devices, and compositions described herein may be used to test individuals in a population of individuals for the presence or absence of neutralizing antibodies to identify those individuals that responded to the vaccine (e.g., produced neutralizing antibodies), and those individuals that did not respond to the vaccine (e.g., did not produce neutralizing antibodies). The population may be a group of individuals that received a vaccine against the pathogen (e.g., SARS-CoV-2). The population may be a group of individuals that received a vaccine during a clinical trial to assess vaccine efficacy. The population may be a group of individuals that received a vaccine as part of a large-scale, mass vaccination campaign. In some embodiments, the methods provided herein assess an individual’s particularized response to a vaccine. The methods provided herein identify those individuals that did not respond to a particular vaccine, inform those individuals that they did not develop immunity to the pathogen, and inform those individuals that they should seek alternative interventions (e.g., alternative vaccines) and/or start or continue safety precautions (e.g., social distancing, the use of PPE, etc.).
[0250] In some emb odiments, the methods may involve administering a vaccine to a population of individuals, and testing each of those individuals for the presence or absence of neutralizing antibodies to assess whether the vaccine conferred immunity again st the pathogen (e.g. , SARS - CoV-2). The individuals, after receiving the vaccine, may submit one or more biological samples to be tested for the presence or absence of neutralizing antibodies as described herein (e.g., by submitting the sample to a centralized testing center). For example, each individual that receives a vaccine may be given a sample collection device (e.g., a blood spot card) for submittinga biological sample fortesting. The sample collection device (e.g., blood spot card) may be given to the individual at the time of vaccination.
[0251] The individual may collect one or more biological samples (e.g., a finger prick to collect a blood sample) and provide the one or more biological samples in the sample collection device (e.g., blood card). This may be done before and after vaccine administration. For example, the one or more biological samples may be collected at a time point before the individual has been vaccinated to establish a baseline level of neutralizing antibodies. After administration of the vaccine, the one or more biological sample may be collected again in the same manner at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 2 months after vaccine administration to determine whether the vaccineinduced a sufficient neutralizing antibody titer to confer protection against the pathogen. Generally, the one or more biological samples are taken after sufficient time has elapsed for the individual to develop neutralizing antibodies (e.g., if the individual responds to the vaccine). To assess the durability of the vaccine, one or more biological samples may be collected in the same manner at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, or more following the vaccine administration.
[0252] The individual may submit the one or more biological samples in the sample collection device (e.g., blood spot card) to a testing center. The testing center may test the one or more biological samples for the presence or absence of neutralizing antibodies (e.g., using the sy stems, devices, and compositions describedherein), and may identify the individual as either a vaccine responder (e.g., the individual developed neutralizing antibodies) or as a vaccine nonresponder (e.g., the individual did not develop neutralizing antibodies). The testing center may provide the individual with a report (or may otherwise inform the individual) indicating the individual’s status as a vaccine responder or as a vaccine non-responder. When the individual is identified as a vaccine responder, the individual may be understood to have developed immunity (e.g., the individual responded to the vaccine) to the pathogen (e.g., SARS-CoV-2). [0253] When the individual is identified as a vaccine non-responder (e.g., the individual did not respond to the vaccine), the individual may be understood to not have developed immunity to thepathogen (e.g., SARS-CoV-2). Alternatively, if durability of an initial response weakens over time, the individual may be understood to no longer have sufficient immunity to the pathogen. In some embodiments, an alternative intervention (e.g., an alternative vaccine), may be recommended to the individual. In some embodiments, additional precautions may be recommended to the individual, such as social distancing, sheltering-in-place, quarantining the use of personal protective equipment (e.g., gloves, face coverings), and the like. In some embodiments, the vaccine has entered the market. In some embodiments, the vaccine is a vaccinein developmentoravaccinethathasnotyetbeen approvedby aregulatory entity, such as the FDA. In some cases, the vaccine is a vaccine that has been licensed for use by the regulatory entity.
Mitigating Risk During Vaccine Usage in a Population Following Market Entry
[0254] In some embodiments, methods are provided herein using the systems, devices, and compositions described herein to mitigate or reduce the risk associated with usage of a commercialized vaccine in a population. The methods provided herein may assess the benefit and the risk of a particular vaccine by identifying the individuals who respond to the vaccine and the individuals who do not respond to the vaccine. In some embodiments, the systems, devices, and compositions described herein may be used to test individuals in a population for the presence or absence of neutralizing antibodies to identify those individuals that responded to a particular vaccine (e.g., produced neutralizing antibodies), and those individuals that did not respond to the vaccine (e.g., did not produce neutralizing antibodies). In some embodiments, the non -responders are identified and informed that they did not develop immunity, thereby removing the risk associated with a particular vaccine. The methods provided herein allow a vaccine developer to leverage the benefits associated with a particular vaccine, including vaccines with limited efficacy, while minimizing or reducing the risks associated with the vaccine. The methods provided herein may be particularly suited for emergent situations in which multiple vaccines are being developed on an accelerated timeline against a pathogen (and may notbe subject to the entire FDA review process).
[0255] As depicted in FIG. 11 A, two vaccines targeting the same pathogen (e.g., SARS-CoV- 2) have been developed and licensed for use. Each of the two vaccines may have different levels of efficacy in a population. For example, 70% of the individuals in a population may respond to vaccination with Vaccine 1 (e.g., the “benefit”), whereas 30% of the individuals in the population may not respond to vaccination with Vaccine 1 (e.g., the “risk”). Vaccine 1 may be deemed “efficacious” because the benefit outweighs the risk (e.g., the number of responders is greater than the number of non-responders). Although there are some individuals for which Vaccine 1 was not effective, Vaccine 1 is developed into a vaccine for widespread use against the pathogen (e.g., SARS-CoV-2). In contrast, 40% of the individuals in a population may respond to vaccination with Vaccine 2 (e.g. , the “benefit”), and 60% of the individuals in the populationmay notrespondto vaccination with Vaccine 2 (e.g., the “risk”). In this scenario, Vaccine 2 may be deemed “not efficacious” because the risk outweighs the benefit (e.g., the number of non-responders is greater than the number of responders). Despite having a benefit for a subset of the population, Vaccine 2 is not used to prevent infection in a population by the pathogen (e.g., SARS-CoV-2).
[0256] In some embodiments, the methods provided herein may be used to identify the risk of a particularvaccine and remove the risk (e.g., by informingthe non-responders of their status).. As depicted in FIG. 11B, the methods provided herein identify the risk of Vaccine 1 (e.g., the 30% of the population who did not respond to Vaccine 1) and those individuals are informed that they did not develop immunity to the pathogen (SARS-CoV-2). In this scenario, Vaccine
1 is still considered to be “efficacious” and is used. Similarly, the methods provided herein identify the risk of Vaccine 2 (e.g., the 60% of the population who did not respond to Vaccine 2) and those individuals are informed that they did not develop immunity to the pathogen (e.g, SARS-CoV-2). In this scenario, the risk is removed because the non-responders are informed that they do not have immunity to the pathogen (and can take precautionary measures such as social distancing, the use of PPE, and the like). In contrast to the scenario depicted in FIG.
I I A, Vaccine 2 is now considered to be “efficacious” and used. Thus, the methods provided herein reduce or remove the risk (e.g., the population of non-responders) associated with a vaccine and increase the chances that a vaccine is used. [0257] The individual may collect one or more biological samples (e.g., a finger prick to collect a blood sample) and provide the one or more biological samples in the sample collection device (e.g., blood card). This may be done before and after vaccine administration. For example, the one or more biological samples may be collected at a time point before the individual has been vaccinated to establish a baseline level of neutralizing antibodies. After administration of the vaccine, the one or more biological sample may be collected again in the same manner at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 2 months after vaccine administration to determine whether the vaccineinduced a sufficient neutralizing antibody titer to confer protection against the pathogen. Generally, the one or more biological samples are taken after sufficient time has elapsed for the individual to develop neutralizing antibodies (e.g., if the individual responds to the vaccine). To assess the durability of the vaccine, one or more biological samples may be collected in the same manner at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, or more following the vaccine administration.
[0258] The individual may submit the one or more biological samples in the sample collection device (e.g., blood spot card) to a testing center. The testing center may test the one or more biological samples for the presence or absence of neutralizing antibodies (e.g., using the systems, devices, and compositions describedherein), and may identify the individual as either a vaccine responder (e.g., the individual developedneutralizing antibodies) or as a vaccine nonresponder (e.g., the individual did not develop neutralizing antibodies). The testing center may provide the individual with a report (or may otherwise inform the individual) indicating the individual’s status as a vaccine responder or as a vaccine non-responder.
[0259] When the individual is identified as a vaccine responder, the individual may be understood to have developed immunity (e.g., the individual responded to the vaccine) to the pathogen (e.g., SARS-CoV-2). When the individual is identified as a vaccine non-responder (e.g., the individual did notrespond to the vaccine), the individual may be understood to not have developed immunity to the pathogen (e.g., SARS-CoV-2). Alternatively, if the durability of the initial response weakens over time, the individual may be understood to no longer have sufficient immunity to the pathogen. In some embodiments, an alternative intervention, such as an alternative vaccine under development, maybe recommended to the individual. In some embodiments, additional precautions may be recommended to the individual, such as social distancing, sheltering-in-place, quarantining, the use of personal protective equipment (e.g, gloves, face coverings), and the like. In some embodiments, the vaccine has entered the market for use. In some embodiments, the vaccine is a vaccine in development or a vaccine that has notyet been approvedby a regulatory entity (e.g., the FDA). In some cases, the vaccine is a vaccine that has been licensed for use by the regulatory entity.
Classifying Vaccines in a Population of Individuals
[0260] In some embodiments, methods are provided using the systems, devices, and compositions described herein to classify a vaccine in a population of individuals. In some embodiments, the methods comprise (a) providing a biological sample from each individual of a population of individuals; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen in the biological sample; (c) classifying each biological sample as having a presence, an absence, or a level of the neutralizing antibody; and (d) classifying the vaccine based on the presence or the level of the neutralizing antibodies measured in (b). In some embodiments, the vaccine is classified by % efficacy in the population (e.g., the % of individuals in a population that responded (e.g., developed neutralizing antibodies)to the vaccine). In some embodiments, the vaccine is at least 50% efficacious in a population (e.g., at least 60%, at least 70%, at least 80%, atleast90%, atleast95%, atleast97%, atleast98%, atleast 99%). In some embodiments, the classification of the vaccine is specific to a particular population class, such as gender, age, and individuals with pre-existing conditions (e.g., immune-compromised). For example, the vaccine may be 50% efficacious in children ages 12 andyounger, whereas the vaccine maybe 75% efficacious in adults ages 65 and older. In some embodiments, measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide-conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen in the biological sample can be performed in one step without the need for washing.
Measuring neutralizing antibodies as a surrogate marker of protection
[0261] In some embodiments, methods are provided using the systems, devices, and compositions described herein to identify the presence and/or a high level of neutralizing antibodies in an individual after administration of a vaccine. In some embodiments, the methods utilize the presence and/or high level of neutralizing antibodies as a surrogate marker of protection in the individual. In some embodiments, the presence and/or high level of neutralizing antibodies in the individual is used to inform a vaccine developer that the individual developed immunity to the pathogen (e.g., SARS-CoV-2). In some embodiments, the presence and/or high level of neutralizing antibodies in the individual is used in place of traditional methods that test efficacy of a vaccine (e.g., a randomized, placebo-controlled clinical trial).
[0262] In some embodiments, the methods comprise (a) providing a biological sample from a subject having been administered a vaccine; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide -conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; (c) classifying the biological sample as having a presence, an absence, or a level of the neutralizing antibody; and (d) identifying the subject as having immunity against the pathogen based on the presence and/or a high level of the neutralizing antibody.
Vaccines
[0263] In some embodiments, the vaccine is for the active immunization for the prevention of disease caused by a pathogen. The pathogen described herein, in some embodiments, is any bacteria, virus, or fungus that causes infection in a mammal. In some embodiments, a pathogen is a virus of any one of the virus described herein. The virus is a DNA virus or an RNA virus. A DNA virus is a single-stranded (ss) DNA virus, a double-stranded (ds) DNA virus, ora DNA virus that contains both ss and ds DNA regions. An RNA virus is a single -stranded (ss) RNA virus or a double-stranded (ds) RNA virus. A ssRNA virus can further be classified into a positive-sense RNA virus or a negative-sense RNA virus.
[0264] In some embodiments, the dsDNA virus is from the family: Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfaviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Mimiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipoviridae, and Tectiviridae. A ssDNA virus is from the family: Anelloviridae, Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae. A DNA virus that contains both ss and ds DNA regions is from the group of pleolipoviruses. In some cases, the pleolipoviruses include Haloarcula hispanica pleomorphic virus 1, Halogeometricum pleomorphic virus 1, Halorubrum pleomorphic virus 1, Halorubrum pleomorphic virus 2, Halorubrum pleomorphic virus 3, and Halorubrum pleomorphic virus 6.
[0265] In some embodiments, the dsRNA virus is from the family: Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megavimaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Rotavirus and Totiviridae. A positive-sense ssRNA virus is from the family: Alphaflexiviridae, Alphatetraviridae, Alvemaviridae, Arteriviridae, Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae, Dicistroviridae, Flaviviridae, Gammaflexiviridae, Iflaviridae, Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae, Roniviridae, Secoviridae, Togaviridae, Tombusviridae, Tymoviridae, and Virgaviridae. A negative -sense ssRNA virus is from the family: Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, and Orthomyxoviridae.
[0266] In some embodiments, the vaccine described herein confers adaptive immunity against the pathogen when administered to a subject. In some embodiments, the vaccine described herein confers adaptive immunity to a subject against pathogenic (e.g., viral) infection caused by the pathogen. In some embodiments, the vaccine described herein induces neutralizing antibody against the pathogen in the subject after administration of the vaccine.
[0267] In some embodiments, the vaccine confers adaptive immunity against a coronavirus. In some instances, the coronavirus is selected from the group consisting of: alphacoronavirus, betacoronavirus, deltacoronavirus, and gammacoronavirus. Examples of alphacoronavirus include, but are not limited to, Bat coronavirus CDPHE15, Bat coronavirus HKU 10, Human coronavirus 229E, Human coronavirus NL63, Miniopterus bat coronavirus 1 , Miniopterus bat coronavirus HKU8, Mink coronavirus 1, Porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2, and Scotophilus bat coronavirus 512. Examples of betacoronavirus include, but are not limited to, Betacoronavirus 1, Hedgehog coronavirus 1, Human coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, Murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Severe acute respiratory syndrome-related coronavirus, Tylonycteris bat coronavirus HKU4. Examples of deltacoronavirus include, but are not limited to, Bulbul coronavirus HKU11, Common moorhen coronavirus HKU21, Coronavirus HKU15, Munia coronavirus HKU13, Night heron coronavirus HKU19, Thrush coronavirus HKU12, White-eye coronavirus HKU16, Wigeon coronavirus HKU20. Examples of gammacoronavirus include, but are not limited to, Avian coronavirus, Beluga whale coronavirus SW1. Additional examples of coronavirus include MERS-CoV, SARS-CoV, and SARS-CoV-2.
[0268] In some embodiments, the vaccine comprises an inactivated virus of any one of the virus described herein. In some embodiments, the vaccine comprises an inactivated coronavirus. In some embodiments, the vaccine comprises an inactivated SARS-CoV-2. In some embodiments, the vaccine comprises formalin -inactivated SARS-CoV-2. In some cases, the vaccine comprises a live -attenuated virus of any one of the virus described herein. In some cases, the vaccine comprises a live-attenuated coronavirus. In some cases, the vaccine comprises a live-attenuated SARS-CoV-2. In some embodiments, the vaccine comprises chemical inactivated SARS-CoV-2. The goal of chemical and physical inactivation of viruses may be to destroy the infectivity of the pathogen without destroying the immunogenicity of important epitopes. In some embodiments, inactivation processes may require confirmation of that the virus infectivity is destroyed. In some embodiments, the inactivation processes may require confirmation that the immunogenicity is intact. In some embodiments, the inactivation process may require that the inactivated pathogen does not pause a health risks to the subject. In some embodiments, chemical inactivation of the virus is complete using various inactivating chemical agents, such as for example formaldehyde, β-Propiolactone (BPL), hydrogen peroxide treatment, zinc-finger reactive treatment. In some embodiments, the vaccine comprises a physically inactivated SARS-CoV-2. In some embodiments, physical inactivation uses heating or pasteurizing of viruses, gamma irradiation, ultraviolet light, among other techniques. In some embodiments, viral inactivation can use chemical or physical methods or a combination of the two. In some embodiments, various methods may be employed to inactivate SARS-CoV-2 virus for vaccine production; these may include methods described in Sanders et al, 2015 (Vaccine Analysis: Strategies, Principles and Controls), which reference is hereby incorporated in its entirety.
[0269] In some embodiments, antigenic peptide is derived from a protein of any one of the pathogens described herein. In some embodiments, the antigen is viral antigen derived from a viral protein, a fragment of a viral protein, or a nucleic acid encoding the viral protein or the fragment of the viral protein. In some embodiments, the viral antigen is a viral antigen of coronavirus. In some embodiments, the viral antigen is a viral antigen of SARS-CoV-2.
[0270] In some embodiments, the vaccine comprises viral antigen for inducing adaptive immunity in a subject. In some embodiments, the viral antigen is a non -mutated antigen. In some embodiments, the viral antigen is derived from publicly disclosed information on the viral genetic information. In some embodiments, the viral antigen is derived from analysis of the viral genome to predict suitable epitopes for T cell activation. In some embodiments, the viral antigen is derived from analysis of the sequence of the viral genome in a MHC-peptide presentation prediction algorithm implemented in a computer processor. In some embodiments, the viral antigen is derived from analysis of the viral sequences in an MHC-peptide presentation prediction algorithm implemented in a computer processor that has been trained by a machine learning software, which predicts the likelihood of binding and presentation of an epitope by an MHC class I or an MHC class II antigen. In some embodiments, the MHC-peptide presentation predictor is neonmhc2.
[0271] In some embodiments, the viral antigen is encoded by a nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS: 7-10 (SARS-CoV-2), 13-16 (SARS-CoV NL630), 17-20 (SARS Co V Tor2). In some embodiments, the viral antigen is encoded by a nucleic acid sequence that is 100% identical to any one of SEQ ID NOS: 7-10, 13-16, or 17-20. In some instances, the viral antigen comprises a peptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 2-6, or 11-12. In some instances, the viral antigen comprises a peptide sequence that is 100% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. In some instances, the viral antigen comprises a peptide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a fragment of any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. In some instances, the viral antigen comprises a peptide sequence that is 100% identical to a fragment of SEQ ID NOS: 2-4, or 11-12
Table 1: Amino Acid Sequence Mutations to SEQ ID NO: 5
Figure imgf000099_0001
Figure imgf000100_0001
[0272] In some embodiments, the viral antigen comprisesan amino acid sequence provided in SEQ ID NO: 5, or a variant thereof. In some embodiments, the viral antigen comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1.
[0273] In some embodiments, the vaccine comprises a viral antigen of SARS-CoV-2. In some embodiments, the viral antigen is selected from the group consisting of: orfla, orflab, Spike glycoprotein (S protein), 3a, 3b, Envelope protein (E protein), Membrane protein (M protein), p6, 7a, 7b, 8b, 9b, Nucleocapsid protein (N protein), orfl4, nspl (leader protein), nsp2, nsp3, nsp4, nsp5 (3C-like proteinase), nsp6, nsp7, nsp8, nsp9, nsplO (growth -factor-like protein), nspl2 (RNA-dependentRNA polymerase, orRdRp), nspl3 (RNA 5 '-triphosphatase), nspl4 (3'-to-5' exonuclease), nspl5 (endoRNAse), and nspl6 (2'-O-ribose methyltransferase). In some embodiments, the viral antigen induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine.
[0274] In some embodiments, the viral antigen comprises Spike glycoprotein (S protein) or a fragment of the Spike glycoprotein. In some embodiments, the Spike glycoprotein or a fragment thereof is a monomer or a trimer. In some embodiments, the Spike glycoprotein or a fragment thereof is prefusion stabilized form of Spike glycoprotein or fragment thereof. In some embodiments, the viral antigen of the Spike glycoprotein or a fragment thereof is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. In some embodiments, the viral antigen of the Spike glycoprotein or a fragment thereof is 100% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115 In some embodiments, the viral antigen of the Spike glycoprotein or a fragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, or 25 or more of the mutations to SEQ ID NO: 5 listed in Table 1. In some embodiments, the viral antigen of the Spike glycoprotein or a fragment thereof comprise an amino acid length at least 5 amino acids, 10 amino acids, 20 amino acids, 25 amino acids, 50 amino acids, 100 amino acids, 200 amino acids, ormore. In embodiments, the viral antigen comprising the Spike glycoprotein or a fragment thereof induces formation of neutralizing antibody againstthe Spike -glycoprotein or a fragmentthereof in a subject after administration of the vaccine. [0275] In some embodiments, the vaccine comprises one or more mutations in the viral antigen that confers increased stability of the viral antigen. In some embodiments, the one or more mutations are in a polypeptide sequence of the S1, S2, or in a region between the S1 and S2 subunits of the spike protein. In some embodiments, the one or more mutations are effective to reduce the likelihood of the dissociation of the S1 and S2 subunits that can happen when these subunits of the viral antigen of the spike protein are produced in vitro. In some embodiments, the one or more mutations arrests the S1 and S2 subunits in a pre-fusion trimer conformation. In some embodiments, the one or more mutations increases stable interaction of the subunits following furin cleavage of in vitro produced spike protein.
[0276] In some embodiments, theone ormoremutations are sufficientto curtailthe challenges posed by singly (in vitro) produced S1 and Su subunits of the spike protein (lack of intact viral production of spike protein) and increase the stability, reduce likelihood of S1 and S2 dissociation, and force the S1 and S2 subunits in a pre-fusion trimer conformation. In some embodiments, the one or more mutations comprises one or more proline mutations in the S2 subunit of the spike protein (e.g., K986P and V987P). In some embodiments, the one or more protein mutations comprise two proline mutations, such as K986P and V987P. In some embodiments, the two or more proline mutations are between the two alpha helices between the S1 and S2 subunit of the spike protein. In some embodiments, the one or more mutations comprises a deletion of the furin cleavage site of the spike protein. In some embodiments, the vaccine comprises Ad26.COV.S. In some embodiments, the vaccine comprises mRNA-1273. In some embodiments, the vaccine comprises BNT162b2.
[0277] In some embodiments, the vaccine comprises displaying, conjugating, or complexing the viral antigen with cell-based carrier, polymer(e.g. polyester), or extra cellular vesicles such as exosomes, microvesicles, retrovirus -like particles, apoptotic bodies, apoptosomes, oncosomes, exoph ers, enveloped viruses, exomeres, or other very large extracellular vesicles. [0278] In some embodiments, the vaccine comprises at least one heterologous polynucleotide encoding the viral antigen described herein. In some embodiments, the heterologous polynucleotide comprises a viral vector or a plasmid. Non -limiting examples of heterologous polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), self -amplifying RNA, uridine containing RNA (uRNA), self-amplifyingmRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell -free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequenceof nucleotides is interrupted by non-nucleotide components. In some embodiments, the viral antigen translated from the heterologous polynucleotide induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine.
[0279] In some embodiments, the heterologous DNA sequence is transcribed into mRNA and then translated into the viral antigen after administration of the vaccine to the subject. In some embodiments, the viral antigen translated from the heterologous DNA sequence induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine. In some embodiments, the viral antigen comprising Spike glycoprotein or a fragment thereof is translated from the heterologous DNA sequence and induce formation of neutralizing antibody against the Spike glycoprotein or fragment thereof in a subject after administration of the vaccine.
[0280] In some embodiments, the vaccine comprises mRNA encoding Spike glycoprotein or a fragment thereof. In some embodiments, the vaccine comprises the heterologous mRNA, where the mRNA is a self-amplifying mRNA (saRNA). In some embodiments, the vaccine comprises the heterologous mRNA, where the mRNA comprises uridine (uRNA). In some embodiments, the heterologous RNA sequence is translated into the viral antigen after administration of the vaccine to the subject. In some embodiments, the viral antigen translated from the heterologous RNA sequence induces formation of neutralizing antibody against the viral antigen in a subject after administration of the vaccine. In some embodiments, the viral antigen comprising Spike glycoprotein or a fragment thereof is translated from the heterologous RNA sequence and induce formation of neutralizing antibody against the Spike glycoprotein or fragment thereof in a subject after administration of the vaccine. In some embodiments, the vaccine is mRNA-1273, which encodes a full-length, prefusion stabilized Spike (S) protein. In some embodiments, the vaccine is BNT162 vaccine, comprising mRNA or modified mRNA to express the Spike (S) protein or a fragment thereof. In some instances, the BNT162 vaccine comprises nucleoside modified mRNA (modRNA), uridine containing mRNA (uRNA), or self -amplifying mRNA (saRNA),
[0281] In some embodiments, the vaccine comprising the at least one heterologous polynucleotide encoding the viral antigen comprises one or more DNA or RNA vectors. In some embodiments, the DNA or RNA vectors is plasmids. In some embodiments, the DNA or RNA vectors is viral vector. Viral vectors, and especially retroviral vectors, are engineered to contain a nucleic acid molecule with a sequence encoding any one of the viral antigens described herein and to be delivered to a target tissue. In some embodiments, the viral vectors are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. Non-limiting examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAVs), pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vectors (HSVs). In some instances, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In some embodiments, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some embodiments, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some embodiments, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In additional embodiments, the viral vector is a recombinant viral vector. In some embodiments, the vaccine is an Ad5 -nCoC vaccine comprising nucleic acid sequence encoding the Spike protein. In some embodiments, the vaccine is AZD-1222 vaccine comprising a replication-deficient chimpanzee adenovirus, ChAdOxl, which is engineered to express the Spike (S) protein. In some embodiments, the vaccine is INO-4800 vaccine comprising pGX DNA plasmid with nucleic acid encoding the Spike (S) protein.
[0282] In some embodiments, the vaccine comprises adjuvant. In some instances, the vaccine comprises immune modulator selected from any one of the biomarkers described herein. Companion or Complementary Diagnostic
[0283] Described herein, in some embodiments, are companion complementary diagnostic testing devices or system and methods of their use. In some embodiments, the companion diagnostic testing device or system is used to determine the efficacy or effectiveness of a vaccine against a pathogen described herein. In some embodiments, the companion diagnostic testing device or system is used to identify a person in need of the vaccine. A “companion diagnostic” as used herein, refers to a diagnostic test that may be required for administration of a therapeutic agent for the treatment or prevention of a disease caused by a pathogen disclosed herein. A “complementary diagnostic,” refers to a diagnostic testthatmay be optional for administration of the therapeutic agent. [0284] In some embodiments, the method determines the efficacy or effectiveness of a vaccine described herein by detecting presence, absence, or quantity of neutralizing antibody against the pathogen in a biological sample from a subject th at was administered the vaccine. In some embodiments, the companion diagnostic method confirms adaptive immunity against a pathogen in a subject or in a population of subjects. In some embodiments, the method confirms the need for vaccination against the pathogen for a subject or for a population of subjects. In some embodiments, the companion diagnostic method confirms the prevalent of infection by the pathogen in a population. In some embodiments, the companion diagnostic method identifies a subject or subjects in a population, who are most likely to benefit from being vaccinated with any one of the vaccine describedherein. In some embodiments, the companion diagnostic method identifies a subject or subjects in a population, who are likely to be at increased risk for serious side effects as a result of being vaccinated. In some embodiments, the companion diagnostic method monitors response to vaccination for the purpose of adjusting the dosage or the frequency of vaccination to achieve improved safety, efficacy, or effectiveness of the vaccine. For example, based on efficacy results, subjects aged 65 or older may receive a dosage that contains two times, three times, four times, five times, six times, seven times, eight times, nine times, or ten or more times the amount of an antigen than a dosage for subjects aged 64 or younger. In some embodiments, the companion diagnostic method comprises utilizing the biomarker panel describedherein.
[0285] Companion diagnostic methods provided herein comprise: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, an absence, or a level of a labeled complex between at least two of (i) a capture molecule, (ii) a peptide -conjugate comprising a peptide derived from a pathogen, and (iii) a neutralizing antibody against the pathogen; and (c) classifying the biological sample as having a presence or quantity of the labeled complex. In some embodiments, the method further comprises identifying a vaccine administered to the subject as effective to induce a sufficient or insufficient adaptive immune response against the pathogen in the subject, provided the biological sample is classified as having the presence or the quantity of the labeled complex between (ii) and (iii) . In some embodiments, the method further comprises identifying the subject as in need for a vaccine against the pathogen, provided the biological sample is classified as having the presence or the quantity of the labeled complex between (i) and (ii). In some embodiments, performing (a)-(c) is required for administering the vaccine to the subject (or a population of subjects). In some embodiments, the method further comprises identifying a vaccine administered to the subject as not effective to induce a sufficient or insufficient adaptive immune response against the pathogen in the subject, provided the biological sample is classified as having the presence or the quantity of the labeled complex between (i) and (ii).
[0286] In some embodiments, the level is high relative to an index or a control. In some embodiments, the vaccine is effective, provided the presence or the level of the labeled complex between the peptide-conjugate and the neutralizing antibody against the pathogen is measured. In some embodiments, the vaccine is not effective, provided the presence or the level of the labeled complex between the peptide -conjugate and the capture molecule is measured.
[0287] In some embodiments, method described herein comprises administering a therapeutic agent to a subject in need thereof, provided the subject is identified as having an acute infection by a pathogen. In some embodiments, the therapeutic agent is effective to reduce or eliminate the acute infection by the pathogen, such as an anti-viral therapeutic agent.
[0288] In some embodiments, the companion diagnostic method is performed prior to treating the subject with the therapeutic agent. In some embodiments, performing the companion diagnostic method is required prior to treating the subject with the therapeutic agent. In some embodiments, the therapeutic agent is an anti-viral therapeutic agent. In some embodiments, the anti-viral therapeutic agent treats a disease or condition associated with SARS-CoV-2. In some embodiments, the pathogen is a virus. In some embodiments, the virus is SARS-CoV-2. [0289] In some embodiments, the companion diagnostic method further comprises identifying the subject as having the acute infection comprises by determining the presence, absence, or a quantity of any one of the biomarkers described herein. In some embodiments, the companion diagnostic method comprises determining the presence, absence, or a quantity of both neutralizing antibody and biomarker. In some embodiments, the biomarker is interleukin 6 (IL- 6).
[0290] Described herein, in some embodiments, are complementary diagnostic testing devices and methods of their use to determine to test the biological sample of the subject, where the subject does not display the symptoms ofbeing infected by or is not exposed to any one of the pathogen described herein. In some embodiments, the complementary diagnostic method determines the benefit-risk decision-making about vaccinating the subject or vaccinating the population, where the difference in benefit-risk is clinically meaningful. In some embodiments, the complementary diagnostic method determines the need to vaccinate a subject or subjects of a population, when the status or prevalence of infection by any one of the pathogen described herein is unknown. In some embodiments, the complementary diagnostic method determines the need to vaccinate or treat the subject or subjects of a population, when the status or prevalence of infection by any one of the pathogen described herein is unknown. For example, a subject or subjects of a population have displayed symptoms of respiratory disease or disorder.
[0291] In some embodiments, the complementary diagnostic method confirmsthe particular pathogen that is causing the respiratory disease or disorder in the absence of a reason to test for that particular pathogen. In some embodiments, the complementary diagnostic method determines the efficacy or effectiveness of a vaccine. For example, a vaccine designed for one disease is showingunexpected protective effect against a different disease caused by a different pathogen. In some embodiments, the complementary diagnostic method identifies a subject or subjects in a population, who are most likely to benefit from being vaccinated with any one of the vaccine described herein. In some embodiments, the complementary diagnostic method identifies a subject or subjects in a population, who are likely to be at increased risk for serious side effects as a result of being vaccinated. In some embodiments, the complementary diagnostic method monitors response to vaccination for the purpose of adjusting the dosage or the frequency of vaccination to achieve improved safety, efficacy, or effectiveness of the vaccine. For example, based onresponse results, subjects aged 65 or older may receive a dosage that contains two times, three times, four times, five times, six times, seven times, eight times, nine times, or ten or more times the amount of an antigen than a dosage for subjects aged 64 or younger. In some embodiments, the complementary diagnostic method comprises utilizing the biomarker panel described herein.
Biomarker Panel
[0292] Described herein, in some embodiments, are methods utilizing the systems, devices, and compositions describe herein for detecting presence, absence, quantity, or activity of a biomarker described herein. In some embodiments, the biomarkers are associated with infection by any one of the pathogens described herein. In some embodiments, the biomarkers are induced by infection by the pathogen. In some embodiments, the biomarkers are cytokines induced by infection. In some embodiments, the biomarkers are serological biomarkers. The quantity or activity of the biomarkers can increase or decrease after the infection. In some embodiments, the biomarkers may comprise a peptide or protein that is encoded by a nucleic acid sequence, or that has an amino acid sequence, that is at least 80% identical to any one of SEQ ID NOS: 21-23, 25-42, 44-46, 49-66, 68-70, 73-90, 92-94, or 97-114. [0293] Biomarker panel methods provided herein comprise: (a) providing a biological sample from a subject in need thereof; (b) measuring a presence, an absence, level, or activity of at least one biomarker; (c) classifying the biological sample as having a presence or level of the biomarker associated or induced by an infection; and optionally (d) measuring a presence, an absence, or quantity of neutralizing antibody induced by the infection. In some embodiments, the method identifies the infection caused by the pathogen based on the quantity or activity of the biomarker. In some embodiments, the method identifies the infection caused by the pathogen based on the quantity or activity of the biomarker in a subject, who does not have neutralizing antibody against the pathogen. In some embodiments, the method confirms the infection caused by the pathogen based on the quantity or activity of the biomarker. In some embodiments, the method confirms the infection caused by the pathogen based on the quantity or activity of the biomarker in a subject, who does not have neutralizing antibody against the pathogen. In some embodiments, the method comprise prognosing an immunity or measuring susceptibility to infection based on detecting presence, absence, level, or activity of the biomarkers described herein. In some embodiments, the method comprise prognosing an immunity or measuring susceptibility to infection based on detecting presence, absence, quantity, or activity of the biomarkers in combination with detecting presence, absence, or quantity of the neutralizing antibodies described herein to confirm an infection caused by any one of the pathogen described herein.
[0294] In some embodiments, the presence, absence, level, or activity of the biomarker is detected. In some embodiments, the method detects presence absence, level, or activity of at least one, two, three, four, five, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more biomarkers of the biomarker panel in a biological sample. In some embodiments, the method detects biomarker comprising a non-peptide coding nucleic acid sequence. In some embodiments, the method detects biomarker comprising a peptide-coding nucleic acid sequence such as mRNA or cDNA. In some embodiments, the method detects biomarker comprising peptide or protein.
[0295] In some instances, the method detects presence, absence, or level, or activity of the biomarker by utilizing a nucleic acid sequence by subjecting a biological sample to a nucleic acid-based detection assay. The nucleic acid-based detection assay comprises quantitative polymerase chain reaction (qPCR), gel electrophoresis (including for e.g., Northern or Southemblot), immunochemistry, in situ hybridization such as fluorescent in situ hybridization (FISH), cytochemistry, or sequencing. The sequencing technique comprises next generation sequencing. In some embodiments, the method involves a hybridization assay such as fluorogenic qPCR (e.g., TaqMan™, SYBR green, SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin), which involves a nucleic acid amplification reaction with a specific primer pair, and hybridization of the amplified nucleic acid probes comprising a detectable moiety or molecule that is specific to a target nucleic acid sequence. In some instances, a number of amplification cycles for detecting a target nucleic acid in a qPCR assay is about 5 to about 30 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is at least about 5 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is at most about 30 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 cycles. For TaqMan™ methods, the probe may be a hydrolysable probe comprising a fluorophore and quencher that is hydrolyzed by DNA polymerase when hybridized to a target nucleic acid. In some cases, the presence of a target nucleic acid is determined when the number of amplification cycles to reach a threshold value is less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 cycles. In some instances, hybridization may occur at standard hybridization temperatures, e.g., between about 35 °C and about 65 °C in a standard PCR buffer. An additional exemplary nucleic acid-based detection assay comprises the use of nucleic acid probes conjugated or otherwise immobilized on a bead, multi -well plate, or other substrate, wherein the nucleic acid probes are configured to hybridize with a target nucleic acid sequence. In some instances, the nucleic acid probe is specific to one or more genetic variants disclosed herein is used. In some instances, the nucleic acid probe specific to a SNP or SNV comprises a nucleic acid probe sequence sufficiently complementary to a risk or protective allele of interest, such that hybridization is specific to the risk or protective allele. In some instances, the nucleic acid probe specific to an indel comprises a nucleic acid probe sequence sufficiently complementary to an insertion of a nucleobase within a polynucleotide sequence flanking the insertion, such that hybridization is specific to the indel. In some instances, the nucleic acid probe specific to an indel comprises a probe sequence sufficiently complementary to a polynucleotide sequence flanking a deletion of a nucleobase within the polynucleotide sequence, such that hybridization is specific to the indel. In some instances, the nucleic acid probe specific to a biomarker comprises a nucleic acid probe sequence sufficiently complementary to the polynucleotide sequence of the biomarker. In some instances, the biomarker comprises a transcribed polynucleotide sequence (e.g., RNA, cDNA). In some embodiments, the nucleic acid probe is, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of atleast about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length and sufficient to specifically hybridize under standard hybridization conditions to the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is immobilized on a solid surface and contacted with a probe, forexample by running the isolated target nucleic acid sequence on an agarose gel and transferring the target nucleic acid sequence from the gel to a membrane, such as nitrocellulose. In some embodiments, the probe(s) are immobilized on a solid surface, for example, in an Affymetrix gene chip array, and the probe(s) are contacted with the target nucleic acid sequence.
[0296] In some instances, the method detects presence, absence, or level, or activity of the biomarker by utilizing a protein-based assay. In some embodiments, the method detects the biomarker by an antibody -based assay, where an antibody specific to the biomarker is utilized. In some embodiments, antibody-based detection method can utilize an antibody that binds to any region of the biomarker. Another exemplary method of detecting the biomarker comprises performing an enzyme-linked immunosorbent assay (ELISA). The ELISA assay is a sandwich ELISA or a direct ELISA. Another exemplary method of detecting the biomarker comprises a single molecule array, e.g., Simoa. Other exemplary methods of detecting biomarkers can include immunohistochemistry, lateral flow assay, gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, or various immunological methods such as fluid or gel precipitation reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), immunofluorescent assays, and Western blotting. In some embodiments, antibodies, or antibody fragments, is used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. The antibody or protein is immobilized on a solid supportfor Western blots and immunofluorescencetechniques. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Exemplary supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
Data Analysis Using Personal Electronic Device [0297] Methods disclosed herein further comprise: (a) capturing an image of a detection zone of the testing device; and (b) analyzing data from the image using one or more computer programs. In some embodiments, the one or more computer programs is run on a computing device described here. In some embodiments, capturing is performed with an imaging device described herein. In some cases, capturing and analyzing are performed by a single personal electronic device, such as a smartphone, tablet, or laptop computer. In some embodiments, capturing and analyzing are performed at the point of need (e.g., same time and place as performing the assay with the testing device).
[0298] In some embodiments, capturing comprises taking a still photograph of a liquid phase, such as a liquid composition disclosed herein. In some embodiments, capturing comprises taking a still photograph of the solid surface of the assay assembly described herein. In some embodiments, capturing comprises taking a video of the solid surface of the assay assembly.
[0299] In some embodiments, analyzing comprises detecting binding between the analyte (antibody against an antigenic peptide from the pathogen) and peptide -conjugate. In some embodiments, analyzing comprises subtracting a background signal, thereby increasing the signal to noise ratio. In some embodiments, analyzing is performed by a data analytics module of an application or computer program of the computing device. In some embodiments, the analyzing by the data analytics module comprises performing machine learning.
Numbered Embodiments'.
[0300] The following non-limiting embodiments provide illustrative examples of the disclosure, but do not limit the scope of the disclosure.
[0301] Embodiment 1. A method of identifying adaptive immunity to a coronavirus in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) introducing the biological sample with (i) a detectable peptide derived from a spike glycoprotein of a coronavirus; and (ii) one or more capture molecules derived from an angiotensin-converting enzyme 2 (ACE2) receptor; and (c) detecting a binding complex between the detectable peptide and the one or more capture molecules .
[0302] Embodiment 2. The method of embodiment 1, wherein steps (a)-(c) are performed at the point of need.
[0303] Embodiment 3. The method of any one of embodiments 1-2, wherein steps (a)-(c) are performed at the point of care.
[0304] Embodiment 4. The method of any one of embodiments 1-3, wherein the detectable peptide, the one or more capture molecules, or both of the detectable peptide and the one or more capture molecules is coupled to a surface, which surface optionally comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane. [0305] Embodiment 5. The method of any one of embodiments 1-5, wherein the surface is a passivated surface.
[0306] Embodiment 6. The method of embodiment 5 , wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (aery lie acid) (PAA), polyacrylamide, poly (N -isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethyleneglycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0307] Embodiment 8. The method of any one of embodiments 4-6, wherein the surfaceis or comprises an interior surface of a capillary.
[0308] Embodiment 9. The method of any one of embodiments 4-7, wherein the surfaceis or comprises an interior surface of a capillary in a capillary flow cell device.
[0309] Embodiment 10. The method of any one of embodiments 4-5, wherein the surface comprises a porous membrane, optionally, comprising cellulose.
[0310] Embodiment 11. The method of any one of embodiments 4-5, wherein the surface comprises a waveguide.
[0311] Embodiment 12. The method of embodiment 11, wherein the waveguide is a planar waveguide.
[0312] Embodiment 13. The method of any one of embodiments 1-12, whereinthe one ormore capture molecules comprises two or more capture molecules.
[0313] Embodiment 14. The method of any one of embodiments 4-13, whereinthe one ormore capture molecules is coupled to the surface by a covalent bond, a linker, or a combination thereof.
[0314] Embodiment 15. The method of embodiment 14, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0315] Embodiment 16. Themethod of anyone of embodiments 1-15, whereinthe one ormore capture molecules is a fusion polypeptide.
[0316] Embodiment 17. The method of embodiment 16, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody. [0317] Embodiment 18. The method of any one of embodiments 1-17, whereinthe one ormore capture molecules is bound by an antibody that is coupled to the surface.
[0318] Embodiment 19. The method of any one of embodiments 1-18, wherein the detectable peptide or the one or more capture molecules or a combination thereof comprises a nanoparticle, a fluorescent metal, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0319] Embodiment 20. The method of any one of embodiments 1-19, wherein the detectable peptide is or comprises a fluorescence resonance energy transfer (FRET) donor or acceptor.
[0320] Embodiment 21 . The method of any one of embodiments 1-20, whereinthe one ormore capture molecules comprises a detectable label coupled thereto.
[0321] Embodiment22. The methodof embodiment21 , wherein the detectable label comprises a FRET donor or acceptor.
[0322] Embodiment 23. The method of any one of embodiments 20-21, wherein the FRET is lanthanide-based FRET (LRET).
[0323] Embodiment 24. The method of embodiment 20 or 22, wherein the FRET donor comprises a luminescent lanthanide.
[0324] Embodiment 25. The method of embodiment 24, wherein the luminescent lanthanide is terbium or europium.
[0325] Embodiment 26. The method of embodiment 24, wherein the FRET acceptor comprises a fluorophore, such as an organic fluorophore.
[0326] Embodiment 27. The methodof any one of embodiments 1-26, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, andplastic. [0327] Embodiment28. The method of any one of embodiments 1-27, wherein the nanoparticle is magnetic.
[0328] Embodiment 29. The methodof any one of embodiments 1-28, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0329] Embodiment 30. The method of any one of embodiments 1-29, further comprises calculating a percent neutralization of binding between the detectable peptide and the one or more capture molecules.
[0330] Embodiment 31. The method of embodiment 30, wherein the percent neutralization is calculated by a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein
ST is the signal detected in (c), SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein.
[0331] Embodiment 32, The method of any one of embodiments 1-30, wherein a percent neutralization of about 62% correlates to about 400 reciprocal titers when measured using a cell-based pseudovirion neutralization assay (PNA).
[0332] Embodiment 33. The method of any one of embodiments 1-29, further comprising comparing a number of the binding complexes to a number of binding complexes in a control sample with a known amount of a neutralizing antibody in it having a known IC50.
[0333] Embodiment 34. The method of embodiment 33, wherein if the number of the binding complexes is low relative to the number of binding complexes in the control sample, then identifying the subject as being immune to an infection by the coronavirus; and if the number of the binding complexes is the same as or high relative to the number of binding complexes in the control sample, then identifying the subject as not being immune to an infection by the coronavirus.
[0334] Embodiment 35. The method of any one of embodiments 30-33, further comprising comparing the percent neutralization for the biological sample to the percent neutralization calculated for a control sample.
[0335] Embodiment 36. The method of embodiment 35, wherein the control sample is a negative sera or blood sample spiked with a known amount of a neutralizing antibody in it having a known IC50, or a positive sera or blood sample obtained from a control subject.
[0336] Embodiments 7. The method of embodiment 35, wherein the control subject is a subject that (a) recovered from an infection by the coronavirus, (b) is vaccinated against the coronavirus, (c) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (d) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus.
[0337] Embodiment 38. The method of any one of embodiments 35-37, wherein if the percent neutralization for the biological sample is low relative to the percent neutralization calculated for the control sample, then identifying the subject as being immune to an infection by the coronavirus; and if the number of the binding complexes is the same as or high relative to the number of binding complexes in the control sample, then identifying the subject as not being immune to an infection by the coronavirus. [0338] Embodiment 39. The method of any one of embodiments 35 -38, further comprising comparing the percent neutralization of the biological sample to the percent neutralization measured with the cell-based PNA, thereby correlating the cell-based PNA with the percent neutralization of the biological sample.
[0339] Embodiment 40. The method of any one of embodiments 35 -39, further comprising detecting a percent inhibition in the biological sample comprises between about 1-100%, 10- 90%, 20-80%, 30-70%, 40-60%, or about 50% inhibition in the biological sample.
[0340] Embodiment 41. The method of any one of embodiments 35 -39, further comprising detecting a percent inhibition in the biological sample comprising between about 30-40%, 40%-50%, 60-70%, 70-80%.
[0341] Embodiment42. Themethod of anyone of embodiments 1-41, wherein the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0342] Embodiment 43. The method of any one of embodiments 1-42, wherein the detectable peptide derived from the spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS- CoV).
[0343] Embodiment 44. The method of embodiment 43, wherein the SARS-CoV is SARS- CoV-2 or a variant or a derivative thereof.
[0344] Embodiment 45. The method of any one of embodiments 43-44, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
[0345] Embodiment 46. The method of any one of embodiments 1-45, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0346] Embodiment 47. The method of any one of embodiments 1-46, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0347] Embodiment 48. The method of any one of embodiments 1-47, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2 [0348] Embodiment 49. The method of any one of embodiments 1-48, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0349] Embodiment 50. The method of any one of embodiments 1-49, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0350] Embodiment 51 . The method of any one of embodiments 1-50, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0351] Embodiment 52. The method of any one of embodiments 1-51, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible using FRET.
[0352] Embodiment 53. The method of embodiment 52, wherein the complex between the peptide-conjugate and the one or more capture molecules emits a first fluorescent signal and a second fluorescent signal, which ratio of the first fluorescent signal and the second fluorescent signal is detectable using FRET.
[0353] Embodiment 54. The method of any one of embodiments 1-53, wherein the binding complexes are optically connected to a planar waveguide optical sensor.
[0354] Embodiment 55. The method of any one of embodiments 4-54, wherein the surface is housed in a cartridge that is disposable.
[0355] Embodiment 56. The method of embodiment 55, wherein the cartridge is made from a polymer material comprising metal, agarose, acrylic, or plastic.
[0356] Embodiment 57. The method of any one of embodiments 1-56, further comprising providing a web -based portal on the personal electronic device.
[0357] Embodiment 58. The method of embodiment 57, wherein method further comprises providing an application on the web-based portal.
[0358] Embodiment 59. The method of embodiment 58, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
[0359] Embodiment 60. The method of any one of embodiments 58-59, wherein method further comprises providing a data analytics module at the application. [0360] Embodiment 61. The method of embodiment 60, further comprising analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
[0361] Embodiment 62. The method of embodiment 61, wherein the positive or the negative result is relative to a threshold number of the binding complexes or a threshold percent neutralization calculated for a control that is indicative of immunity against the coronavirus.
[0362] Embodiment 63. The method of embodiment 62, wherein the threshold percent neutralization is equal to or about 60%.
[0363] Embodiment 64. The method of any one of embodiments 60-63, further comprising analyzing the result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules on the surface.
[0364] Embodiment 65. The method of any one of embodiments 60-64, further comprising analyzingthe result, by the data analytics module, to determine a ratio of the binding complexes to unbound detectable peptide or the one or more capture molecules, wherein a high ratio of the binding complexes to unbound detectable peptide or the one or more capture molecules indicates a low level of immunity, and a low ratio of the binding complexes to unbound detectable peptide or the one or more capture molecules indicates a high level of immunity.
[0365] Embodiment 66. The method of any one of embodiments 60-65, further comprising normalizing, by the data analytics module, the result by subtracting background noise.
[0366] Embodiment 67. The method of any one of embodiments 60-66, further comprising identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
[0367] Embodiment 68. The method of embodiment 67, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
[0368] Embodiment 69. The method of any one of embodiments 60-68, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
[0369] Embodiment 70. The method of any one of embodiments 60-69, further comprising providing a data store that is a cloud-based data store or a web-based data store, or a local data store. [0370] Embodiment 71. The method of any one of embodiments 60-71, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
[0371] Embodiment 72. The method of embodiment 71 , wherein the external datais data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0372] Embodiment 73. The method of any one of embodiments 71-72, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
[0373] Embodiment 74. The method of any one of embodiments 71-73, further comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0374] Embodiment 75. The method of any one of embodiments 1-74, wherein the biological sample comprises one or more antibodies specific to the detectable peptide.
[0375] Embodiment 76. The method of any one of embodiments 1-74, wherein the biological sample does not consist of one or more antibodies specific to the detectable peptide.
[0376] Embodiment 77. The method of any one of embodiments 75-76, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
[0377] Emb odiment 78. The method of any one of embodiments 1-77, wherein the subj ect was, or is, exposed to the coronavirus.
[0378] Embodiment 79. The method of any one of embodiments 1-77, wherein exposure of the subject to the coronavirus is unknown.
[0379] Embodiment 80. The method of any one of embodiments 1-79, wherein the method is performed on a plurality of biological samples obtained from a plurality of subjects.
[0380] Embodiment 81. The method of embodiments 80, further comprising identifying a level of the adaptive immunity of the plurality of subjects to the coronavirus.
[0381] Embodiment 82. The method of any one of embodiments 80-81, further comprising monitoring a spread of infection of the plurality of subjects by the coronavirus.
[0382] Embodiment 83. The method of any one of embodiments 1-82, wherein the subject was administered a vaccine against the coronavirus. [0383] Embodiment 84. The method of embodiment 83, further comprising determining that the vaccine is effective to substantially immunize the subject against the coronavirus, based, at least in part on the binding complex detected in (c).
[0384] Embodiment 85. The method of any one of embodiments 1-84, wherein the biological sample comprises blood, urine, saliva, or feces.
[0385] Embodiment 86. The method of embodiment 85, wherein the bloodis capillary blood. [0386] Embodiment 87. The method of embodiment 86, wherein the capillary blood is obtained from the subject by a prick of the subject’ s finger.
[0387] Embodiment 88. The method of any one of embodiments 85-87, wherein method further comprises separating serum from the blood in the biological sample.
[0388] Embodiment 89. The method of any one of embodiments 1-88, wherein the detecting in (c) comprises capturing an image of the surface with an imaging device to detect the binding complex between the detectable peptide and the one or more capture molecules.
[0389] Embodiment 90. The method of embodiments 89, wherein the imaging device is the personal electronic device.
[0390] Embodiment 91. The method of embodiment 90, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
[0391] Embodiment 92. The method of any one of embodiments 1-91, further comprising calculating a percent neutralization on a scale of 0-100% based on the binding complex detected in (c), wherein:
(i) a percent neutralization of 0% correlates with 0 antibody titers using the cell -based PNA;
(ii) a percent neutralization of 62% correlates with a 400 antibody titers using the cellbased PNA; or
(iii) a percent neutralization of 100% correlates with above 800 antibody titers using the cell-based PNA, or any combination thereof.
[0392] Embodiment 93. The method of any one of embodiments 1-92, wherein the method is performed without a wash step to wash away unbound detectable peptide, one or more capture molecules, or any combination thereof.
[0393] Embodiment 94. The method of any one of embodiments 1-93, wherein the method is performed in solution.
[0394] Embodiment 95. The method of any one of embodiments 1-94, wherein the introducing in (a) is performed in a well or container. [0395] Embodiment 96. The method of any one of embodiments 1-95, further comprising repeating the method on a plurality of biological samples obtained from the subject, wherein at least two biological samples of the plurality of biological samples are obtained at different time points.
[0396] Embodiment 97. The method of any one of embodiments 1-96, wherein the subject has an underlying immune-comprising condition comprising a pregnancy, an immune-mediated disease, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof.
[0397] Embodiment 98. The method of any one of embodiments 1-97, wherein the subject is pregnant or breastfeeding.
[0398] Embodiment 99. The method of any one of embodiments 1-98, wherein the subject is receiving an immunosuppressant therapy.
[0399] Embodiment 100. The method of embodiment 99, wherein the immunosuppressant therapy comprises a biologic drug (e.g. , adalimumab (Humira®) and infliximab (Remicade®)), calcineurin inhibitors (e.g., tacrolimus (Envarsus XR® or Protopic) and cyclosporine (Gengraf®, Neoral® or Sandimmune®)), corticosteroids (e.g., prednisone), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolate mofetil (CellCept®)), janus kinase inhibitors (e.g., tofacitinib (Xeljanz®)), mechanistic target of rapamycin (mTOR) inhibitors (e.g., sirolimus (Rapamune®)), or monoclonal antibodies (e.g., basiliximab (Simulect®)), or any combination thereof.
[0400] Embodiment 101. The method of any one of embodiments 1-100, wherein the method is performed with an accuracy comprising greater than or equal to or about 50%, 60%, 70%, 80%, 90%, or 99%.
[0401] Embodiment 102. The method of any one of embodiments 1-101, wherein the method is performed with a specificity comprisinggreaterthan or equal to about 50%, 60%, 70%, 80%, 90%, or 99%.
[0402] Embodiment 103. The method of any one of embodiments 1-102, wherein the method is performed with a sensitivity comprisinggreaterthan or equal to about 50%, 60%, 70%, 80%, 90%, or 99%.
[0403] Embodiment 104. The method of any one of embodiments 1 -102, wherein the method is performed with a percent coefficient of variation (CV) of less than or equal to about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. [0404] Embodiment 105. The method of any one of embodiments 101-104, wherein the accuracy is measured by:
(a) introducing a reference sample with (i) the detectable peptide; and (ii) the one or more capture molecules, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC50; and
(b) detecting a binding complex between the detectable peptide and the one or more capture molecules; and
(c) determine an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50.
[0405] Embodiment 106. The method of any one of embodiments 1-105, further comprising administering to the subject a vaccine against the coronavirus, provided the percent neutralization calculated for the subjectbased on the detecting in (c) is lower than 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%..
[0406] Embodiment 107. The method of embodiment 106, wherein the vaccine is a full dose or booster dose of the vaccine.
[0407] Embodiment 108. The method of any one of embodiments 1-107, selectingthe subject for treatment with a vaccine against the coronavirus, provided the percent neutralization calculated for the subject based on the detecting in (c) is lower than 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
[0408] Embodiment 110. The method of any one of embodiments 1-109, further comprising identifying the subject as being sufficiently immune to an infection by the coronavirus, provided the percent neutralization calculated for the subject based on the detecting in (c) is higher than 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%.
[0409] Embodiment 11 1. The method of any one of embodiments 1-110, further comprising:
(a) identifying the subject as needing a dose of a vaccine against the coronavirus, provided the percentneutralization calculated for the subjectbased on the detectingin (c) is lowerthan 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%;
(b) suspending a treatment regimen comprising the immunosuppressant therapy for a period of time during which period of time, the subject is administered the dose of the vaccine.
[0410] Embodiment 112. The method of any one of embodiments 1-113, wherein the subject is a pediatric subject. KITS
[0411] Described herein, in some embodiments, are kits for obtaining a biological sample or detecting an analyte in the biological sample. In some embodiments, the kits may comprise a biological sample collection device, such as, for example, a transdermal puncture device (e.g, finger prick lance) or other sample collection device (e.g., a swab). In some embodiments, the kits may comprise a dried blood spot (DBS) card described herein for storing the biological sample. In some embodiments, the kits comprise an assay assembly described herein, such as a lateral flow assay or an ELISA that is capable of detecting neutralizing antibodies against a pathogen of interest in the biological sample.
[0412] In some embodiments, the kits described herein comprise instructions. In some embodiments, the instructions provide information about howto obtain the biological sample. In some embodiments, the instructions comprise information about how to discard the biological sample. In some embodiments, the kits comprise information about how to download a mobile application for a personal electronic device that captures and analyzes the results of the testing device. In some embodiments, the kits comprise a survey having one or more clinical questions pertaining to a symptom of an infection by a pathogen of interest. In some embodiments, the survey is on the back of the DBS card of the kit.
EMBODIMENTS
[0413] The following non-limiting embodiments provide illustrative examples of the disclosure, but do not limit the scope of the disclosure.
[0414] Embodiment 1. A system comprising: (a) one or more capture molecules derived from an angiotensin-converting enzyme 2 (ACE2) receptor; and (b) apeptide-conjugate comprising: (i) a peptide derived from a spike glycoprotein of a coronavirus; and (ii) a detectable moiety.
[0415] Embodiment 2. The system of embodiment 1 further comprises (a) a surface; and (b) an imaging device configured to capture an image of a complex between the peptide -conjugate and the one or more capture molecules on the surface when the complex is coupled to the surface.
[0416] Embodiment 3. The system of any one of embodiments 1-2 further comprises an imaging device configured to capture an image of a complex between the peptide -conjugate and the one or more capture molecules.
[0417] Embodiment 4. The system of any one of embodiments 1-3 comprises a container comprising (a) and (b), wherein the container is portable. [0418] Embodiment 5. The system of any one of embodiments 1-4 is a point of need system. [0419] Embodiment 6. The system of any one of embodiments 1-5 is a point of care system.
[0420] Embodiment 7. The system of any one of embodiments 1-6, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
[0421] Embodiment 8. The system of any one of embodiments 1-7, wherein the surface is a passivated surface.
[0422] Embodiment 9. The system of any one of embodiments 1 -8, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (aery lie acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly glutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0423] Embodiment 10. The system of any one of embodiments 1-9, wherein the complex is coupled to the surface.
[0424] Embodiment 11 . The system of any one of embodiments 1-10, wherein, the complex is coupled to the surface by a covalent bond, a linker, or a combination thereof.
[0425] Embodiment 12. The system of any one of embodiments 1-11, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0426] Embodiment 13. The system of any one of embodiments 1-12, wherein the one or more capture molecules comprises two or more capture molecules.
[0427] Embodiment 14. The system of any one of embodiments 1-13, wherein the one or more capture molecules is coupled to the surface.
[0428] Embodiment 15. The system of any one of embodiments 1-14, whereinthe one or more capture molecules is coupled to the surface by a covalent bond, a linker, or a combination thereof.
[0429] Embodiment 16. The system of any one of embodiments 1-15, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0430] Embodiment 17. The system of any one of embodiments 1-16, whereinthe one or more capture molecules is a fusion polypeptide. [0431] Embodiment 18. The system of any one of embodiments 1- 17, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
[0432] Embodiment 19. The system of any one of embodiments 1-18, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
[0433] Embodiment 20. The system of any one of embodiments 1- 19, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0434] Embodiment21 . The system of any one of embodiments 1-20, whereinthe nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, andplastic. [0435] Embodiment 22. The system of any one of embodiments 1-21, whereinthe nanoparticle is magnetic.
[0436] Embodiment 23. The system of any one of embodiments 1-22, the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0437] Embodiment24. The system of any one of embodiments 1-23, wherein, the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0438] Embodiment 25. The system of any one of embodiments 1-24, wherein, the peptide derived from a spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV). [0439] Embodiment 26. The system of any one of embodiments 1-25, wherein the SARS-CoV is SARS-CoV-2.
[0440] Embodiment 27. The system of any one of embodiments 1-26, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
[0441] Embodiment 28. The system of any one of embodiments 1-27, wherein the at least a portion of the spike protein comprises a subunit 1 of the spike protein.
[0442] Embodiment 29. The system of any one of embodiments 1-28, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein. [0443] Embodiment 30. The system of any one of embodiments 1-29, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
[0444] Embodiment 31 . The system of any one of embodiments 1-30, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0445] Embodiment 32. The system of any one of embodiments 1-31, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 4
[0446] Embodiment 33. The system of any one of embodiments 1-32, wherein the complex between the peptide-conjugate and the one or more capture molecules on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence. [0447] Embodiment 34. The system of any one of embodiments 1-33, where the system further comprises a housing at least partially enclosing the surface.
[0448] Embodiment 35. The system of any one of embodiments 1-34, wherein the system further comprises a sample receptor configured to receive a biological sample from a subject. [0449] Embodiment 36. The system of any one of embodiments 1-35, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface.
[0450] Embodiment 37. The system of any one of embodiments 1-36, wherein the biological sample comprises one or more antibodies specific to the peptide.
[0451] Embodiment 38. The system of any one of embodiments 1-37, wherein the biological sample does not consist of one or more antibodies specific to the peptide. Embodiment 10. the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D. Embodiment 10. the subject was, oris, exposed to the coronavirus.
[0452] Embodiment 39. The system of any one of embodiments 1-38, wherein exposure of the subject to the coronavirus is unknown.
[0453] Embodiment 40. The system of any one of embodiments 1-39, wherein the subject was administered a vaccine against the coronavirus. [0454] Embodiment 41 . The system of any one of embodiments 1-40, wherein the biological sample comprises blood, urine, saliva, or feces.
[0455] Embodiment 42. The system of any one of embodiments 1-41, wherein the blood is capillary blood.
[0456] Embodiment 43. The system of any one of embodiments 1-42, wherein the system further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
[0457] Embodiment 44. The system of any one of embodiments 1-43, wherein the sample receptor comprises a filter to separate serum from the blood.
[0458] Embodiment 45. The system of any one of embodiments 1-44, wherein the system further comprises a data store for storing data from the image that is captured by the imaging device.
[0459] Embodiment 46. The system of any one of embodiments 1-45, wherein the data store is a cloud-based or a web-based data store, or a local data store.
[0460] Embodiment 47. The system of any one of embodiments 1-46, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
[0461] Embodiment 48. The system of any one of embodiments 1-47, wherein the external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0462] Embodiment 49. The system of any one of embodiments 1-48, wherein the external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
[0463] Embodiment 50. The system of any one of embodiments 1-49, wherein the system further comprises an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0464] Embodiment 51. The system of any one of embodiments 1-50, wherein the imaging device is a personal electronic device.
[0465] Embodiment 52. The system of any one of embodiments 1-51, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
[0466] Embodiment 53. The system of any one of embodiments 1-52, wherein the personal electronic device comprises a web-based portal. [0467] Embodiment 54. The system of any one of embodiments 1-53, wherein the web-based portal utilizes an application.
[0468] Embodiment 55. The system of any one of embodiments 1-54, wherein the application is configured to receive data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, and external data from one or more external device.
[0469] Embodiment 56. The system of any one of embodiments 1-55, wherein the application comprises a data analytics module configured to analyze the result by: (a) determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity; (b) determining an absolute number of complexes between the peptide-conjugate and the ACE2 receptor on the surface; or (c) determining a level of binding between the peptide-conjugate and the ACE2, wherein a high level of binding indicates a low level of immunity, and a low lev el of binding indicates a high level of immunity.
[0470] Embodiment 57. The system of any one of embodiments 1-56, wherein the positive or the negative result is relative to a threshold number of complexes between the peptide- conjugate and the ACE2 receptor on the surface.
[0471] Embodiment 58. The system of any one of embodiments 1-57, wherein the threshold number is predetermined relative to an index a control.
[0472] Embodiment 59. The system of any one of embodiments 1-58, wherein the data analytics module is further configuredto normalize the resultby subtractingbackground noise. [0473] Embodiment 60. The system of any one of embodiments 1-59, wherein the data analytics module is further configuredto identify a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
[0474] Embodiment 61. The system of any one of embodiments 1-60, wherein the data analytics module utilizes geofencing from coordinates of the personal electronic device to identify the geographical location.
[0475] Embodiment 62. The system of any one of embodiments 1-61, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
[0476] Embodiment 63. The system of any one of embodiments 1-62, wherein system further comprises one or more capture molecules specific to one or more antibodies against the coronavirus. [0477] Embodiment 64. The system of any one of embodiments 1-63, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
[0478] Embodiment 65. The system of any one of embodiments 1-64, wherein system further comprises a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
[0479] Embodiment 66. The system of any one of embodiments 1-65, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen-binding fragment. Embodiment 10. the antibody or antigen-binding fragment is monoclonal, antibody or antigen-binding fragment is polyclonal.
[0480] Embodiment67. Aspects disclosed herein comprise system comprising: (a) one or more capture molecules derived from a spike glycoprotein of a coronavirus; (b) a peptide-conjugate comprising: (i) a peptide derived from angiotensin -converting enzyme 2 (ACE2) receptor, and (ii) a detectable moiety.
[0481] Embodiment 68. The system of embodiment 67, wherein the system further comprises (a) a surface; and (b) an imaging device configured to capture an image of a complex between the peptide-conjugate and the one or more capture molecules on the surface when the complex is coupled to the surface.
[0482] Embodiment 69. The system of any one of embodiments 67-68, wherein the system further comprises an imaging device configured to capture an image of a complex between the peptide-conjugate and the one or more capture molecules.
[0483] Embodiment 70. The system of any one of embodiments 67-69, wherein the system further comprises a container comprising (a) and (b), wherein the container is portable.
[0484] Embodiment 71 . The system of any one of embodiments 67-70, wherein the system is a point of need system.
[0485] Embodiment 72. The system of any one of embodiments 67-71, wherein the point of need is a point of care system.
[0486] Embodiment 73. The system of any one of embodiments 67-72, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
[0487] Embodiment 74. The system of any one of embodiments 67-73, wherein the surface is a passivated surface.
[0488] Embodiment 75. The system of any one of embodiments 67-74, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly (aery lie acid) (PAA), polyacrylamide, poly(N -isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxyl ethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), poly glutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0489] Embodiment 76. The system of any one of embodiments 67-75, wherein the complex is coupled to the surface.
[0490] Embodiment 77. The system of any one of embodiments 67-76, wherein the complex is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0491] Embodiment 78. The system of any one of embodiments 67-77, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0492] Embodiment 79. The system of any one of embodiments 67-78, wherein the one or more capture molecules comprises two or more capture molecules.
[0493] Embodiment 80. The system of any one of embodiments 67-79, wherein the one or more capture molecules is coupled to the surface.
[0494] Embodiment 81. The system of any one of embodiments 67-80, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0495] Embodiment 82. The system of any one of embodiments 67 -81, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0496] Embodiment 83. The system of any one of embodiments 67-82, wherein the one or more capture molecules is a fusion polypeptide.
[0497] Embodiment 84. The system of any one of embodiments 67-83, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
[0498] Embodiment 85. The system of any one of embodiments 67-84, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
[0499] Embodiment 86. The system of any one of embodiments 67-85, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0500] Embodiment 87. The system of any one of embodiments 67-86, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic. [0501] Embodiment 88. The system of any one of embodiments 67-87, wherein the nanoparticle is magnetic.
[0502] Embodiment 89. The system of any one of embodiments 67-88, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0503] Embodiment 90. The system of any one of embodiments 67-89, wherein the peptide derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1
[0504] Embodiment 91. The system of any one of embodiments 67-90, wherein the one or more capture molecules derived from a spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
[0505] Embodiment 92. The system of any one of embodiments 67-91, wherein the SARS- CoV is SARS-CoV-2.
[0506] Embodiment 93. The system of any one of embodiments 67-92, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
[0507] Embodiment 94. The system of any one of embodiments 67-93, wherein the at least a portion of the spike protein comprises a subunit 1 of the spike protein.
[0508] Embodiment 95. The system of any one of embodiments 67-94, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0509] Embodiment 96. The system of any one of embodiments 67-95, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
[0510] Embodiment 97. The system of any one of embodiments 67-96, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0511] Embodiment 98. The system of any one of embodiments 67-97, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0512] Embodiment 99. The system of any one of embodiments 67-98, wherein the complex between the peptide-conjugate and the one or more capture molecules on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence. [0513] Embodiment 100. The system of any one of embodiments 67-99, wherein system further comprises one or more capture molecules specific to one or more antibodies against the coronavirus. Embodiment 101 . The system of any one of embodiments 67-100, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
[0514] Embodiment 102. The system of any one of embodiments 67-101, wherein system further comprises a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
[0515] Embodiment 103. The system of any one of embodiments 67-102, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen -binding fragment.
[0516] Embodiment 104. The system of anyone of embodiments 67-103, wherein the antibody or antigen-binding fragment is monoclonal, antibody or antigen-binding fragment is polyclonal.
[0517] Embodiment 105. The system of any one of embodiments 67-104, wherein system further comprises a housing at least partially enclosing the surface.
[0518] Embodiment 106. The system of any one of embodiments 67-105, wherein system further comprises a sample receptor configured to receive a biological sample from a subject. [0519] Embodiment 107. The system of any one of embodiments 67-106, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface.
[0520] Embodiment 108. The system of any one of embodiments 67-107, wherein the biological sample comprises one or more antibodies specific to the peptide.
[0521] Embodiment 109. The system of any one of embodiments 67-108, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
[0522] Embodiment 110. The system of any one of embodiments 67-109, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D. [0523] Embodiment 111. The system of any one of embodiments 67 -110, wherein the subject was, or is, exposed to the coronavirus.
[0524] Embodiment 112. The system of any one of embodiments 67-111, wherein exposure of the subject to the coronavirus is unknown.
[0525] Embodiment 113. The system of any one of embodiments 67-112, wherein the subject was administered a vaccine against the coronavirus.
[0526] Embodiment 114. The system of any one of embodiments 67-113, wherein the biological sample comprises blood, urine, saliva, or feces.
[0527] Embodiment 115. The system of any one of embodiments 67-114, wherein the blood is capillary blood.
[0528] Embodiment 116. The system of any one of embodiments 67-115, wherein system further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
[0529] Embodiment 117. The system of any one of embodiments 67-116, wherein the sample receptor comprises a filter to separate serum from the blood.
[0530] Embodiment 118. The system of any one of embodiments 67-117, wherein system further comprises a data store for storing data from the image that is captured by the imaging device.
[0531] Embodiment 119. The system of any one of embodiments 67-118, wherein the data store is a cloud-based or a web-based data store, or a local data store.
[0532] Embodiment 120. The system of any one of embodiments 67-119, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
[0533] Embodiment 121. The system of any one of embodiments 67-120, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0534] Embodiment 122. The system of any one of embodiments 67-121, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
[0535] Embodiment 123. The system of any one of embodiments 67-122, wherein system further comprises an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device. [0536] Embodiment 124. The system of any one of embodiments 67-123, wherein the imaging device is a personal electronic device.
[0537] Embodiment 125. The system of any one of embodiments 67-124, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
[0538] Embodiment 126. The system of any one of embodiments 67-125, wherein the personal electronic device comprises a web -based portal.
[0539] Embodiment 127. The system of any one of embodiments 67-126, wherein the webbased portal utilizes an application.
[0540] Embodiment 128. The system of any one of embodiments 67-127, wherein the application is configured to receive data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, and external data from one or more external device.
[0541] Embodiment 129. The system of any one of embodiments 67-128, wherein the application comprises a data analytics module configured to analyze the result by: (a) determining whether the resultis a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity; (b) determining an absolute number of complexes between the peptide-conjugate and the ACE2 receptor on the surface; or (c) determining a level of binding between the peptide -conjugate and the ACE2, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
[0542] Embodiment 130. The system of any one of embodiments 67-129, wherein the data analytics module is further configured to analyze the result by determining whether the result is a positive result or a negative result, wherein a positive result indicates a presence of an acute infection by the coronavirus, and a negative results indicates an absence of the acute infection by the coronavirus.
[0543] Embodiment 131. The system of any one of embodiments 67-130, wherein the positive or the negative result is relative to a threshold number of complexes between the peptide- conjugate and the ACE2 receptor on the surface.
[0544] Embodiment 132. The system of any one of embodiments 67-131, wherein the positive or the negative result is relative to a threshold number of complexes between the one or more capture molecules specific to the one or more antibodies against the coronavirus.
[0545] Embodiment 133. The system of any one of embodiments 67-132, wherein the threshold number is predetermined relative to an index a control. [0546] Embodiment 134. The system of any one of embodiments 67-133, wherein the data analytics module is further configured to normalize the resultby subtractingbackground noise. [0547] Embodiment 135. The system of any one of embodiments 67-134, wherein the data analytics module is further configuredto identify a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
[0548] Embodiment 136. The system of any one of embodiments 67-135, wherein the data analytics module is further configuredto identify a geographical location comprised of subjects for which a positive result was determined to identify regions of infection or re-infection, or to recommend further testing.
[0549] Embodiment 137. The system of any one of embodiments 67-136, wherein the data analytics module utilizes geofencing from coordinates of the personal electronic device to identify the geographical location.
[0550] Embodiment 138. The system of any one of embodiments 67-137, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
[0551] Embodiment 139. Aspects disclosed herein provide a method of identifying adaptive immunity to a coronavirus in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) producing a mixture by introducing the biological sample with a detectable peptide derived from a spike glycoprotein of a coronavirus; (c) bringing the mixture into contact with one or more capture molecules derived from an angiotensin -converting enzyme 2 (ACE2) receptor; (d) detecting a number of binding complexes between the detectable peptide and the one or more capture molecules; (e) if the number of the binding complexes is low relative to an index or a control, then identifyingthe subject as being immune to an infection by the coronavirus; and (f) if the number of the binding complexes is high relative to an index ora control, then identifyingthe subject as notbeingimmuneto an infection by the coronavirus.
[0552] Embodiment 140. The method of embodiment 139, wherein steps (a)-(f) are performed at the point of need.
[0553] Embodiment 141 . The method of any one of embodiments 139-140, wherein steps (a)- (f) are performed at the point of care.
[0554] Embodiment 142. The method of any one of embodiments 139-141, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane. [0555] Embodiment 143. The method of any one of embodiments 139- 142, wherein the surface is a passivated surface.
[0556] Embodiment 144. The method of any one of embodiments 139-143, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(m ethyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0557] Embodiment 145. The method of any one of embodiments 139-144, wherein the one or more capture molecules comprises two or more capture molecules.
[0558] Embodiment 146. The method of any one of embodiments 139-145, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0559] Embodiment 147. The method of any one of embodiments 139-146, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0560] Embodiment 148. The method of any one of embodiments 139-147, wherein the one or more capture molecules is a fusion polypeptide.
[0561] Embodiment 149. The method of any one of embodiments 139-148, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
[0562] Embodiment 150. The method of any one of embodiments 139-149, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
[0563] Embodiment 151. The method of any one of embodiments 139-150, wherein the detectable peptide comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0564] Embodiment 152. The method of any one of embodiments 139-151, wherein The method of any one of embodiments 139-192, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
[0565] Embodiment 153. The method of any one of embodiments 139-152, wherein the nanoparticle is magnetic. [0566] Embodiment 154. The method of any one of embodiments 139-153, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0567] Embodiment 155. The method of any one of embodiments 139-154, wherein the detectable peptide derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0568] Embodiment 156. The method of any one of embodiments 139-155, wherein the one or more capture molecules derived from the spike glycoprotein of a coronavirus comprises atleast a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
[0569] Embodiment 157. The method of any one of embodiments 139-156, wherein the SARS- CoV is SARS-CoV-2.
[0570] Embodiment 158. The method of any one of embodiments 139-157, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
[0571] Embodiment 159. Themethodof any oneof embodiments 139-158, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0572] Embodiment 160. Themethodof any oneof embodiments 139-159, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0573] Embodiment 161. Themethodof any oneof embodiments 139-160, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
[0574] Embodiment 162. Themethodof any oneof embodiments 139-161, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0575] Embodiment 163. Themethodof any oneof embodiments 139-162, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115 [0576] Embodiment 164. The method of any one of embodiments 139-163, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0577] Embodiment 165. The method of any one of embodiments 139-164, wherein the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0578] Embodiment 166. The method of any one of embodiments 139-165, wherein the detectable peptide derived from the spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
[0579] Embodiment 167. The method of any one of embodiments 139-166, wherein the SARS- CoV is SARS-CoV-2.
[0580] Embodiment 168. The method of any one of embodiments 139-167, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
[0581] Embodiment 169. Themethodof any oneof embodiments 139-168, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0582] Embodiment 170. Themethodof any oneof embodiments 139-169, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0583] Embodiment 171. Themethodof any oneof embodiments 139-170, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
[0584] Embodiment 172. Themethodof any oneof embodiments 139-171, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0585] Embodiment 173. Themethodof any oneof embodiments 139-172, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115 [0586] Embodiment 174. The method of any one of embodiments 139-173, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0587] Embodiment 175. The method of any one of embodiments 139-174, wherein method further comprises providing a web -based portal on the personal electronic device.
[0588] Embodiment 176. The method of any one of embodiments 139-175, wherein method further comprises providing an application on the web-based portal.
[0589] Embodiment 177. The method of any one of embodiments 139-176, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
[0590] Embodiment 178. The method of any one of embodiments 139-177, wherein method further comprises providing a data analytics module at the application.
[0591] Embodiment 179. The method of any one of embodiments 139-178, wherein method further comprises analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
[0592] Embodiment 180. The method of any one of embodiments 139-179, wherein the positive or the negative result is relative to a threshold number of complexes between the detectable peptide and the one or more capture molecules on the surface.
[0593] Embodiment 181. The method of any one of embodiments 139-180, wherein the threshold number is predetermined relative to an index a control.
[0594] Embodiment 182. The method of any one of embodiments 139-181, wherein method further comprises analyzingthe result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules on the surface.
[0595] Embodiment 183. The method of any one of embodiments 139-182, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of bindingbetween the peptide-conjugate andthe ACE2, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
[0596] Embodiment 184. The method of any one of embodiments 139-183, wherein method further comprises normalizing, by the data analytics module, the result by subtracting background noise. [0597] Embodiment 185. The method of any one of embodiments 139-184, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
[0598] Embodiment 186. The method of any one of embodiments 139-185, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
[0599] Embodiment 187. The method of any one of embodiments 139-186, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
[0600] Embodiment 188. The method of any one of embodiments 139-187, wherein method further comprises providing a data store that is a cloud-based data store or a web-based data store, or a local data store.
[0601] Embodiment 189. The method of any one of embodiments 139-188, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
[0602] Embodiment 190. The method of any one of embodiments 139-189, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0603] Embodiment 191. The method of any one of embodiments 139-190, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
[0604] Embodiment 192. The method of any one of embodiments 139-191, wherein comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device. [0605] Embodiment 193. Aspects disclosed herein provide a method of identifying adaptive immunity to a coronavirus in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) producing a mixture by introducing the biological sample with a detectable peptide derived from an angiotensin -converting enzyme 2 (ACE2) receptor; (c) bringing the mixture into contact with one or more capture molecules derived from a spike glycoprotein of a coronavirus; (d) detecting a number of binding complexes between the detectable peptide and the one or more capture molecules; (e) if the number of the binding complexes is low relative to an index or a control, then identifyingthe subject as being immune to an infection by the coronavirus; and (f) if the number of the binding complexes is high relativeto an index ora control, then identifyingthe subject as notbeingimmuneto an infection by the coronavirus.
[0606] Embodiment 194. The method of embodiment 193, wherein steps (a)-(f) are performed at the point of need.
[0607] Embodiment 195. The method of any one of embodiments 193-194, wherein steps (a)- (f) are performed at the point of care.
[0608] Embodiment 196. The method of any oneof embodiments 193-195, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
[0609] Embodiment 197. The method of any oneof embodiments 193-196, wherein the surface is a passivated surface.
[0610] Embodiment 198. The method of any one of embodiments 193- 197, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(m ethyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0611] Embodiment 199. The method of any one of embodiments 193-198, wherein the one or more capture molecules comprises two or more capture molecules.
[0612] Embodiment 200. The method of any one of embodiments 193-199, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0613] Embodiment 201 . The method of any one of embodiments 193-200, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0614] Embodiment 202. The method of any one of embodiments 193-201, wherein the one or more capture molecules is a fusion polypeptide.
[0615] Embodiment 203. The method of any one of embodiments 193-202, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
[0616] Embodiment 204. The method of any one of embodiments 193-203, wherein the one or more capture molecules is bound by an antibody th at is coupled to the surface. [0617] Embodiment 205. The method of any one of embodiments 193-204, wherein the detectable peptide comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0618] Embodiment 206. The method of any one of embodiments 193-205, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
[0619] Embodiment 207. The method of any one of embodiments 193-206, wherein the nanoparticle is magnetic.
[0620] Embodiment 208. The method of any one of embodiments 193-207, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0621] Embodiment 209. The method of any one of embodiments 193-208, wherein the detectable peptide derived from the ACE2 receptor comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0622] Embodiment 210. The method of any one of embodiments 193-209, wherein the one or more capture molecules derived from the spike glycoprotein of a coronavirus comprises atleast a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS-CoV).
[0623] Embodiment 211. The method of any one of embodiments 193-210, wherein the SARS- CoV is SARS-CoV-2.
[0624] Embodiment 212. The method of any one of embodiments 193-211, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
[0625] Embodiment213. Themethodof any oneof embodiments 193-212, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0626] Embodiment214. Themethodof any oneof embodiments 193-213, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0627] Embodiment215. Themethodof any oneof embodiments 193-214, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2 [0628] Embodiment216. Themethodof any oneof embodiments 193-215, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0629] Embodiment217. Themethodof any oneof embodiments 193-216, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0630] Embodiment 218. The method of any one of embodiments 193-217, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0631] Embodiment 219. The method of any one of embodiments 193-218, wherein the biological sample comprises one or more antibodies specific to the peptide.
[0632] Embodiment 220. The method of any one of embodiments 193-219, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
[0633] Embodiment 221. The method of any one of embodiments 193-220, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
[0634] Embodiment222. Themethodof any one of embodiments 193-221, wherein the subject was, or is, exposed to the coronavirus.
[0635] Embodiment 223. The method of any one of embodiments 193-222, wherein exposure of the subject to the coronavirus is unknown.
[0636] Embodiment224. Themethodof any one of embodiments 193-223, wherein the subject is a plurality of subjects.
[0637] Embodiment 225. The method of any one of embodiments 193-224, wherein method further comprises identifying adaptive immunity of the plurality of subjects to the coronavirus. [0638] Embodiment 226. The method of any one of embodiments 193-225, wherein method further comprises monitoring a spread of infection of the plurality of subjects by the coronavirus.
[0639] Embodiment227. Themethodof any one of embodiments 193-226, wherein the subject was administered a vaccine against the coronavirus.
[0640] Embodiment 228. The method of any one of embodiments 193-227, wherein method further comprises determining that the vaccine is effective to substantially immunize the subject against the coronavirus, provided the number of the binding complexes is low relative to an index or a control.
[0641] Embodiment 229. The method of any one of embodiments 193-228, wherein the biological sample comprises blood, urine, saliva, or feces.
[0642] Embodiment 230. The method of any one of embodiments 193-229, wherein the blood is capillary blood.
[0643] Embodiment 231. The method of any one of embodiments 193-230, wherein the capillary blood is obtained from the subject by a prick of the subject’ s finger.
[0644] Embodiment 232. The method of any one of embodiments 193-231, wherein method further comprises separating serum from the blood in the biological sample.
[0645] Embodiment 233. The method of any one of embodiments 193-232, wherein detecting in (d) comprises capturing an image of the surface with an imaging device to detect a number of binding complexes between the detectable peptide and the one or more capture molecules.
[0646] Embodiment 234. The method of any one of embodiments 193-233, wherein the imaging device is a personal electronic device.
[0647] Embodiment 235. The method of any one of embodiments 193-234, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
[0648] Embodiment 236. The method of any one of embodiments 193-235, wherein method further comprises providing a web -based portal on the personal electronic device.
[0649] Embodiment 237. The method of any one of embodiments 193-236, wherein method further comprises providing an application on the web-based portal.
[0650] Embodiment 238. The method of any one of embodiments 193-237, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
[0651] Embodiment 239. The method of any one of embodiments 193-238, wherein method further comprises providing a data analytics module at the application.
[0652] Embodiment 240. The method of any one of embodiments 193-239, wherein method further comprises analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity. [0653] Embodiment 241. The method of any one of embodiments 193-240, wherein the positive or the negative result is relative to a threshold number of complexes between the detectable peptide and the one or more capture molecules on the surface.
[0654] Embodiment 242. The method of any one of embodiments 193-241, wherein the threshold number is predetermined relative to an index a control.
[0655] Embodiment 243. The method of any one of embodiments 193-242, wherein method further comprises analyzingthe result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules on the surface.
[0656] Embodiment 244. The method of any one of embodiments 193-243, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of bindingbetween the peptide-conjugate andthe ACE2, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
[0657] Embodiment 245. The method of any one of embodiments 193-244, wherein method further comprises normalizing, by the data analytics module, the result by subtracting background noise.
[0658] Embodiment 246. The method of any one of embodiments 193-245, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result was determined to detect a presence of herd immunity to the coronavirus, or recommend further testing.
[0659] Embodiment 247. The method of any one of embodiments 193-246, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
[0660] Embodiment 248. The method of any one of embodiments 193-247, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
[0661] Embodiment 249. The method of any one of embodiments 193-248, wherein method comprises further comprise providing a data store that is a cloud-based data store or a webbased data store, or a local data store.
[0662] Embodiment 250. The method of any one of embodiments 193-249, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data. [0663] Embodiment 251. The method of any one of embodiments 193-250, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0664] Embodiment 252. The method of any one of embodiments 193-251, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
[0665] Embodiment 253. The method of any one of embodiments 193-252, wherein comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device. [0666] Embodiment 254. Aspects disclosed herein provide a method comprising: (a) obtaining a biological sample from the subject; (b) determiningwhetherthe subjecthas an acute infection by a pathogen by: (i) introducing the biological sample to a mixture comprising one or more capture molecules specific to an antibody against the pathogen; (ii) detecting a number of binding complexes between the one or more capture molecules specific to the antibody against the pathogen, wherein if the number of the binding complexes is high relative to an index or a control, then identifying the subject as having the acute infection by the coronavirus; or (c) determiningwhetherthe subjecthas a sufficient adaptive immunity against the pathogen by: (i) introducing the biological sample with a second mixture comprising detectable peptide; (ii) bringing the second mixture into contact with one or more capture molecules specific to the detectable peptide; (iii) detecting a number of binding complexes between the detectable peptide and the one or more capture molecules specific to the peptide, wherein if the number of the binding complexes is low, then identifying the subject as being immune to an infection by the coronavirus.
[0667] Embodiment 255. The method of embodiment 254, wherein if the number of the binding complexes is high relative to an index or a control, then identifying the subject as not being immune to an infection by the coronavirus.
[0668] Embodiment 256. The method of any one of embodiments 254-255, wherein the first mixture and the second mixture is a single mixture.
[0669] Embodiment 257. The method of any one of embodiments 254-256, wherein the one or more capture molecules comprises two or more capture molecules.
[0670] Embodiment 258. The method of any one of embodiments 254-257, wherein the one or more capture molecules comprises a first antibody, a second antibody or a third antibody, wherein the first antibody is specific to an immunoglobulin G antibody against SARS-CoV-2, the second antibody is specific to an immunoglobulin M antibody against SARS-CoV-2, and the third antibody is specific to an immunoglobulin A of the SARS-CoV-2.
[0671] Embodiment 259. The method of any one of embodiments 254-258, wherein steps (a)- (c) are performed at the point of need.
[0672] Embodiment 260. The method of any one of embodiments 254-259, wherein steps (a)- (c) are performed at the point of care.
[0673] Embodiment 261. The method of any one of embodiments 254-260, wherein the one or more capture molecules specific to the antibody against the pathogen are coupled to a surface. [0674] Embodiment 262. The method of any one of embodiments 254-261, wherein the one or more capture molecules specific to the labeled peptide are coupled to the surface.
[0675] Embodiment263. The method of any oneof embodiments 254-262, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
[0676] Embodiment 264. The method of any oneof embodiments 254-263, wherein the surface is a passivated surface.
[0677] Embodiment 265. The method of any one of embodiments 254-264, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0678] Embodiment 266. The method of any one of embodiments 254-265, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0679] Embodiment 267. The method of any one of embodiments 254-266, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0680] Embodiment 268. The method of any one of embodiments 254-267, wherein the one or more capture molecules is a fusion polypeptide.
[0681] Embodiment 269. The method of any one of embodiments 254-268, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody. [0682] Embodiment 270. The method of any one of embodiments 254-269, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
[0683] Embodiment 271. The method of any one of embodiments 254-270, wherein the detectable peptide comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0684] Embodiment 272. The method of any one of embodiments 254-271, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
[0685] Embodiment 273. The method of any one of embodiments 254-272, wherein the nanoparticle is magnetic.
[0686] Embodiment 274. The method of any one of embodiments 254-273, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0687] Embodiment 275. The method of any one of embodiments 254-274, wherein the detectable peptide is derived from the ACE2 receptor and comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0688] Embodiment 276. The method of any one of embodiments 254-275, wherein the one or more capture molecules specific to the detectable peptideis derived from the spike glycoprotein of a coronavirus and comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS-CoV).
[0689] Emb odiment277. Them eth odofanyone ofembodiments254-276, wh erein the S ARS - Co V is SARS-CoV-2.
[0690] Embodiment 278. The method of any one of embodiments 254-277, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
[0691] Embodiment279. Themethodof any oneof embodiments 254-278, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0692] Embodiment280. Themethodof any oneof embodiments 254-279, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0693] Embodiment281. Themethodof any oneof embodiments 254-280, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID
NO: 2
[0694] Embodiment282. Themethodof any oneof embodiments 254-281, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0695] Embodiment283. Themethodof any oneof embodiments 254-282, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0696] Embodiment 284. The method of any one of embodiments 254-283, wherein the complex between the peptide-conjugate and the one or more capture molecules is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0697] Embodiment 285. The method of any one of embodiments 254-284, wherein the one or more capture molecules specific to the detectable peptide is derived from the ACE2 receptor and comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0698] Embodiment 286. The method of any one of embodiments 254-285, wherein the detectable peptide is derived from the spike glycoprotein of a coronavirus and comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome-associated coronavirus (SARS-CoV).
[0699] Embodiment287. Themethodof any one ofembodiments 254-286, wherein the SARS- CoV is SARS-CoV-2.
[0700] Embodiment 288. The method of any one of embodiments 254-287, wherein an infection in a human subjectby the SARS-CoV-2 causes coronavirus disease of 2019 (COVID- 19) in the human subject.
[0701] Embodiment289. Themethodof any oneof embodiments 254-288, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0702] Embodiment290. Themethodof any oneof embodiments 254-289, wherein the atleast a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0703] Embodiment291. Themethodof any oneof embodiments 254-290, wherein the atleast a portion of the spike protein comprises an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID
NO: 2
[0704] Embodiment292. Themethodof any oneof embodiments 254-291, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0705] Embodiment293. Themethodof any oneof embodiments 254-292, wherein the atleast a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0706] Embodiment 294. The method of any one of embodiments 254-293, wherein the complex between the detectable peptide and the one or more capture molecules specific to the detectable peptide is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0707] Embodiment 295. The method of any one of embodiments 254-294, wherein the biological sample comprises one or more antibodies specific to the peptide.
[0708] Embodiment 296. The method of any one of embodiments 254-295, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
[0709] Embodiment 297. The method of any one of embodiments 254-296, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
[0710] Embodiment 298. The method of any one of embodiments 254-297, wherein pathogen is a coronavirus.
[0711] Embodiment 299. Themethodof any one ofembodiments 254 -298, wherein the subject was, or is, exposed to the coronavirus.
[0712] Embodiment 300. The method of any one of embodiments 254-299, wherein exposure of the subject to the coronavirus is unknown.
[0713] Embodiment 301. The method of any one of embodiments 254-300, wherein the subject is a plurality of subjects.
[0714] Embodiment 302. The method of any one of embodiments 254-301, wherein method further comprises identifying adaptive immunity of the plurality of subjects to the coronavirus. [0715] Embodiment 303. The method of any one of embodiments 254-302, wherein method further comprises monitoring a spread of infection of the plurality of subjects by the coronavirus.
[0716] Embodiment 304. The method of anyone of embodiments 254-303, wherein the subject was administered a vaccine against the coronavirus.
[0717] Embodiment 305. The method of any one of embodiments 254-304, wherein method further comprises determining that the vaccine is effective to substantially immunize the subject against the coronavirus, provided the number of the binding complexes between the detectable peptide and the one or more capture molecules specific to the detectable peptide is low relative to an index or a control.
[0718] Embodiment 306. The method of any one of embodiments 254-305, wherein the biological sample comprises blood, urine, saliva, or feces.
[0719] Embodiment 307. The method of any one of embodiments 254-306, wherein the blood is capillary blood.
[0720] Embodiment 308. The method of any one of embodiments 254-307, wherein the capillary blood is obtained from the subject by a prick of the subject’ s finger.
[0721] Embodiment 309. The method of any one of embodiments 254-308, wherein method further comprises separating serum from the blood in the biological sample.
[0722] Embodiment 310. The method of any one of embodiments 254-309, wherein detecting in (d) comprises capturing an image of the surface with an imaging device to detect a number of binding complexes between the detectable peptide and the one or more capture molecules.
[0723] Embodiment 311. The method of any one of embodiments 254-310, wherein the imaging device is a personal electronic device.
[0724] Embodiment 312. The method of any one of embodiments 254-311, wherein the personal electronic device is a smart phone, tablet, body camera, web camera, or personal computer.
[0725] Embodiment 313. The method of any one of embodiments 254-312, wherein method further comprises providing a web -based portal on the personal electronic device.
[0726] Embodiment 314. The method of any one of embodiments 254-313, wherein method further comprises providing an application on the web-based portal.
[0727] Embodiment 315. The method of any one of embodiments 254-314, wherein method further comprises receiving data, by the application, the data comprising a result from the image captured by the personal electronic device, a geolocation of the personal electronic device, or external data from one or more external devices.
[0728] Embodiment 316. The method of any one of embodiments 254-315, wherein method further comprises providing a data analytics module at the application.
[0729] Embodiment 317. The method of any one of embodiments 254-316 wherein method further comprises analyzing the result, by the data analytics module, to determining whether the result is a positive result or a negative result, wherein a positive results indicates immunity and a negative results indicates a lack of immunity.
[0730] Embodiment 318. The method of any one of embodiments 254-317, wherein the positive or the negative result (e.g., immune or not immune) is relative to a threshold number of complexes between the detectable peptide and the one or more capture molecules.
[0731] Embodiment 319. The method of any one of embodiments 254-318, wherein the threshold number is predetermined relative to an index a control.
[0732] Embodiment 320. The method of any one of embodiments 254-319, wherein the positive or the negative result (e.g., acute infection present or not present) is relative to a threshold number of complexes between the one or more capture molecules specific to the antibody against the pathogen and the antibody against the pathogen.
[0733] Embodiment 321 . The method of any one of embodiments 254-320, wherein method further comprises analyzingthe result, by the data analytics module, to determining an absolute number of complexes between the detectable peptide and the one or more capture molecules specific to the detectable peptide, or the antibody against the pathogen and the one or more capture molecules specific to the antibody.
[0734] Embodiment 322. The method of any one of embodiments 254-321, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of binding between the detectable peptide and the one or more capture molecules specific to the detectable peptide, wherein a high level of binding indicates a low level of immunity, and a low level of binding indicates a high level of immunity.
[0735] Embodiment 323. The method of any one of embodiments 254-322, wherein method further comprises analyzingthe result, by the data analytics module, to determine a level of binding between the one or more capture molecules specific to the antibody against the pathogen and the antibody against the pathogen, wherein a high level of binding indicates a presence of an acute infection by the pathogen. [0736] Embodiment 324. The method of any one of embodiments 254-323, wherein method further comprises normalizing, by the data analytics module, the result by subtracting background noise.
[0737] Embodiment 325. The method of any one of embodiments 254-324, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result for immunity was determined to detect a presence of herd immunity to the pathogen, or recommend further testing.
[0738] Embodiment 326. The method of any one of embodiments 254-325, wherein method further comprises identifying, by the data analytics module, a geographical location comprised of subjects for which a positive result for an acute infection was determined to detect a presence of infectivity or re-infectivity by the pathogen, or recommend further testing.
[0739] Embodiment 327. The method of any one of embodiments 254-326, wherein identifying the geographical location comprises utilizing geofencing from coordinates of the personal electronic device.
[0740] Embodiment 328. The method of any one of embodiments 254-327, wherein the data analytics module utilizes a machine learning algorithm, artificial intelligence, or both.
[0741] Embodiment 329. The method of any one of embodiments 254-328, wherein method further comprises providing a data store that is a cloud-based data store or a web-based data store, or a local data store.
[0742] Embodiment 330. The method of any one of embodiments 254-329, wherein the data comprises one or more of geolocation of the imaging device, a result from the image captured by the imaging device, and external data.
[0743] Embodiment 331. The method of any one of embodiments 254-330, wherein external data is data from an external device selected from a diagnostic device, a prognostic device, or a health or fitness tracking device.
[0744] Embodiment 332. The method of any one of embodiments 254-331, wherein external data comprises body temperature, heart rate variability, resting heart rate, sleep quality, and sleep quantity.
[0745] Embodiment 333. The method of any one of embodiments 254-332, wherein comprising receiving, from an external device, the external data, wherein the external device is selected from a diagnostic device, a prognostic device, or a health or fitness tracking device. [0746] Embodiment 334. Aspects disclosed herein provide a device comprising: (a) a liquid composition comprising a peptide-conjugate comprising: (i) a peptide derived from a spike gly coprotein of a coronavirus; and (ii) a detectable moiety; and (b) a surface submerged in the liquid composition, the surface comprising one or more capture molecules coupled to the surface, the one or more capture molecules derived from an angiotensin -converting enzyme 2 (ACE2) receptor.
[0747] Embodiment 335. The device of embodiment 334, wherein the surface is a surface of a container, wherein the container contains (a) and (b).
[0748] Embodiment 336. The device of any one of embodiments 334-335, wherein the device is portable.
[0749] Embodiment 337. The device of any one of embodiments 334-336, wherein the device is a point of need device.
[0750] Embodiment 338. The device of any one of embodiments 334-337, wherein the point of need is a point of care.
[0751] Embodiment 339. The device of any one of embodiments 334-338, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
[0752] Embodiment 340. The device of any one of embodiments 334-339, wherein the surface is a passivated surface.
[0753] Embodiment 341. The device of any one of embodiments 334-340, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly(m ethyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0754] Embodiment 342. The device of any one of embodiments 334-341, wherein the one or more capture molecules comprises two or more capture molecules.
[0755] Embodiment 343. The device of any one of embodiments 334-342, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0756] Embodiment 344. The device of any one of embodiments 334-343, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0757] Embodiment 345. The device of any one of embodiments 334-344, wherein the one or more capture molecules is a fusion polypeptide. [0758] Embodiment 346. The device of any one of embodiments 334-345, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody.
[0759] Embodiment 347. The device of any one of embodiments 334-346, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
[0760] Embodiment 348. The device of any one of embodiments 334-347, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0761] Embodiment 349. The device of any one of embodiments 334-348, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
[0762] Embodiment 350. The device of any one of embodiments 334-349, wherein the nanoparticle is magnetic.
[0763] Embodiment 351. The device of any one of embodiments 334-350, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0764] Embodiment 352. The device of any one of embodiments 334-3514, wherein the one or more capture molecules derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1.
[0765] Embodiment 353. The device of any one of embodiments 334-352, wherein the peptide derived from the spike glycoprotein of a coronavirus comprises at least a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV). [0766] Embodiment 354. The device of any one of embodiments 334-353, wherein the SARS- CoV is SARS-CoV-2.
[0767] Embodiment 355. The device of any one of embodiments 334-354, wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
[0768] Embodiment 356. The device of any one of embodiments 334-355, wherein the at least a portion of the spike protein comprises a subunit 1 of the spike protein.
[0769] Embodiment 357. The device of any one of embodiments 334-356, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein. [0770] Embodiment 358. The device of any one of embodiments 334-357, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
[0771] Embodiment 359. The device of any one of embodiments 334-358, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0772] Embodiment 360. The device of any one of embodiments 334-359, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0773] Embodiment 361. The device of any one of embodiments 334-360, wherein the complex between the peptide-conjugate and the one or more capture molecules receptor on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0774] Embodiment 362. The device of any one of embodiments 334-361, wherein the one or more capture molecules is specific to one or more antibodies against the coronavirus.
[0775] Embodiment 363. The device of any one of embodiments 334-362, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
[0776] Embodiment 364. The device of any one of embodiments 334-363, wherein device further comprise a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
[0777] Embodiment 365. The device of any one of embodiments 334-364, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen -binding fragment.
[0778] Embodiment 366. The device of any one of embodiments 334-365, wherein the antibody or antigen -binding fragment is monoclonal, antibody or antigen -binding fragment is polyclonal.
[0779] Embodiment 367. The device of any one of embodiments 334-366, wherein device further comprises a housing at least partially enclosing the surface.
[0780] Embodiment 368. The device of any one of embodiments 334-367, wherein device further comprises a sample receptor configured to receive a biological sample from a subject. [0781] Embodiment 369. The device of any one of embodiments 334-368, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface.
[0782] Embodiment 370. The device of any one of embodiments 334-369, wherein the biological sample comprises one or more antibodies specific to the peptide.
[0783] Embodiment 371. The device of any one of embodiments 334-370, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
[0784] Embodiment 372. The device of any one of embodiments 334-371, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
[0785] Embodiment 373. The device of any one of embodiments 334-372, wherein the subject was, or is, exposed to the coronavirus.
[0786] Embodiment 374. The device of any one of embodiments 334-373, wherein exposure of the subject to the coronavirus is unknown.
[0787] Embodiment 375. The device of any one of embodiments 334-374, wherein the subject was administered a vaccine against the coronavirus.
[0788] Embodiment 376. The device of any one of embodiments 334-375, wherein the biological sample comprises blood, urine, saliva, or feces.
[0789] Embodiment 377. The device of any one of embodiments 334-376, wherein the blood is capillary blood.
[0790] Embodiment 378. The device of any one of embodiments 334-377, wherein device further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
[0791] Embodiment 379. The device of any one of embodiments 334-378, wherein the sample receptor comprises a filter to separate serum from the blood.
[0792] Embodiment 380. The device of any one of embodiments 334-379, wherein the device is a single integrated device.
[0793] Embodiment 381. Aspects disclosed herein provide a device comprising: (a) a liquid composition comprising a peptide-conjugate comprising: (i) a peptide derived from an angiotensin-converting enzyme 2 (ACE2) receptor; and (ii) a detectable moiety; and (b) a surface submerged in the liquid composition, the surface comprising one or more capture molecules coupled to the surface, the one or more capture molecules derived from a spike glycoprotein of a coronavirus. [0794] Embodiment 382. The device of embodiment 381, wherein the surface is a surface of a container, wherein the container contains (a) and (b).
[0795] Embodiment 383. The device of any one of embodiments 381-382, wherein the device is portable.
[0796] Embodiment 384. The device of any one of embodiments 381-383, wherein the device is a point of need device.
[0797] Embodiment 385. The device of any one of embodiments 381-384, wherein the point of need is a point of care.
[0798] Embodiment 386. The device of any one of embodiments 381-385, wherein the surface comprises a material selected from the group consisting of a metal, a plastic, glass, and a nitrocellulose membrane.
[0799] Embodiment 387. The device of any one of embodiments 381-386, wherein the surface is a passivated surface.
[0800] Embodiment 388. The device of any one of embodiments 381-387, wherein the passivated surface comprises a polymer layer comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N- isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2 -hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly -lysine, poly -glucoside, streptavidin, and dextran.
[0801] Embodiment 389. The device of any one of embodiments 381-388, wherein the one or more capture molecules comprises two or more capture molecules.
[0802] Embodiment 390. The device of any one of embodiments 381-389, wherein the one or more capture molecules is coupled to the surface by a covalentbond, a linker, or a combination thereof.
[0803] Embodiment 391 . The device of any one of embodiments 381-390, wherein the linker is a chemical linker, a peptide linker, or a combination thereof.
[0804] Embodiment 392. The device of any one of embodiments 381-391, wherein the one or more capture molecules is a fusion polypeptide.
[0805] Embodiment 393. The device of any one of embodiments 381-392, wherein the fusion polypeptide comprises at least a portion of a fragment crystallizable region (Fc) region of a monoclonal antibody. [0806] Embodiment 394. The device of any one of embodiments 381-393, wherein the one or more capture molecules is bound by an antibody that is coupled to the surface.
[0807] Embodiment 395. The device of any one of embodiments 381-394, wherein the peptide- conjugate comprises a nanoparticle, a fluorescent dye, an enzymatic label, or a colorimetric label, or a combination thereof.
[0808] Embodiment 396. The device of any one of embodiments 381-395, wherein the nanoparticle comprises a material selected from the group consisting of a metal, agarose, acrylic, and plastic.
[0809] Embodiment 397. The device of any one of embodiments 381-396, wherein the nanoparticle is magnetic.
[0810] Embodiment 398. The device of any one of embodiments 381-397, wherein the nanoparticle is conjugated to the peptide by a linker comprising a chemical linker, a peptide linker, or a combination thereof.
[0811] Embodiment 399. The device of any one of embodiments 381-398, wherein the peptide derived from the ACE2 receptor comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 1
[0812] Embodiment 400. The device of any one of embodiments 381-399, wherein the one or more capture molecules derived from the spike glycoprotein of a coronavirus comprises atleast a portion of a spike protein derived from Severe acute respiratory syndrome -associated coronavirus (SARS-CoV).
[0813] Embodiment 401. The device of any one of embodiments 381-400, wherein the SARS- CoV is SARS-CoV-2.
[0814] Embodiment402. The device of any one of embodiments 381-401 , wherein an infection in a human subject by the SARS-CoV-2 causes coronavirus disease of 2019 (COVID-19) in the human subject.
[0815] Embodiment 403. The device of any one of embodiments 381-402, wherein the atleast a portion of the spike protein comprises a subunit 1 of the spike protein.
[0816] Embodiment 404. The device of any one of embodiments 381-403, wherein the at least a portion of the spike protein comprises a receptor binding domain (RBD) of the subunit 1 of the spike protein.
[0817] Embodiment 405. The device of any one of embodiments 381-404, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 2
[0818] Embodiment 406. The device of any one of embodiments 381-405, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to SEQ ID NO: 3
[0819] Embodiment 407. The device of any one of embodiments 381-406, wherein the at least a portion of the spike protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to any one of SEQ ID NOS: 4-6, 11-12, 24, 47-48, 71-72, 95-96, or 115
[0820] Embodiment 408. The device of any one of embodiments 381-407, wherein the complex between the peptide-conjugate and the one or more capture molecules receptor on the surface is visible on the surface using color, reflectance, fluorescence, bioluminescence, or chemiluminescence.
[0821] Embodiment 409. The device of any one of embodiments 381-408, wherein the one or more capture molecules is specific to one or more antibodies against the coronavirus.
[0822] Embodiment 410. The device of any one of embodiments 381-409, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is labeled.
[0823] Embodiment 411 . The device of any one of embodiments 381-410, wherein device further comprises a labeled secondary capture molecule specific to the one or more capture molecules specific to one or more antibodies against the coronavirus.
[0824] Embodiment 412. The device of any one of embodiments 381-411, wherein the one or more capture molecules specific to one or more antibodies against the coronavirus is an antibody or antigen -binding fragment.
[0825] Embodiment 413. The device of any one of embodiments 381-412, wherein the antibody or antigen -binding fragment is monoclonal, antibody or antigen -binding fragment is polyclonal.
[0826] Embodiment 414. The device of any one of embodiments 381-413, wherein device further comprises a housing at least partially enclosing the surface.
[0827] Embodiment 415. The device of any one of embodiments 381-414, wherein device further comprises a sample receptor configured to receive a biological sample from a subject.
[0828] Embodiment 416. The device of any one of embodiments 381-415, wherein the sample receptor is mechanically coupled to a housing at least partially enclosing the surface. [0829] Embodiment 417. The device of any one of embodiments 381-416, wherein the biological sample comprises one or more antibodies specific to the peptide.
[0830] Embodiment 418. The device of any one of embodiments 381-417, wherein the biological sample does not consist of one or more antibodies specific to the peptide.
[0831] Embodiment 419. The device of any one of embodiments 381-418, wherein the one or more antibodies belong to one or more immunoglobulin classes comprising immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin E, and immunoglobulin D.
[0832] Embodiment 420. The device of any one of embodiments 381-419, wherein the subject was, or is, exposed to the coronavirus.
[0833] Embodiment 421. The device of any one of embodiments 381-420, wherein exposure of the subject to the coronavirus is unknown.
[0834] Embodiment 422. The device of any one of embodiments 381-421, wherein the subject was administered a vaccine against the coronavirus.
[0835] Embodiment 423. The device of any one of embodiments 381-422, wherein the biological sample comprises blood, urine, saliva, or feces.
[0836] Embodiment 424. The device of any one of embodiments 381-423, wherein the blood is capillary blood.
[0837] Embodiment 425. The device of any one of embodiments 381-424, wherein device further comprises a transdermal puncture device configured to obtain the capillary blood from the subject.
[0838] Embodiment 426. The device of any one of embodiments 381-425, wherein the sample receptor comprises a filter to separate serum from the blood.
[0839] Embodiment 427. The device of any one of embodiments 381-426, wherein he device is a single integrated device.
[0840] Embodiment 428. Aspects disclosed herein provide a kit comprising: (a) a lateral flow assay assembly comprising: a composition comprising: a first peptide or protein derived from an ACE2, a portion thereof; or a second peptide or protein derived from a spike glycoprotein of SARS-CoV-2 or variant thereof, or a portion thereof, the first peptide or protein or the second peptide or protein comprising a detectable moiety; and a porous membrane comprising: (1) a test zone, wherein the test zone comprises: one or more capture molecules coupled to the porous membrane at the test zone, the one or more capture molecules comprising: (i) a third peptide or protein derived from the spike glycoprotein of the SARS-CoV-2 or variant thereof, or a portion thereof; (ii) a fourth peptide or protein derived from the ACE2, or portion thereof; or (iii) a primary capture molecule specific to (i), (ii), or a protein tag conjugated thereto; and (b) instructions for assaying a biological sample with the lateral flow assay assembly to detect a presence, an absence, or a quantity of neutralizing antibodies against SARS-CoV-2 orvariant thereof in the biological sample, or a component thereof.
[0841] Embodiment 429. The kit of embodiment 428, wherein the kit further comprises a sample receptor mechanically coupled to the porous membrane, the sample receptor configured to contain the biological sample.
[0842] Embodiment 430. The device of any one of embodiments 428 -429, wherein the kit further comprises instructions for: obtaining the biological sample; assaying the biological sample with the lateral flow assay assembly to detect a presence, an absence, or a quantity of a binding complex between the composition and the one or more capture molecules in the biological sample, or component thereof; and classifying the biological sample as havingthe presence, the absence, or the quantity of the neutralizing antibodies against the SARS-CoV-2 or variant thereof.
[0843] Embodiment 431. The device of any one of embodiments 428 -430, wherein the kit further comprises instructions for downloading a mobile application on a personal electronic device, the mobile application configuredto analyze image data from an image of the test zone of the lateral flow assay assembly to classify the biological sample as havingthe presence, the absence, or the quantity of neutralizing antibodies against the SARS-CoV-2 or variant thereof. [0844] Embodiment 432. The device of any one of embodiments 428 -431, wherein the kit further comprises: (a) an second primary capture molecule specific to one or more antibodies against the SARS-CoV-2 orvariantthereof, wherein the one or more antibodies against SARS- CoV-2 or variant thereof comprises an immunoglobulin G, an immunoglobulin M, an immunoglobulin A, or a combination thereof; and (b) instructions for: detecting a presence, an absence, or a quantity of a binding complex between the second primary capture molecules and the one or more antibodies against the SARS-CoV-2 or variant th ere of in the biological sample or a component thereof, by bringing the biological sample or component thereof into contact with the second primary capture molecule in (a); and classifying the biological sample as having a presence, an absence, or a quantity of the one or more antibodies against the SARS- CoV-2 or variant thereof in the biological sample or component thereof based on the presence, the absence or the quantity of the binding complex that is detected.
[0845] Embodiment 433. Aspects disclosed herein provide a method comprising: step (a) providing a biological sample from a subject; step (b) analyzing the biological sample, or first component thereof, to detect a presence, an absence, or a quantity of neutralizing antibodies against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or variant thereof by : contacting the first component of the biological sample to a first peptide or protein derived from a spike protein of the SARS-CoV-2 or variantthereof, or a portion thereof, in the presence of a second peptide or protein derived from an angiotensin converting enzyme 2 (ACE2), or a portion thereof, wherein the first peptide or protein or the second peptide or protein comprises a detectable moiety; and detecting the presence, the absence, or the quantity of one or more neutralizing antibodies in the biological sample that functionally block binding between the first peptide or protein and the second peptide or protein by detecting a binding complex between the first peptide or protein and the second peptide or protein in the presence of the biological sample; step (c) classifying the biological sample, or first component thereof, as having the presence, the absence, or the quantity of neutralizing antibodies to the SARS-CoV- 2 or variantthereof, based, at least partially, on analyzing in step (b); step (d) analyzing the biological sample, or second component thereof, to detect a presence, an absence, or a quantity of one or more antibodies against SARS-CoV-2 or variant thereof by: contacting the biological sample, or second component thereof, to a capture molecule specific to the one or more antibodies against SARS-CoV-2 or variant thereof, wherein the capture molecule comprises a detectable moiety; and detectingthe presence, the absence, orthe quantity of a binding complex between the capture molecule and the one or more antibodies; and step (e) classifying the biological sample, or second component thereof, as having the presence, the absence, or the quantity of the one or more antibodies against SARS-CoV-2 or variant thereof, at least partially, on analyzing in step (d).
[0846] Embodiment 434. The method of embodiment 433, wherein the classifying in step (c) comprises classifying the biological sample from the subject as positive for the presence of neutralizing antibodies against the SARS-CoV-2 or variant thereof, provided an absence of the binding complex between the first peptide or protein and the second peptide or protein is detected.
[0847] Embodiment 435. The method of any one of embodiments 433-434, wherein the classifying in step (e) comprises classifying the biological sample from the subject as positive for exposure to SARS-CoV-2 or variantthereof, provided the presence of the binding complex between the capture molecule and the one or more antibodies is detected, or providing the quantity of the binding complex between the capture molecule and the one or more antibodies is above an index or a control. [0848] Embodiment 436. The method of any one of embodiments 433-435, wherein the steps (a)-(e) are performed at a point of need or a point of care.
[0849] Embodiment 437. The method of any one of embodiments 433-436, wherein step (b) does not utilize an immortalized cell or an immortalized cell culture.
[0850] Embodiment 438. The method of any one of embodiments 433-437, wherein the biological sample of the method comprises whole blood, blood plasma, or serum.
[0851] Embodiment 439. The method of any one of embodiments 433-428, wherein the analyzing in step (b) and analyzing in step (d) are performed with a single integrated device.
[0852] Embodiment 440. The method of any one of embodiments 433-439, wherein the steps (c) and (e) are performed by an application that runs on an electronic device, the application comprising a mobile application or a web application.
[0853] Embodiment 441. The method of any one of embodiments 433-440, wherein the application of the method is configured to generate a report comprising a status of the biological sample, the status comprising the presence, the absence, or the quantity of the neutralizing antibodies against SARS-CoV-2 or variant thereof in the biological sample.
[0854] Embodiment 442 The method of any one of embodiments 433-441, wherein the application of the method is configured to generate a report comprising a status of the biological sample, the status comprising the presence, the absence, or the quantity of the one or more antibodies against the SARS-CoV-2 or variant thereof, wherein the one or more antibodies against SARS-CoV-2 comprises immunoglobulin G, immunoglobulin M, immunoglobulin A, or a combination thereof.
[0855] Embodiment 443. Aspects disclosed herein provide a system comprising: a first testing device or module to detect a presence, an absence, or a quantity of neutralizing antibodies against SARS-CoV-2 or variant thereof, the first testing device or module comprising: a composition comprising: a first peptide or protein derived from an ACE2, or portion thereof; and a second peptide or protein derived from a spike glycoprotein of the SARS-CoV-2 or variant thereof, or a portion thereof, the first peptide or protein or the second peptide or protein comprises a detectable moiety; and a test zone for visualization of the detectable moiety; and a second testing device or module to measure a presence, an absence, or a quantity of one or more antibodies against SARS-CoV-2, or variant thereof, the second testing device or module comprising: a first surface; and one or more capture molecules coupled to a region of the first surface, the one or more capture molecules comprising: a binding domain specific to one or more antibodies against SARS- CoV-2 or variant thereof; and a detectable moiety.
[0856] Embodiment 444. The system of embodiment 443, wherein the system further comprises an imaging device operatively coupled to the first device, the imaging device configured to capture an image of the test zone.
[0857] Embodiment 445. The system of any one of embodiments 443-444, wherein the system further comprises an imaging device operatively coupled to the second device, the imaging device configured to capture an image of the region of the first surface.
[0858] Embodiment 446. The system of any one of embodiments 443-445, wherein the system further comprises an electronic device comprising one or more processors operatively coupled to the imaging device configured to run an algorithm to generate a first classification of the biological sample as having the presence, the absence, or the quantity of the neutralizing antibodies to the SARS-CoV-2 or variant thereof.
[0859] Embodiment 447. The system of any one of embodiments 443-446, wherein the system further comprises an electronic device comprising one or more processors operatively coupled to the imaging device configured to run an algorithm to generate a second classification of the biological sample as having the presence, the absence, or the quantity of the one or more antibodies against the SARS-CoV-2 or variant thereof, wherein the one or more antibodies against SARS-CoV-2 or variant thereof comprises immunoglobulin G, immunoglobulin M, immunoglobulin A, or a combination thereof.
[0860] Embodiment 448. The system of any one of embodiments 443-447, wherein the system further comprises an application that runs on an electronic device, the application configured to display on a graphical user interface a report comprising a status of the biological sample, the status comprising one or more of: the presence, the absence, or the quantity of the neutralizing antibodies against SARS-CoV-2 or variant thereof; and the presence, the absence, or the quantity of the one or more antibodies against SARS-CoV-2 or variant thereof, wherein the one or more antibodies against SARS-CoV-2 or variant thereof comprises immunoglobulin G, immunoglobulin M, immunoglobulin A, or a combination thereof.
[0861] Embodiment 449. The system of any one of embodiments 443-448, wherein the test zone of the system is positioned at a second surface and wherein either of the first peptide or protein and the second peptide or protein is coupled to the second surface at the test zone directly or indirectly. [0862] Embodiment 450. The system of any one of embodiments 443-449, wherein the first testing module and the second testing module of the system are in a single integrated device, and wherein the first surface and the second surface are the same surface.
[0863] Embodiment 451 . The system of any one of embodiments 443-450, wherein the first testing device and the second testing device are portable.
[0864] Embodiment 452. The system of any one of embodiments 443-451, wherein the first testing device or module does not consist of an immortalized cell or an immortalized cell culture.
[0865] Embodiment 453. Aspects disclosed herein provide a system for point of need or point of care comprising a testing device to detect neutralizing antibodies against a SARS-CoV-2 or variant thereof to aid in the diagnosis of a disease or a condition caused by SARS-CoV-2 or variant thereof, the testing device comprising a composition comprising: a first peptide or protein derived from an ACE2, or portion thereof; and a secondpeptide or protein derived from a spike glycoprotein of the SARS-CoV-2 or variant thereof, or portion thereof, wherein at least one of the first peptide or protein and the second peptide or protein is labeled with a detectable moiety.
[0866] Embodiment 454. The system of embodiment 453, wherein the system further comprises an application configured to run on the electronic device, the application configured to generate a classification of a biological sample as having a presence, an absence, or a quantity of neutralizing antibodies against the SARS-CoV-2 or variant thereof based on a presence, an absence, or a quantity of a binding complex between the first peptide or protein and the second peptide or protein in the presence of the biological sample.
[0867] Embodiment 455. The system of any one of embodiments 453-454, wherein the classification of the biological sample is indicative of at least one of: (a) a diagnosis related to the disease or the condition caused by SARS-CoV-2; (b) a prognosis related to adaptive immunity of the subject against an infection by the SARS-CoV-2 or variant thereof, or the disease or the condition caused by the SARS-CoV-2; and (c) a measure of susceptibility of the subject to an infection by the SARS-CoV-2.
[0868] Embodiment 456. The system of any one of embodiments 453-455, wherein the system further comprises a vaccine composition against the SARS-CoV-2 or variant thereof, wherein the testing device is capable of identifying the subject as being in need of treatment with the vaccine composition based on any one of (a)-(c). [0869] Embodiment 457. The system of any one of embodiments 453-456, wherein the testing device is a point of need or a point of care device.
[0870] Embodiment 458. The system of any one of embodiments 453-457, wherein the system does not consist of an immortalized cell or an immortalized cell culture.
[0871] Embodiment 459. The system of any one of embodiments 453-458, wherein the electronic device of the system is a personal electronic device comprising a smartphone, a tablet, or a personal computer.
[0872] Embodiment 460. Aspects disclosed herein provide a system comprising a lateral flow assay assembly comprising: (i) a composition comprising: a first peptide or protein derived from an ACE2, a portion thereof; or a second peptide or protein derived from a spike glycoprotein of SARS-CoV-2 or variant thereof, or a portion thereof, the first peptide or protein or the second peptide or protein comprising a detectable moiety; and (ii) a porous membrane comprising a test zone, wherein the test zone comprises one or more capture molecules coupled to the porous membrane atthe test zone, the one or more capture molecules comprising: a third peptide or protein derived from the spike glycoprotein of the SARS-CoV- 2 or variant thereof, or a portion thereof; a fourth peptide or protein derived from the ACE2, or portion thereof; or a primary capture molecule specific to (i), (ii), or a protein tag conjugated thereto.
[0873] Embodiment 461 . The system of embodiment 460, wherein the lateral flow assay assembly of the system is portable.
[0874] Embodiment 462. The system of any one of embodiments 460-461, wherein the system further comprises an application configuredto run on a personal electronic device, the personal electronic device comprising a camera to capture an image of the test zone before or after a biological sample obtained from a subject is applied to the porous membrane, wherein the application is configured to generate a classification of the biological sample as having a presence, an absence, or a quantity of neutralizing antibodies against the SARS-CoV-2 or variant thereof based on a presence, an absence, or a quantity of a binding complex between the composition and the one or more capture molecules detected in the biological sample using the lateral flow assay assembly.
[0875] Embodiment 463. The system of any one of embodiments 460-462, wherein the one or more capture molecules of the system comprises the primary capture molecule specific to (i) the third peptide or protein or the protein tag conjugated thereto, or (ii) the fourth peptide or protein or the protein tag conjugated thereto. [0876] Embodiment 464. The system of any one of embodiments 460-463, wherein the composition of the system comprises the second peptide or protein, and wherein the one or more capture molecules comprises (i) the fourth peptide or protein, or (ii) the primary capture molecule specific to the fourth peptide or protein, or the protein tag conjugated thereto.
[0877] Embodiment 465. The system of any one of embodiments 460-464, wherein the system further comprises a labeled secondprimary capture molecule specific to one or more antibodies against the SARS-CoV-2 or variant thereof, the one or more antibodies comprising an immunoglobulin G, immunoglobulin M, an immunoglobulin A, or a combination thereof.
[0878] Embodiment466. The system of any one of embodiments 460-465, wherein the labeled second primary capture molecule is coupled to the porous membrane at the test zone.
[0879] Embodiment 467. Aspects disclosed herein provide a system comprising: (a) a first testing device or module to detect a presence, an absence, or a quantity of neutralizing antibodies against SARS-CoV-2 or variant thereof, the first testing device or module comprising: (i) a composition comprising: (1) a first peptide or protein derived from an ACE2, or portion thereof; and (2) a second peptide or protein derived from a spike glycoprotein of the SARS-CoV-2 or variant thereof, or a portion thereof, the first peptide or protein or the second peptide or protein comprises a detectable moiety; and (ii) a test zone for visualization of the detectable moiety ; and (b) a second testing device or module to measure a presence, an ab sence, or a quantity of one or more antibodies against SARS-CoV-2, or variant thereof, the second testing device or module comprising: a first surface; and one or more capture molecules coupled to a region of the first surface, the one or more capture molecules comprising: a binding domain specific to one or more antibodies again st SARS-CoV-2 orvariantthereof; and a detectable moiety.
[0880] Embodiment 468. The system of embodiment 467, wherein the system further comprises an application that runs on an electronic device, the application configured to generate a classification of the biological sample, the classification comprising one or more of : the presence, the absence, or the quantity of the neutralizing antibodies against SARS-CoV- 2 orvariantthereof; and the presence, the absence, orthe quantity of the one ormore antibodies against SARS-CoV-2 or variant thereof, wherein the one or more antibodies against SARS- CoV-2 orvariantthereof comprises immunoglobulin G, immunoglobulin M, immunoglobulin A, or a combination thereof. [0881] Embodiment 469. The system of any one of embodiments 467-468, wherein the test zone is positioned at a second surface and wherein either of the first peptide or protein and the second peptide or protein is coupled to the second surface at the test zone directly or indire ctly. [0882] Embodiment 470. The system of any one of embodiments 467-469, wherein the first testing module and the second testing module are in a single integrated device, and wherein the first surface and the second surface are the same surface.
[0883] Embodiment 471. The system of any one of embodiments 467-470, wherein the first testing device and the second testing device are portable.
[0884] Embodiment 472. The system of any one of embodiments 467-471, wherein the first testing device or module does not consist of an immortalized cell or an immortal cell culture. [0885] Embodiment 473. Aspects disclosed herein provide a system for point of need or point of care comprising a testing device to detect neutralizing antibodies against a SARS-CoV-2 or variant thereof to aid in the diagnosis of a disease or a condition caused by SARS-CoV-2 or variant thereof, the testing device comprising a composition comprising: a first peptide or protein derived from an ACE2, or portion thereof; and a secondpeptide or protein derived from a spike glycoprotein of the SARS-CoV-2 or variant thereof, or portion thereof, wherein at least one of the first peptide or protein and the second peptide or protein is labeled with a detectable moiety.
[0886] Embodiment 474. The system of embodiment 473, wherein the system further comprises an application configured to run on the electronic device, the application configured to generate a classification of a biological sample as having a presence, an absence, or a quantity of neutralizing antibodies against the SARS-CoV-2 or variant thereof based on a presence, an absence, or a quantity of a binding complex between the first peptide or protein and the second peptide or protein in the presence of the biological sample.
[0887] Embodiment 475. The system of any one of embodiments 473-474, wherein the classification of the biological sample is indicative of at least one of: (a) a diagnosis related to the disease or the condition caused by SARS-CoV-2; (b) a prognosis related to adaptive immunity of the subject against an infection by the SARS-CoV-2 or variant thereof, or the disease or the condition caused by the SARS-CoV-2; and (c) a measure of susceptibility of the subject to an infection by the SARS-CoV-2.
[0888] Embodiment 476. The system of any one of embodiments 473-475, wherein the system further comprises a vaccine composition against the SARS-CoV-2 or variant thereof, wherein the testing device is capable of identifying the subject as being in need of treatment with the vaccine composition based on any one of (a)-(c).
[0889] Embodiment 477. The system of any one of embodiments 473-476, wherein the testing device is a point of need or a point of care device.
[0890] Embodiment 478. The system of any one of embodiments 473-477, wherein the system does not consist of an immortalized cell or an immortalized cell culture.
[0891] Embodiment 479. The system of any one of embodiments 473-478, wherein the electronic device of the system is a personal electronic device comprising a smartphone, a tablet, or a personal computer.
[0892] Embodiment 480. Aspects disclosed herein provide a system comprising a lateral flow assay assembly comprising: (i) a composition comprising: a first peptide or protein derived from an ACE2, a portion thereof; or a second peptide or protein derived from a spike glycoprotein of SARS-CoV-2 or variant thereof, or a portion thereof, the first peptide or protein or the second peptide or protein comprising a detectable moiety; and (ii) a porous membrane comprising a test zone, wherein the test zone comprises one or more capture molecules coupled to the porous membrane atthe test zone, the one or more capture molecules comprising: a third peptide or protein derived from the spike glycoprotein of the SARS-CoV- 2 or variant thereof, or a portion thereof; a fourth peptide or protein derived from the ACE2, or portion thereof; or a primary capture molecule specific to (i), (ii), or a protein tag conjugated thereto.
[0893] Embodiment 481 . The system of embodiment 480, wherein the lateral flow assay assembly of the system is portable.
[0894] Embodiment 482. The system of any one of embodiments 480-481, wherein the system further comprises an application configuredto run on a personal electronic device, the personal electronic device comprising a camera to capture an image of the test zone before or after a biological sample obtained from a subject is applied to the porous membrane, wherein the application is configured to generate a classification of the biological sample as having a presence, an absence, or a quantity of neutralizing antibodies against the SARS-CoV-2 or variant thereof based on a presence, an absence, or a quantity of a binding complex between the composition and the one or more capture molecules detected in the biological sample using the lateral flow assay assembly.
[0895] Embodiment 483. The system of any one of embodiments 480-482, wherein the one or more capture molecules comprises the primary capture molecule specificto (i) the third peptide or protein or the protein tag conjugated thereto, or (ii) the fourth peptide or protein or the protein tag conjugated thereto.
[0896] Embodiment 484. The system of any one of embodiments 480-483, wherein the composition comprises the second peptide or protein, and wherein the one or more capture molecules comprises (i) the fourth peptide or protein, or (ii) the primary capture molecule specific to the fourth peptide or protein, or the protein tag conjugated thereto.
[0897] Embodiment 485. The system of any one of embodiments 480-484, wherein the system further comprises a labeled secondprimary capture molecule specific to one or more antibodies against the SARS-CoV-2 or variant thereof, the one or more antibodies comprising an immunoglobulin G, immunoglobulin M, an immunoglobulin A, or a combination thereof.
[0898] Embodiment486. The system of any one of embodiments 480 -485, wherein the labeled second primary capture molecule is coupled to the porous membrane at the test zone.
[0899] Embodiment 487. Aspects disclosed herein provide a system comprising: (a) a first testing device or module to detect a presence, an absence, or a quantity of neutralizing antibodies against SARS-CoV-2 or variant thereof, the first testing device or module comprising: (i) a composition comprising: (1) a first peptide or protein derived from an ACE2, or portion thereof; and (2) a second peptide or protein derived from a spike glycoprotein of the SARS-CoV-2 or variant thereof, or a portion thereof, the first peptide or protein or the second peptide or protein comprises a detectable moiety; and (ii) a test zone for visualization of the detectable moiety; and (b) a second testing device or module to measure a presence, an ab sence, or a quantity of one or more antibodies against SARS-CoV-2, or variant thereof, the second testing device or module comprising: a first surface; and one or more capture molecules coupled to a region of the first surface, the one or more capture molecules comprising: a binding domain specific to one or more antibodies against SARS-CoV-2 orvariantthereof; and a detectable moiety.
[0900] Embodiment 488. The system of embodiment 487, wherein the system further comprises an application that runs on an electronic device, the application configured to generate a classification of the biological sample, the classification comprising one or more of : the presence, the absence, or the quantity of the neutralizing antibodies against SARS-CoV- 2 orvariantthereof; and the presence, the absence, orthe quantity of the one or more antibodies against SARS-CoV-2 or variant thereof, wherein the one or more antibodies against SARS- CoV-2 orvariantthereof comprises immunoglobulin G, immunoglobulin M, immunoglobulin A, or a combination thereof. [0901] Embodiment 489. The system of any one of embodiments 487-488, wherein the test zone is positioned at a second surface and wherein either ofthe first peptide or protein and the second peptide or protein is coupled to the second surface at the test zone directly or indirectly. [0902] Embodiment 490. The system of any one of embodiments 487-489, wherein the first testing module and the second testing module are in a single integrated device, and wherein the first surface and the second surface are the same surface.
[0903] Embodiment 491. The system of any one of embodiments 487-490, wherein the first testing device and the second testing device are portable.
[0904] Embodiment 492. The system of any one of embodiments 487-491, wherein the first testing device or module does not consist of an immortalized cell or an immortalized cell line. [0905] Embodiment 493. Any of the preceding embodiments, wherein an age of the subject is at least or about 50 years old.
[0906] Embodiment 494. Any of the preceding embodiments, wherein the subject is administered a commercialized vaccine.
[0907] Embodiment 495. Any of the preceding embodiments, wherein detecting a number of binding complexes between the detectable peptide and the one or more capture molecules is performed in less than 20 minutes from the time the biological sample from the subject is contacted with the detectable peptide and the one or more capture molecules.
[0908] Embodiment 496. Any of the preceding embodiments, wherein the biological sample comprises a volume of less than 1 milliliter.
[0909] Embodiment 497. Any of the preceding embodiments, wherein the systems and methods are computer-automated.
[0910] Embodiment 498. Any of the preceding embodiments, wherein detecting a number of binding complexes between the detectable peptide and the one or more capture molecules can be performed in a single step without the need for washing.
[0911] Embodiment 499. Any of the preceding embodiments, wherein detecting a number of binding complexes between the detectable peptide and the one or more capture molecules specific to the peptide can be performed in a single step without the need for washing.
[0912] Embodiment 500. A method of measuring a presence, an absence, or a quantity of neutralizing antibodies against a coronavirus that functionally block binding between a spike protein of the coronavirus and Angiotensin -converting enzyme 2 (ACE2). The method of comprises: providing a biological sample obtained from one or more finger pricks of a subject that was administered a vaccine against the coronavirus; bringing at least a portion of the biological sample into contact with: (i) a first peptide or protein comprising the ACE2 polypeptide or a portion thereof; and (ii) a second peptide or protein comprising the spike protein or a portion thereof, wherein: (1) the first peptide or protein and the second peptide or protein form a complex in an absence of neutralizing antibodies in the biological sample that block binding between the spike protein and ACE2 under conditions otherwise suitable for binding; and (2) the first peptide or protein, the second peptide or protein, or a combination thereof comprises a detectable moiety; detecting an absence, a presence, or a quantity of the complex formed in (b); and (d) measuring the presence, the absence, or the quantity of the neutralizing antibodies againstthe coronavirus, whichis inversely correlated with the presence, the absence, or the quantity of the complex detected in (c), respectively.
[0913] Embodiment 501. The method of embodiment 500, wherein the biological sample comprises capillary blood.
[0914] Embodiment 502. The method of any of embodiments 500-501, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0915] The Embodiment 503. The method of any of embodiments 500-502, wherein the vaccine againstthe coronavirus is approved by a regulatory agency for prevention of a disease caused by the coronavirus.
[0916] Embodiment 504. The method of any of embodiments 500-503, further comprising administering a second dose of the vaccine againstthe coronavirus to the human subject based, at least in part, on measuring in (d).
[0917] Embodiment 505. The method of any of embodiments 500-504, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
[0918] Embodiment 506. The method of any of embodiments 500-505, wherein the coronavirus is a Severe acute respiratory syndrome -related (SARS) virus.
[0919] Embodiment 507. The method of embodiment 506, wherein the SARS is SARS-CoV- 2.
[0920] Embodiment 508. The method of any of embodiments 500-507, wherein the human subject is at least or equal to about 50 years of age.
[0921] Embodiment 509. The method of any of embodiments 500-508, further comprising prior to (b): (e) eluting the at least the portion of the biological sample from a dried blood spot card prior to (b). [0922] Embodiment 510. A method for detecting a presence, an absence, or a quantity of neutralizing antibodies against a coronavirus in biological samples obtained from a population of human subjects vaccinated against the coronavirus, the method comprising: (a) providing the biological samples obtained from the population of human subjects vaccinated against the coronavirus, wherein the biological samples have an average volume of less than 1 milliliter when obtained; (b) introducing at least a portion of the biological samples to a testing device or module to detect neutralizing antibodies against the coronavirus that functionally block binding between a spike protein of the coronavirus and Angiotensin -converting enzyme 2 (ACE2), wherein the testing device or module comprises: (i) a first peptide or protein comprising the ACE2 polypeptide or a portion thereof that binds to the spike protein or a portion thereof; and (ii) a second peptide or protein comprising the spike protein or the portion thereof, wherein (i), (ii), or a combination of (i) and (ii) comprises a detectable label; and (c) detecting, with the testing device or module, a presence, an absence, or a quantity of a complex formed between (i) and (ii) in the presence of the biological samples, wherein the presence, the absence, orthe quantity of the complexis inversely correlated with the presence, the absence, or the quantity of the neutralizing antibodies against the coronavirus in the biological samples, respectively.
[0923] Embodiment 511. The method of embodiment 510, wherein the biological samples comprise capillary blood.
[0924] Embodiment 512. The method of any of embodiments 510-511, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0925] Embodiment 513. The method of any of embodiments 510-512, wherein the human subjects are vaccinated against the coronavirus with a vaccine that is approved by a regulatory agency for prevention of a disease caused by the coronavirus.
[0926] Embodiment 514. The method of any of embodiments 510-513, further comprising administering a second dose of the vaccine against the coronavirus to at least one of the human subjects based, at least in part, on detecting in (c).
[0927] Embodiment 515. The method of any of embodiments 510-514, further comprising prior to (b): (d) eluting the at least the portion of the biological samples from one or more dried blood spot cards prior to (b).
[0928] Embodiment 516. The method of any of embodiments 510-515, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo. [0929] Embodiment 517. The method of any of embodiments 510-516, wherein the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus.
[0930] Embodiment 518. The method of embodiment 517, wherein the SARS is SARS-CoV- 2.
[0931] Embodiment 519. The method of any of embodiments 510-518, wherein the human subjects are at least or equal to about 50 years of age.
[0932] Embodiment 520. A method for detecting a neutralizing antibody against the spike (S) protein of a coronavirus (CoV) in a biological sample from a human subject that was vaccinated against the CoV, the method comprising: (a) obtaining the biological sample obtained from the human subject that was vaccinated against the CoV with a vaccine against the CoV; (b) analyzing at least a portion of the biological sample to detect a level of the neutralizing antibody against the coronavirus that blocks binding between the S protein of the CoV (CoV S) and Angiotensin-converting enzyme 2 (ACE2) in the biological sample by:
[0933] (i) contacting the biological sample with (1) a first protein comprising ACE2, or a fragment thereof, that binds to the CoV S protein, or a fragment thereof, and (2) a second protein comprising the CoV S protein, or the fragment thereof, that binds to the ACE2, or the fragment thereof, under conditions that permit binding between the ACE2 or fragment thereof, and the CoV S protein or fragment thereof, to produce an ACE2-CoV S complex, wherein the first protein or the second protein is labeled directly or indirectly with a detection reagent; and (ii) detecting a signal corresponding to a level of the ACE2-CoV S complex in the biological sample wherein the signal is inversely correlated with the level of the neutralizing antibody in the biological sample; and (c) detecting the level of the neutralizing antibody in the biological sample based, at least in part, on the signal detected in (b)(ii), wherein performing steps (b) to (c) are performed without a washing step.
[0934] Embodiment 521. The method of embodiment 520, wherein the biological sample comprises capillary blood.
[0935] Embodiment 522. The method of any of embodiments 520-521, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0936] Embodiment 523. The method of any of embodiments 520-522, wherein the vaccine against the coronavirus is approved by a regulatory agency for prevention of a disease caused by the coronavirus. [0937] Embodiment 524. The method of any of embodiments 520-523, further comprising administering a second dose of the vaccine against the coronavirus to the human subject based, at least in part, on detecting in (c).
[0938] Embodiment 525. The method of any of embodiments 520-524, further comprising prior to (b): (d) eluting the at least the portion of the biological sample from a dried blood spot card prior to (b).
[0939] Embodiment 526. The method of any of embodiments 520-525, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
[0940] Embodiment 527. The method of any of embodiments 520-526, wherein the coronavirus is a Severe acute respiratory syndrome -related (SARS) virus.
[0941] Embodiment 528. The method of embodiment 527, wherein the SARS is SARS-CoV- 2.
[0942] Embodiment 529. The method of any of embodiments 520-528, wherein the human subject is at least or equal to about 50 years of age.
[0943] Embodiment 530. A method of assessing adaptive immunity against a coronavirus in subject, the method comprising: (a) bringing a biological sample of the subject into contact with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from the coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies thatblockbindingbetweenthe ACE2 polypeptideand the spike protein; (b) measuring percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample when the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is a signal from the binding complex, SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein; and (c) comparing the percent inhibition of the binding measured in (b) to a percent inhibition of binding of the first peptide or protein to the second peptide or protein of in a control sample or index, wherein the control or the index is derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus.
[0944] Embodiment 531 . The method of embodiment 530, wherein the biological sample has an average volume of less than 1 milliliter when obtained from the subject.
[0945] Embodiment 532. The method of embodiment 530 or embodiment 531, wherein the biological sample is not a liquid when it is provided in (a).
[0946] Embodiment 533. The method of any one of embodiments 530-532, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0947] Embodiment 534. The method of any one of embodiments 530-533, wherein the one or more control subjects received a vaccine against the coronavirus within one month of comparing in (d).
[0948] Embodiment 535. The method of any one of embodiment 530-534, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
[0949] Embodiment 536. The method of any one of embodiment 530-535, wherein the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus.
[0950] Embodiment 537. The method of embodiment 536, wherein the SARS virus is SARS- CoV-2 or a variant thereof.
[0951] Embodiment 538. The method of any one of embodiment 530-537, wherein the subject is a human subject that has an age comprising more than or equal to about 50 years of age.
[0952] Embodiment 539. The method of any one of embodiment 530-538, further comprising eluting the at least the portion of the biological sample from a dried blood spot card.
[0953] Embodiment 540. The method of any one of embodiment 530-539, wherein the subject is unvaccinated against the coronavirus.
[0954] Embodiment 541. A method comprising: identifying a subject as having adaptive immunity against a coronavirus relative to a control subject that is vaccinated against the coronavirus, wherein the identifying comprises: (a) bringing a biological sample obtained from the subject into contact with (1) a firstpeptide orprotein comprisingan Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (2) a second peptide or protein comprising a spike protein or a portion thereof derived from the coronavirus under conditions sufficientto form abinding complex between the firstpeptideor protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring a percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample; and (c) comparing the percent inhibition of the binding to a percent inhibition of binding of the first peptide or protein to the second peptide or protein in a control or an index, wherein the control or the index is derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus. [0955] Embodiment 542. The method of embodiment 541, wherein the biological sample has an average volume of less than 1 milliliter when obtained from the subject.
[0956] Embodiment 543. The method of embodiment 541 or embodiment 542, wherein the biological sample comprises capillary blood.
[0957] Embodiment 544. The method of any one of embodiments 541-543, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0958] Embodiment 545. The method of any one of embodiments 541-543 wherein the control subject received a vaccine against the coronavirus within one month of comparing in (c).
[0959] Embodiment 546. The method of any one of embodiments 541-545, further comprising: (d) identifyingthe adaptive immunity againstthe coronavirus of the subject as beinglower than the adaptive immunity of the control or the index; and (e) administering a vaccine againstthe coronavirus to the subject.
[0960] Embodiment 547. The method of any one of embodiments 541-546, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
[0961] Embodiment 548. The method of any one of embodiments 541-547, wherein the coronavirus is a Severe acute respiratory syndrome -related (SARS) virus.
[0962] Embodiment 549. The method of embodiment 548, wherein the SARS virus is SARS- CoV-2, or a variant thereof.
[0963] Embodiment 550. The method of any one of embodiments 541-549, wherein the human subject has an age comprising greater than or equal to about 50 years of age.
[0964] Embodiment 551. The method of any one of embodiments 541-550, further comprising eluting the biological sample from a dried blood spot card.
[0965] Embodiment 552. The method of any one of embodiments 541-551, whereinthe subject is unvaccinated againstthe coronavirus. [0966] Embodiment 553. A method of preparing biological sample from a subject useful for analyzing an adaptive immunity against a coronavirus in the subject, the method comprising: (a) eluting the biological sample from a biological sample storage medium; (b) contacting the biological sample with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof of the coronavirus under conditions sufficient for binding between the ACE2 polypeptide and the spike protein; (c) detecting a signal correspondingto a binding complex between the firstpeptide or protein and the second peptide or protein; (d) measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein when the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is the signal detected in (c), SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein.
[0967] Embodiment 554. The method of embodiment 553, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
[0968] Embodiment 555. The method of embodiment 553 or embodiment 554, wherein the biological sample comprises capillary blood.
[0969] Embodiment 556. The method of any one of embodiments 553-555, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0970] Embodiment 557. The method of any one of embodiments 553-556, wherein the one or more control subjects received a vaccine against the coronavirus within one month of comparing in (d).
[0971] Embodiment 558. The method of any one of embodiments 553-557, further comprising administering a vaccine against the coronavirus to the subject, provided the percent inhibition measured in (b) is lower than the control of the index.
[0972] Embodiment 559. The method of any one of embodiments 553-558, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
[0973] Embodiment 560. The method of any one of embodiments 553-559, wherein the coronavirus is a Severe acute respiratory syndrome -related (SARS) virus.
[0974] Embodiment 561 . The method of embodiment 560, wherein the SARS virus is SARS- CoV-2, or a variant thereof. [0975] Embodiment 562. The method of any one of embodiments 553-561, wherein the human subject is at least or equal to about 50 years of age.
[0976] Embodiment 563. The method of any one of embodiments 553-562, wherein the biological sample storage medium is a blood spot card.
[0977] Embodiment 564. A method of analyzing a biological sample, the method comprising: (a) bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an accuracy of at least about 80%.
[0978] Embodiment 565. The method of embodiment 564, wherein the subject is vaccinated against the coronavirus.
[0979] Embodiment 566. The method of embodiment 564, wherein the subject is unvaccinated against the coronavirus.
[0980] Embodiment 567. The method of any one of embodiments 564-566, wherein the coronavirus comprises SARS-CoV-2 or a variant thereof.
[0981] Embodiment 568. The method of any one of embodiments 564-567, wherein the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide.
[0982] Embodiment569. Themethodof any oneof embodiments 564-568, wherein the second protein or peptide comprises the S1 subunit of the spike protein.
[0983] Embodiment 570. The method of any one of embodiments 564-569, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
[0984] Embodiment 571. The method of any one of embodiments 564-570, wherein the biological sample comprises capillary blood.
[0985] Embodiment 572. The method of any one of embodiments 564-571, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[0986] Embodiment 573. The method of embodiment 564, further comprising administering a vaccine against the coronavirus to the subject based on measuring in (b).
[0987] Embodiment 574. The method of embodiment 564, wherein the spike protein of the coronavirus comprises a receptor binding domain specificfor binding with ACE2 in vivo. [0988] Embodiment 575. The method of any one of embodiments 564 -574, wherein the subject is an age comprising greater than or equal to about 50 years of age.
[0989] Embodiment 576. The method of any one of embodiments 564-575, further comprising detecting a signal from a detectable moiety coupled to the binding complex, wherein the signal comprises a fluorescence resonance energy transfer (FRET) signal.
[0990] Embodiment 577. The method of any one of embodiments 564-576, further comprising eluting the at least the portion of the biological sample from a dried blood spot card.
[0991] Embodiment 578. The method of any one of embodiments 564-577, wherein the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is a signal from the binding complex, SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein.
[0992] Embodiment 579. The method of any one of embodiment 564-578, wherein the accuracy is measured by : (a) introducing a reference sample with (i) the first peptide or protein; and (ii) the second peptide or protein, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC50; and (b) detecting a binding complex between the first peptide or protein and the second peptide or protein; and (c) determining an IC50 for the neutralizing antibody, wherein the IC50 is approximately the known IC50.
[0993] Embodiment 580. A method of analyzing a biological sample, the method comprising: (a) bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein; (b) measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an sensitivity of at least about 80%.
[0994] Embodiment 581. The method of embodiment 580, wherein the subject is vaccinated against the coronavirus.
[0995] Embodiment 582. The method of embodiment 580, wherein the subject is unvaccinated against the coronavirus. [0996] Embodiment 583. The method of any one of embodiment 580-582, wherein the coronavirus comprises SARS-CoV-2 or a variant thereof.
[0997] Embodiment 584. The method of any one of embodiments 580-583, wherein the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof.
[0998] Embodiment585. Themethodof any oneof embodiments 580-584, wherein the second protein or peptide comprises the S1 subunit of the spike protein.
[0999] Embodiment 586. The method of any one of embodiments 580-585, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
[1000] Embodiment 587. The method of any one of embodiments 580-586, wherein the biological sample comprises capillary blood.
[1001] Embodiment 588. The method of any one of embodiments 580-587, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[1002] Embodiment 589. The method of embodiment 580, further comprising administering a vaccine against the coronavirus to the subject based on measuring in (b).
[1003] Embodiment 590. The method of embodiment 580, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
[1004] Embodiment 591. Themethodof anyone of embodiments 580-590, wherein the subject is an age comprising greater than or equal to about 50 years of age.
[1005] Embodiment 592. The method of any one of embodiments 580-591, further comprising eluting the at least the portion of the biological sample from a dried blood spot card.
[1006] Embodiment 593. The method of any one of embodiments 580-592, wherein the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is a signal from the binding complex, SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization of binding between the first peptide or protein and the second peptide or protein.
[1007] Embodiment 594. A method of analyzing a biological sample, the method comprising: (a) bringing at least a portion of a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies thatblockbindingbetweenthe ACE2 polypeptideand the spike protein; (b) measuringa percentinhibition of bindingbetween the firstpeptide or protein andthe second peptide orprotein within the biological sample with percent coefficient of variation of lessthan 10%.
[1008] Embodiment 595. The method of embodiment 594, wherein the subject is vaccinated against the coronavirus.
[1009] Embodiment 596. The method of embodiment 594, wherein the subject is unvaccinated against the coronavirus.
[1010] Embodiment 597. The method of any one of embodiments 594-596, wherein the coronavirus comprises SARS-CoV-2 or a variant thereof.
[1011] Embodiment 598. The method of any one of embodiment 594-597, wherein the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof.
[1012] Embodiment599. Themethodof any oneof embodiments 594-598, wherein the second protein or peptide comprises the S1 subunit of the spike protein.
[1013] Embodiment 600. The method of any one of embodiments 594-599, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
[1014] Embodiment 601 . The method of any one of embodiments 594-600, wherein the biological sample comprises capillary blood.
[1015] Embodiment 602. The method of any one of embodiments 594-601, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
[1016] Embodiment 603. The method of any one of embodiments 594-602, further comprising administering a vaccine against the coronavirus to the subject based on measuring in (b).
[1017] Embodiment 604. The method of any one of embodiments 594-74603 wherein the spike protein of the coronavirus comprises a receptor binding domain specific forbinding with ACE2 in vivo.
[1018] Embodiment 605. Themethodof any one of embodiments 594-604, wherein the subject is an age comprising greater than or equal to about 50 years of age.
[1019] Embodiment 606. The method of any one of embodiments 594-605, further comprising eluting the biological sample from a dried blood spot card.
[1020] Embodiment 607. The method of any one of embodiments 594-606, wherein the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein ST is a signal from the binding complex, SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and S0 is a signal expected for zero neutralization ofbindingbetweenthefirstpeptideorprotein and the second peptide or protein.
[1021] Embodiment 608. The method of any of embodiments 530-607, wherein the percent inhibition is predictive of a clinical endpoint when the subject received a pre-approved or licensed vaccine against the coronavirus.
[1022] Embodiment 609. The method of embodiment 608, wherein the pre-approved or licensed vaccine comprises Ad26.COV.S, mRNA-1273 or BNT162b2, or any combination thereof.
[1023] Embodiment 610. The method of any of embodiments 530-609, wherein the percent inhibition is predictive of individual protection against another infection by the coronavirus.
[1024] Embodiment 611. The method of any of embodiments 530-610, further comprising administering to the subject a vaccine booster against the coronavirus or a second dose of a vaccine against the coronavirus, when the percent inhibition is below low relative to a control of the index derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated againstthe coronavirus, (iii) has recovered fromthe infection by the coronavirus and is vaccinated againstthe coronavirus, or (iv) is vaccinated againstthe coronavirus and has received at least one booster vaccine against the coronavirus.
[1025] Embodiment 612. The method of any of embodiments 530-611, wherein the subject does not have any underlying immune-compromising condition.
[1026] Embodiment613. The method of any of embodiments 530 -612, wherein the subject has an underlying immune-compromising condition.
[1027] Embodiment 614. The method of embodiment 611 or 612, wherein the underlying immune-comprising condition comprises a pregnancy, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof.
[1028] Embodiment 615. The method of anyone of embodiments 530-614, wherein the subject is breastfeeding.
[1029] Embodiment 616. The method of any one of embodiments 530-615, wherein further comprising detecting one or more signals from one or more detectable moieties coupled to the binding complex. [1030] Embodiment 617. The method of embodiment 616, wherein the one or detectable moieties comprises a nanoparticle, a fluorescent metal, a fluorescent dye, an enzymatic label, or a colorimetric label, or any combination thereof.
[1031] Embodiment 618. The method of embodiment 616 or 617, wherein the detecting the one or more signals is performed using fluorescent resonance energy transfer (FRET).
[1032] Embodiment 619. The method of embodiment 618, wherein the FRET comprises lanthanide-based FRET (LRET).
[1033] Embodiment 620. The method of any one of embodiments 616-619, wherein the one or more detectable moieties comprises a first detectable moiety coupled to the first peptide or protein, and a second detectable moiety coupled to the second peptide or protein.
[1034] Embodiment 621 . The method of embodiment 620, wherein the first detectable moiety comprises a FRET acceptor and the second detectable moiety comprises a FRET donor.
[1035] Embodiment 622. The method of embodiment 620, wherein: (a) the first detectable moiety comprises a luminescent lanthanide, and the second detectable moiety comprises a fluorophore; or (b) the second detectable moiety comprises the luminescent lanthanide, and the first detectable moiety comprises the fluorophore.
[1036] Embodiment 623. The method of embodiment 622, wherein the luminescent lanthanide comprises terbium or europium.
[1037] Embodiment 624. The method any one of embodiments 622-623, wherein the luminescent lanthanide is a FRET donor, and the fluorophore is a FRET acceptor.
[1038] Embodiment 625. The method of any one of embodiments 530-624, further comprising determining that the biological sample comprises about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell-based pseudovirion neutralization assay (PNA), when the percent neutralization measured is about 62%.
[1039] Emb odiment 626. The method of emb odiment 625 , further comprising determining that the subject is adequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of about 62%.
[1040] Embodiment 627. The method of any one of embodiments 530-626, further comprising determining that the biological sample comprises less than about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell -based pseudovirion neutralization assay (PNA), when the percent neutralization measured is less than about 62%. [1041] Emb odiment 628. The method of emb odiment 627, further comprising determining that the subject is inadequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of less than about 62%.
[1042] Embodiment 629. The method of any one of embodiments 530-628, wherein the method is performed without a wash step towash away the firstpeptide or protein or the second peptide or protein that are unbound following the bringing in (a).
[1043] Embodiment 630. The method of any one of embodiments 530-629, wherein the bringing the biological sample into contact with the first peptide or protein and the second peptide or protein is performed in a container.
[1044] Embodiment 631. The method of embodiment 630, wherein the container is optically clear.
[1045] Embodiment 632. The method of embodiment 630 or 631, wherein the container is a disposable cartridge.
[1046] Embodiment 633. The method of any one of embodiments 530-632, further comprising repeating the method on a plurality of biological samples obtained from the subject, wherein at least two biological samples of the plurality of biological samples are obtained at different time points.
[1047] Embodiment 634. The method of any one of embodiments 530-633, wherein the subject is receiving an immunosuppressant therapy.
[1048] Embodiment 635. The method of embodiment 634, further comprising: (a) identifying the subject as needing a dose of a vaccine against the coronavirus, provided the percent neutralization is lower than 62%; and (b) suspending a treatment regimen comprising the immunosuppressant therapy for a period of time during which period of time, the subject is administered the dose of the vaccine.
DEFINITIONS
[1049] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [1050] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[1051] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.
[1052] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.
[1053] As used herein, “allowed” or “allowance” in connection with a vaccine composition refers to a vaccine composition that is legally permitted by a relevant regulatory entity to be marketed for use to prevent a disease caused by a pathogen in an intended subject. In some embodiments, an allowed vaccine is approved or licensed for human use. In some embodiments, an allowed vaccine is approved, conditionally approved, or indexed for animal use. In some embodiments, the intended subject is a human. In some embodiments, the intended subject is an animal, such as a household pet or a farm animal.
[1054] As used herein, the phrases “at least one”, “one or more”, and “and/or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
[1055] Any systems, methods, software, compositions, and platforms described herein are modular and not limited to sequential steps. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.
[1056] The term “analyte” refers to a substance whose chemical constituents or activity is measured. In some embodiments, the analyte comprises an activity of a neutralizing antibody. In some embodiments, the activity is blocking binding between a pathogen of interest (e.g., SARS-CoV-2) and a cognate receptor (e.g., ACE2). In some embodiments, the analyte is a complex comprisingthe spike protein boundto the neutralizing antibody atthe receptor binding region of the spike protein.
[1057] The term “cloud” refers to shared or sharable storage of electronic data. The cloud may be used for archiving electronic data, sharing electronic data, and analyzing electronic data.
[1058] As used herein, the terms, “clinic,” “clinical setting,” “laboratory” or “laboratory setting” refer to a hospital, a clinic, a pharmacy, a research institution, a pathology laboratory, or other commercial business setting where trained personnel are employed to process and/or analyze biological and/or environmental samples. These terms are contrasted with point of care, a remote location, a home, a school, and otherwise non -business, non-institutional setting
[1059] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing is relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
[1060] The terms “increased,” or “increase” are used herein to generally mean an increase by a statically significant amount. In some embodiments, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5 -fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
[1061] The terms, “decreased” or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some embodiments, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease is, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.
[1062] The terms “subject,” or “individual,” are often usedinterchangeably herein. A “subject" is a biological entity containing expressed genetic materials. The biological entity is a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject is tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. The subject may be suspected of being at high risk for a disease. In some cases, the subject is not diagnosed with the disease. In some embodiments, the subject is a “patient,” that has been diagnosed with the disease. In some embodiments, the subject is a child. In some embodiments, the subject is an adolescent. In some embodiments, the subject is an adult. In some embodiments, the age of the subjectis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years. In some embodiments, the age of the subjectis between 18 and 50 years. In some embodiments, the subject is more than or equal to about 50 years old (e.g., 65 years old). In some embodiments, the subject is between 50-100, 55-95, 60-90, 65-85, or 70-80 years old.
[1063] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.
[1064] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results includebut are not limited to a therapeutic b enefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylacticbenefit, a subjectatrisk of developing a particular disease, or to a subject rep orting one or more of the physiological symptoms of a disease may undergo treatment, eventhough a diagnosis of this disease may not have been made.
[1065] The term “adaptive immune response” or “adaptive immunity” as used herein refers to the components of the immune response that respond in an antigen -restricted way. In some embodiments, the adaptive immune response encompasses cellular immune responses attributable to T lymphocytes and humoral or antibody response attributable to B cells and plasma cells. In some embodiments, the antibody response comprises neutralizing antibody response. A “cellular immune response” is indicated by any one or more of the following: cytokine/chemokine release by T cells; T-cell homing to secondary lymphoid organs; T-cell proliferation; and cytotoxic T-cell responses. Several methods are suitable to verify an antigenspecific cellular immune response, including ex vivo antigen stimulation assays of T lymphocytes and in vivo assays, such as tetramer staining of T lymphocytes. An “antibody response” is indicated at least by any one or more of the following: B cell proliferation, B-cell cytokine/chemokine release, B-cell homing to secondary lymphoid organs, antibody secretion, isotype switching to IgG type antibodies, or plasma cell differentiation. An antibody response may be verified by several methods, but a predominant method is the detection of antigenspecific antibodiesin the serum or plasma of an individual. In some embodiments, the antibody comprises neutralizing antibody.
[1066] An “adjuvant” as described herein refers to a substance that, in combination with an antigen, promotes an adaptive immune response to the antigen. In some embodiments, the adjuvant is an immune stimulatory compound. In some embodiments, the adjuvant comprises analgesic adjuvants. In some embodiments, the adjuvant comprises inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, or calcium phosphate hydroxide. In some embodiments, the adjuvant comprises mineral oil or paraffin oil. In some embodiments, the adjuvant comprises bacterial products such as inactivated Bordetella pertussis, Mycobacterium bovis, tor oxoids. In some embodiments, the adjuvant comprises nonbacterial organics like squalene. In some embodiments, the adjuvant comprises the use of delivery systems such as detergents (Quil A). In some embodiments, the adjuvant comprises plant saponins such as saponin derived from Quillaja, soybean, or Polygala senega. In some embodiments, the adjuvant comprises Freund's complete adjuvant or Freund's incomplete adjuvant. In some embodiments, the adjuvant comprises food -based oil like peanut oil.
[1067] An “immune stimulatory compound” refers to a substance that specifically interacts with the innate immune system to initiate a “danger signal” that ultimately leads to the development of the adaptive components of the immune response (e.g., B cell, T cells). Immune stimulatory compounds include, without limitation, pathogen-associated molecular patterns (PAMPs) such as dsRNA, lipopolysaccharide, and CpG DNA, either naturally occurring or synthetic. Immune stimulatory compounds may be agonists of various innate immune receptors including Toll-like receptors (TLRs), NOD-like receptors, RIG-1 or MDA- 5 receptors, C-type lectin receptors, or the STING pathway.
[1068] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
EXAMPLES
[1069] The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concept(s).
Example 1: Detecting Adaptive Immunity in a Healthcare Worker
[1070] A healthcare worker, responding to an urgent public health crisis, needs to know whether she is immune to an infection of a pathogen of interest so that she may serve others in the community at risk for infection. In this example, the pathogen of interest is SARS-CoV-2, and the healthcare worker is exposed to SARS-CoV-2. Optionally, the healthcare worker has recovered from coronavirus disease of 2019 (COVID-19).
[1071] While at home, the healthcare worker utilizes the point of need testing device described herein to obtain capillary blood by pricking her finger. She applies the capillary blood to the sample receptor component of the testing device, where it separates the blood serum (containing the analyte of interest) and other blood components. The serum flows downstream from the sample receptor to a test zone of the testing device, where the serum is mixed with a fluid composition comprising a peptide-conjugate. In this example, the peptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOS: 2-6, 11-12, 24, 47-48, 71-72, 95-96, or 115. The peptide-conjugate comprises a detectable moiety comprising a gold microsphere. Within 10-20 minutes a signal develops in the detectable zone of the testing device.
[1072] The healthcare worker takes a picture of the detectable zone using her smartphone, which is equipped with a mobile application that will interpret the results from the testing device. The mobile application displays the result via the graphical user interface of the smartphone and indicates to the healthcare worker that she is sufficiently immune to the pathogen. She returns to work immediately.
Example 2: Detecting Adaptive Immunity to Pathogen in Blood or Blood Plasma
[1073] A donation of blood or blood plasma is tested for a presence of neutralizing antibodies that functionally block binding between a pathogen of interest and an pathogen recognition receptor. In this example, the test is performed at the point of need (e.g., a blood bank) by a technician. In this example, the pathogen of interest is SARS-CoV-2.
[1074] At the blood donation site, the technician utilizes the point of need testing device described herein to test a sample of the blood. The technician applies the blood to the sample receptor component of the testing device, where it separates the blood serum (containing the analyte of interest) and other blood components. The serum flows downstream fromthe sample receptor to a test zone of the testing device, where the serum is mixed with a fluid composition comprising the peptide-conjugate from Example 1. Within 10-20 minutes a signal develops in the detectable zone of the testing device.
[1075] The technician takes a picture of the detectable zone using a tablet, which is equipped with a mobile application that will interpret the results from the testing device. The mobile application displays the result via the graphical user interface of the tablet and indicatesto the technician that the blood sample contains neutralizing antibodies that block binding between the spike protein of SARS-CoV-2 and human ACE2.
[1076] The technician sends the blood sample to a research laboratory to determine whether the serum isolated from this blood (containing the neutralizing antibodies) is suitable for use in convalescent plasma therapy.
Example 3. Vaccine Development Tool and Methods of Use
[1077] A pharmaceutical company is developing a vaccine to a pathogen of interest. In this example, the pathogen of interest is SARS-CoV-2. The pharmaceutical company wants to know whether the vaccine induces the production of antibodies which block the interaction between the spike protein of SARS-Co-V-2 and the human ACE2 receptor, that mediates infection in vivo. The pharmaceutical company utilizes the testing device described herein to test the vaccine by testing a biological sample from an animal (e.g., mammal) inoculated with the vaccine. [1078] A biological sample is obtained from a mammal that is exposed to SARS-Co-V-2, and is administered the vaccine. A researcher at the pharmaceutical company applies the biological sample to the sample receptor component of the testing device, where it separates the blood plasma/serum (containing the analyte of interest) and other blood components. The serum flows downstream from the sample receptor to a test zone of the testing device, where the serum is mixed with a fluid composition comprising the peptide-conjugate from Example 1. Within 10-20 minutes a signal develops in the detectable zone of the testing device.
[1079] The researcher takes an image of the detectable zone using an imaging device, which is equipped with an application that will interpret the results from the testing device. The application displays the result via the graphical user interface of the tablet and indicates to 1he researcher that biological sample contains antibodies that block the interaction between the spike protein of SARS-Co-V-2 and the human ACE2 receptor, which means that vaccine was effective. The vaccine moves on for further research and development.
Example 4. Identifying Herd Immunity using Artificial Intelligence
[1080] A governmental agency, respondingto a global pandemic, is monitoring a population of citizens to determine whether a threshold adaptive immunity neutralizing a pathogen of interest is present in the population, such that incidences of infection are drastically reduced (also referred to as “herd immunity”). The agency provides citizens testing devices, such as those described herein; and a free mobile App that is downloaded to their mobile device. Citizens in a given population utilize the testing device, take a picture of the testing zone of the testing device using their mobile device.
[1081] The data is anonymized and uploaded into the cloud. Data includes GPS data from the mobile devices and the data from the picture that was taken for each citizen. The data is analyzed by cloud-computing by machine learning, and results are accessible to the agency via a web-portal. The results are used to identify populations of citizens that are immune from SARS-CoV-2. Geofencing is used to create geographical boundaries around where those populations reside. When a threshold number of citizens with adaptive immunity neutralizing SARS-CoV-2 is detected conferring herd immunity.
Example 5. Rapid Deployment of Neutralization Assay for Newly Discovered Virus
[1082] A new strain of SARS-CoV-2 is discovered that has an amino acid sequence encoding the RBD comprising two amino acid substitutions, relative to SEQ ID NO: 3, 5, or 11. Referring to FIG. 8A-8B, components of the testing device described herein that were previously assembled include a surface comprising a capture molecule coupled to the surface, and a peptide-conjugate comprising a peptide derived from the ACE2 receptor and a detectable moiety (e.g., ACE2-Au).
[1083] Within weeks of the discovery of the new strain of SARS-CoV-2, a second peptide- conjugate is manufactured, the second peptide-conjugate comprising the variant RBD of the new strain of SARS-CoV-2 and conjugated to an epitope tag. The second peptide-conjugate is coupled to the surface via the surface-bound capture molecules specific to the epitope tag.
[1084] An individual in need of the testing device buys the testing device online, and downloadsthe mobile application on her iPhone. The testing device is mailed to the individual, and includes instructions for how to use the device and the testing device. Per the instructions provided, the individual pricks her finger to obtain capillary blood, places the drop of blood in the sample receptor of the testing device, and tilts the testing device sufficient to permit the fluid in the sample device to flow from the sample receptor to the opposite end of the testing device. If the individual has developed sufficient immunity to SARS-CoV-2, then no signal will be observed (FIG. 8B). If the individual has not developed sufficient immunity to SARS- CoV-2, then a signal will be observed (FIG. 8A).
Example 6. Screening for a Population at Risk of Infection
[1085] A population comprising a plurality of subjects is at risk of a SARS-CoV-2 infection due to an outbreak. Accordingly, immunity of the population is determined by utilizing the systems, devices, compositions, and methods described herein. Blood cards are distributed to the subjects for collection of biological sample comprising capillary blood. The healthcare workers perform finger-prick to drop the capillary blood into each slot of the dried blood spot card. After collection of biological samples, the dried blood spot cards are sent by mail to a centralized processing and testing facility, where the blood cards are processed by automated robotics. The blood samples are tested for presence or quantity of neutralizing antibody against at least one antigen of SARS-CoV-2. Most of the subjects are determined to have sufficient quantity of neutralizing antibody, which indicates that most the subjects can carry on with normal daily activities. Some of the subjects have absence or insufficient quantity of neutralizing antibody. These subjects are asked to take shelter -in-place measure and will be vaccinated with any one of the vaccines described herein to induce neutralizing antibody. [1086] In addition to determining the immunity due to the SARS-CoV-2 outbreak, the population is routinely screened at a time interval of once every three months. Such routine screening of the population identifies the percentage of the subjects within the population with sufficient quantity of neutralizing antibody against SARS-CoV-2. If the percentage of the subjects with sufficient quantity of neutralizing antibody is above the percentage of the herd immunity (e.g. over 70%), then the subjects with absence or insufficient quantity of neutralizing antibody do not need to be vaccinated.
[1087] The routine screening also identifies if any of the subjects are infected. The infected subjects can be asymptomatic but contagious. The routine screening is also conducted after the subjects are exposed to SARS-CoV-2. Those exposed subjects with an initial absence of neutralizing antibody against SARS-CoC-2 are retested at least one week, at least one month, or at least one year after the last exposure. Screening of the subjects for neutralizing antibody for SARS-CoV-2 identifies the subjects with the neutralizing antibody who should undergo an evaluation for assessing the symptoms or severity of the SARS-CoV-2 infection.
Example 7. Longitudinal System for Screening
[1088] Efficacy and durability of a vaccine allowed or approved for the prevention of a disease caused by SARS-CoV-2 is assessed by assaying a biological sample from a subject who was administered the vaccine with the confirmatory diagnostic testing device described herein. The confirmatory diagnostic testing devices measures neutralizing antibodies in biological samples obtained from the subject at various time points following vaccine administration.
[1089] A biological sample is obtained from the subject prior to vaccine administration. The biological may be obtained by the subject (e.g., finger prick) and deposited on a biological sample medium (e.g., a dried blood spot card) to be stored and transported to a laboratory for processing. In this instance, the biological sample is obtained by finger pricking the subject for the biological sample comprising capillary blood. The biological may be obtained by the subject (e.g., finger prick) and deposited on a biological sample medium (e.g., a dried blood spot card) to be stored and transported to a laboratory for processing. The biological sample is tested for neutralizing antibody against antigen of SARS-CoV-2. The confirmatory diagnostic confirms absence of the neutralizing antibody in the biological sample and the subject needs to be vaccinated against SARS-CoV-2.
[1090] One month after vaccination, a biological sample comprising the capillary blood is obtained from the subject finger pricking at home. The biological sample is sent back to the laboratory for testing via the use of a blood card. The confirmatory diagnostic confirms sufficient quantity of neutralizing antibody in the biological sample, which indicates that the subject has developed adaptive immunity against SARS-CoV-2. The confirmatory diagnostic also confirms the efficacy and effectiveness of the vaccine by detecting presence and sufficient quantity of neutralizing antibody in the biological sample.
[1091] The subject is tested again at two months after vaccination, at four months after vaccination, at six months after vaccination, at one year after vaccination, at two years after vaccination, at three years after vaccination, at four years after vaccination to confirm that the vaccinated subject has maintained the sufficient quantity of the neutralizing antibody for adaptive immunity against SARS-CoV-2.
[1092] At the testing at five years aftervaccination, the test reveals thatvaccinated subjectnow has insufficient quantity of the neutralizing antibody and is in need of a booster vaccine against SARS-CoV-2.
Example 8. Vaccine Confirmatory Diagnostic - Vaccine Comprising mRNA-1273
[1093] A clinical trial is being conducted to test efficacy and effectiveness of a SARS-CoV-2 vaccine comprising an mRNA encoding the full-length, prefusion stabilized Spike (S) protein (mRNA-1273). Subjects are enrolled into different cohorts to receive different dosages of the vaccine comprising the mRNA-1273. Subjects will receive the vaccine on Days 1 and 29 in 1he deltoid muscle and will be followed through 12 months post second vaccination (Day 394). Follow-up visits will occur 1, 2, and 4 weeks post each vaccination (Days 8, 15, 29, 36, 43, and 57), as well as 3, 6, and 12 months post second vaccination (Days 119, 209, and 394). The primary objective is to evaluate the safety and reactogenicity of a 2 -dose vaccination schedule of mRNA-1273, given 28 days apart, across 5 dosages in healthy adults.
[1094] Primary outcome measures: solicited local and systemic adverse reactions (ARs) [ Time Frame: 7 days post-vaccination]; unsolicited adverse events (AEs) [ Time Frame: 28 days postvaccination ]; medically -attended adverse events (MAAEs) [ Time Frame: Month 0 through Month 13 ]; serious adverse events (SAEs) [ Time Frame: Month 0 through Month 13 ]; change in the measure of clinical safety laboratory values in Cohort 2 from baseline [ Time Frame: Through 1 month after last vaccination ]; the number and percentage of participants with abnormalities in blood pressure, temperature, HR or respiratory rate will be assessed. [ Time Frame: Through 1 year after last vaccination ]; the number and percentage of participants with abnormalities in physical examinationswill be assessed [ Time Frame: Through 1 year after last vaccination ]; evaluate immunogenicity of mRNA-1273 by titer of SARS-CoV-2- specific binding antibody (bAb) measured by enzyme-linked immunosorbent assay (ELISA) [ Time Frame: Through 1 year after the final dose ],
[1095] Secondary outcome measures: titer of SARS-CoV-2-specific neutralizing antibody (nAb) using the testing devices described herein [ Time Frame: Through 1 year post last vaccination ]; and seroconversion as measured by an increase of SARS-CoV-2 -specific neutralizing antibody (nAb) titer using the testing devices described herein [ Time Frame: Through 1 year post last vaccination ].
[1096] Secondary outcome measures for this clinical study show that the mRNA-1273 vaccine induces neutralizing antibodies against SARS-CoV-2, indicating that mRNA-1273 is effective to confer adaptive immunity against SARS-CoV-2. This result is strongly correlative of protection against a future infection by SARS-CoV-2.
[1097] Durability of the mRNA-1273 vaccine is measured continuously throughout 1 year post the last vaccination. Seroconversion measured using the testing devices described show consistent neutralizing antibodies levels overtime, indicating that the mRNA-1273 vaccine is durable. The efficacy and durability of the mRNA-1273 vaccine is continuously monitored using the testing devices described herein during Phase 0, Phase I, Phase II, Phase III, or Phase IV or after Phase IV of the clinical trial.
[1098] Upon approval of the mRNA- 1273 vaccine, the vaccine will b e administered to subj ects in the population. Confirmatory diagnostic utilizing the systems, devices, methods, and compositions described herein will be performed to determine if the vaccinated subj ects will develop and maintain sufficient quantity of neutralizing antibody. Confirmatory diagnostic will be performed at one month, two months, six months, one year, two years, three years, and four years after vaccination. Subjects with insufficient quantity of neutralizing antibody will be advised to receive a booster vaccine.
[1099] In some embodiments, such an example applies to measuring vaccine efficacy for any one of: a SARS-CoV-2 vaccine comprising Bacille Calmette-Guerin (BCG) (a vaccine originally developed for tuberculosis), a SARS-CoV-2 vaccine comprising Ad5-nCoV ( a recombinant coronavirus vaccine using adenovirus Type 5 vector), a SARS-CoV-2 vaccine comprising AZDI 222 (ChAdOxl nCoV-19) (a replication-deficient chimpanzee adenovirus, ChAdOxl, which is engineered to express the Spike (S) protein of SARS-CoV-2), a SARS- CoV-2 vaccine comprising INO-400 (pGXDNA plasmid with nucleic acid encoding the Spike (S) protein of SARS-CoV-2 as the insert), a SARS-CoV-2 vaccine comprising BNT162 (a SARS-CoV-2 anti-viral RNA vaccine comprising mRNA or modified mRNA to express the Spike (S) protein or a fragment thereof of SARS-CoV-2), a SARS-CoV-2 vaccine comprising an inactivated SARS-CoV-2 virus, a SARS-CoV-2 vaccine comprising PiCoVacc (a formalin- inactivated SARS-CoV-2 virus), a SARS-CoV-2 vaccine comprising bacTRL-Spike (comprisingbifidobacterial engineered to expressthe Spike protein of SARS-CoV-2), a SARS- CoV-2 vaccine comprising NVX-CoV2373 (a multiple recombinant nanoparticle vaccine comprisinga prefusion form of the Spike protein of SARS-CoV-2), or any combinationthereof.
Example 9. Confirmatory Diagnostic for Vaccine Licensed for Use - mRNA1273
[1100] After licensure or approval of the mRNA1272 vaccine, the vaccine is administered to a population of subjects to confer adaptive immunity against SARS-CoV-2 in the population. After vaccination, the population is routinely screened with the confirmatory diagnostic testing system and devices described herein. One month after vaccination, the subjects are asked to submit biological samples comprising capillary blood. The subjects perform finger -prick at home to drop the blood droplets onto a blood card. The blood card contains multiple slots for multiple assays to be performed. The subjects drop the blood droplets onto each slot of the blood card and mail the blood card to a centralized facility. The blood cards are processed and assayed with the systems, devices, methods, and compositions described herein at the centralized facility to detect presence, absence, and quantity of neutralizing antibody induced by the mRNA1273 vaccine. At one month after vaccination, 100% of the tested subjects are confirmed to have sufficient quantity of neutralizing antibody against SARS-CoV-2 infection.
[1101] The subjects of the population are recommended to be tested using the confirmatory diagnostic testing device or system at two months after vaccination, at six months after vaccination, at one year after vaccination, at two years after vaccination, at three years after vaccination, and at every year after to ensure that the subjects maintain sufficient quantity of neutralizingantibody. The confirmatory diagnostic also identifiesthe subjects with insufficient quantity of neutralizing antibody at any of the time point for which the confirmatory diagnostic is performed. These subjects are advised to receive a booster vaccine.
[1102] In addition to determining the presence, absence, or quantity of neutralizing antibody in the vaccinated subjects over time, the confirmatory diagnostic also determines the percentage of the vaccinated subjects who have sufficient neutralizing antibody. 100% of the tested subjects are confirmed to have sufficient quantity of neutralizing antibody after one year. At two years after vaccination, 93% of the tested subjects are confirmed to have sufficient quantity of neutralizing antibody. 93% is above a threshold of herd immunity. Accordingly, other than advising the 7% of the subjects without sufficient quantity of neutralizing antibody to receive a booster vaccine, no additional step is taken. At three years after vaccination, only 53 % of the tested subj ects are confirmed to have sufficient neutralizing antibody, which is now below the threshold of herd immunity. Accordingly, in addition to strongly urging the subjects with insufficient quantity of neutralizing antibody to receive a booster vaccine, all subjects are asked to observe social distancing rules until the percentage of the population with sufficient quantity of neutralizing antibody is above the threshold of herd immunity again.
Example 10. Identifying Population in Need of Vaccination
[1103] A population of subjects is in need of a vaccine against a pathogen (e.g., SARS-CoV- 2), but there is a limited capacity to vaccinate each subject at the same time. The testing device or system described herein is used to identify subjects that lack a sufficient adaptive immune response to the pathogen, thereby identifying the subjects as being in need of the vaccine. Optionally, individuals who belong to a high risk category (e.g. , preexisting conditions, old age, and the like) may be prioritized for vaccination with the vaccine.
Example 11. Vaccine Confirmatory Diagnostic - Vaccine Comprising AV-COVID-19
[1104] A clinical trial is being conducted to test efficacy and effectiveness of a SARS-CoV-2 vaccine consisting of autologous dendritic cells loaded with antigens from SARS-CoV-2 to prevent COVID-19 in adults. Primary outcome measures: Confirm safety [ Time Frame: 6 months ], confirm safety of AV-COVID-19 by adverse event monitoring.
[1105] Secondary outcome measures: titer of SARS-CoV-2-specific neutralizing antibody (nAb) using the testing devices described herein [ Time Frame: Through 1 year post last vaccination ]; and seroconversion as measured by an increase of SARS-CoV-2 -specific neutralizing antibody (nAb) titer using the testing devices described herein [ Time Frame: Through 1 year post last vaccination ]; Suggestion of efficacy [ Time Frame: 6 months ], measurement of IgG and IgM in subject blood; Optimal dose of SARS-CoV-2 antigen [ Time Frame: 6 months ]; measurement of IgG and IgM in subject blood; Advantage of administering vaccine admixed with GM-CSF [ Time Frame: 6 months ], measurement of IgG and IgM in subject blood; Frequency of detecting IgG against SARS-CoV-2 in blood after vaccination [ Time Frame: 6 months ], measurement of IgG and IgM in subject blood. [1106] Durability of the AV-COVID- 19 vaccine is measured continuously throughout 1 year post the last vaccination. Seroconversion measured using the testing devices described show consistent neutralizing antibodies levels overtime, indicating that the AV-COVID- 19 vaccine is durable. The efficacy anddurability of the AV -COVID- 19 vaccine is continuously monitored using the testing devices described herein during Phase 0, Phase I, Phase II, Phase III, or Phase IV or after Phase IV of the clinical trial.
[1107] Upon approval of the AV-COVID-19 vaccine, the vaccine will be administered to subjects in the population. Confirmatory diagnostic utilizing the systems, devices, methods, and compositions described herein will be performed to determine if the vaccinated subjects will develop and maintain sufficient quantity of neutralizing antibody. Confirmatory diagnostic will be performed at one month, two months, six months, one year, two years, three years, and fouryears after vaccination. Subjects with insufficient quantity of neutralizing antibody will be advised to receive a booster vaccine.
Example 12. Veterinary Use of Confirmatory Diagnostic Test
[1108] A veterinarian wishes to know whether an animal subject (e.g., a farm animal or a household pet) responded to administration of a new vaccine against SARS-CoV-2. A biological sample is obtained from the animal subject before administration of the vaccine to assess the baseline level of neutralizing antibodies against SARS-CoV-2. A biological sample is obtained from the animal subject at least 2-4 weeks following the administration of the vaccine. The testing device and systems described herein are used to measure a presence or requisite level of neutralizing antibodies against SARS-CoV-2 as compared to the baseline level of neutralizing antibodies measured before administration of the vaccine.
Example 13. Automated and Centralized Sample Processing and Assaying
[1109] Biological samples are received at a testing facility equipped with a centralized and automated system for assaying the biological samples. Each biological sample is contained in a biological sample medium (BSM), such as a dried blood spot card. The automated system, as shown in FIG. 13, comprises a Biological Sample Processor 1302 and an Assay Assembly Block 1309, which modules and submodules are operatively connected with a mechanical arm that is controlled by a centralizing computing system programmed to input the BSMs into the biological sample processor 1302 and transfer the purified biological sample to the Assay Assembly Block 1309 without human intervention. The biological sample receiver 1303 receives BSM and a barcode on the BSM is optionally scanned by the biological sample scanner 1304. The patient data (e.g., name, preexisting condition, address, healthcare insurance provider, etc.) is stored in a database and later optionally associated with the Assay Results 1312 by one or more computer processors in the centralized computing system . The biological sample separator 1305 (e.g., a mechanical hole punch) punches a hole in the BSM to remove a segment of the biological sample, which segment falls into a well in a 96 well plate (“elution plate”). The biological sample imager 1306 is an optical apparatus that images the bottom (or sides) of the well to ensure that the segment is correctly placed into the well. The biological sample extractor 1307 adds elution buffer (phosphate-buffered saline) to the well. The 96 well plate is gently agitated at a temperature of around 4 degrees Celsius for a period oftime to elute the biological sample from the segment. The biological sample is optionally purified by the biological sample purifier 1308, such as isolating blood plasma or serum from a whole blood sample. The eluent is transferred to the Assay Assembly Block 1309, where the eluent from each well of the 96 well plate are diluted 4 times (e.g., 1 :80, 1 :160, 1 : 320, 1 : 640) and transferred into a 384 well plate (“source plate”) by the biological sample diluter 1310. The 1536 well plate is transferred to the biological sample assay conductor 1311, which runs the assay described herein (e.g., FRET, ELISA, etc.) to measure neutralizing antibodies againstthe coronavirus in dilutions. A higher signal intensity correlates with absence or low levels of neutralizing antibodies in the biological sample. The assay results 1312 are stored in a database and analyzed by one or more computer processors in the centralizing computing system. The one or more computer processors are configured to receive the results from the 4 dilutions per biological sample, averages the normalized results from the 4 dilutions per biological sample, and compares the average to a predetermined cutoff for neutralizing antibody titers indicative of CoVS-ACE2 binding inhibition (as measured, for example, using a cellularly based assay as demonstrated in Tan et al., A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2- -spike protein protein interaction, Nature Biotechnology 38, 1073-1078 (2020)), and optionally associates the results from the test to the patient data.
Example 14: Longitudinal Study of Elderly Subjects in Assisted Living Facilities
[1110] A study group of elderly subjects in assisted living facilities, ages 75 and above, will be monitored over a period of time to measure the durability and efficacy of a vaccine against a coronavirus (e.g., SARS-CoV-2). In addition to the elderly subjects who may be residents of the assisted living facility, the study may also include the staff and visitors of the assisted living facility. The environment of the study is chosen as an assisted living facility, as the subjects have high exposure to one another and are all in a patient population with lower immunity to the coronavirus due to immunosenescence. The study will determine if the vaccine has induced a sufficient level of neutralizing antibodies (nAbs) to protect the study subjects from later infection of the coronavirus, or provide a reduced severity of symptoms from the coronavirus if infection were to occur.
[1111] Before the subjects are administered a vaccine, a baseline titer of coronavirus -specific nAbs will be determined for each subject using the testing devices described herein. The subjects will then be administered a vaccine against the coronavirus and tested again to determine a titer of nAbs. Once the vaccine is administered, the subjects will then be tested regularly in monthly intervals to determine a titer of nAbs present in the patients’ system as time passes.
[1112] For each test, the subjects will be asked to submit biological samples comprising capillary blood. Blood cards will be distributed to the subjects for collection of the biological samples. The subjects, or healthcare workers, will perform a finger-prick to drop the capillary blood into each slot of the dried blood spot card. After collection of biological samples, the dried blood spot cardswill then be sent by mail to a centralized processing and testing facility, where the blood cards are processed, as described in Example 22.
[1113] The data in the study will show the efficacy of the vaccine to protect elderly subjects against the coronavirus. The data will show that, for elderly subjects, confidence intervals are wider, and total nAb titers are lower, indicating that patients in this elderly subgroup are in need of close monitoring. The data in this study is also applicable for patients ages 50 and older.
[1114] The data will also show that, for elderly patients ages 65 and older, an increased dose of the coronavirus vaccine is desired to provide adequate protection against the coronavirus. For example, the increased dose of the vaccine may include four times the antigen as a coronavirus vaccine for patients ages 64 and younger.
[1115] The level of nAbs is also correlated to negative PCR test results. The study will show that a high nAbs titer in the subjects is indicative of a negative PCR test result. Example 15: Study Showing the Effects of Obesity on Patients Diagnosed with a
Coronavirus
[1116] A study group of obese subjects will be monitored over a period of time to measure the durability and efficacy of a vaccine against a coronavirus (e.g., SARS-CoV-2) for obese patients. The study will determine the effect obesity has on patients diagnosed with a coronavirus.
[1117] Before the subjects are administered a vaccine, a baseline titer of coronavirus -specific nAbs will be determined for each subject using the testing devices described herein. The subjects will then be administered a vaccine against the coronavirus and tested again to determine a titer of nAbs. Once the vaccine is administered, the subjects will then be tested regularly in monthly intervals to determine a titer of nAbs present in the patients’ system as time passes.
[1118] For each test, the subjects will be asked to submit biological samples comprising capillary blood. Blood cards will be distributed to the subjects for collection of the biological samples. The subjects, or healthcare workers, will perform a finger-prick to drop the capillary blood into each slot of the dried blood spot card. After collection of biological samples, the dried blood spot cardswill then be sent by mail to a centralized processing and testing facility, where the blood cards are processed, as describedin Example 13.
[1119] The data will show that obese patients diagnosed with a coronavirus have an increased risk of death as compared to diagnosed patients who are not obese. The data will also show that obesity causes an increased risk of death for a coronavirus patient, and that the increased risk of death applies to all obese coronavirus patients, irrespective of the patient’s race.
Example 16: Study of Subject with a High Risk of Exposure to a Coronavirus
[1120] A study group of subjects with a high risk of exposure to a coronavirus will be monitored over a period of time to measure the durability and efficacy of a vaccine against a coronavirus (e.g., SARS-CoV-2). The subjects may be at a high risk of exposure to a coronavirusbecauseof congregate living circumstances. For example, the subjects may include the staff, guests, and visitors living on a cruise ship, in dormitories, or other congregate living environments. The study may also include subjects with a high risk of exposure due to their working environment. The study may follow healthcare workers, meat processing workers, food industry workers, public school workers, retail workers, and workers in other high- population-density work environments. The environment of the study is chosen as congregate living and/or high-population -density work environments, as the subjects have high exposure to one another. The study will determine if the vaccine has induced a sufficient level of neutralizing antibodies (nAbs) to protect the study subjects from later infection of the coronavirus, or provide a reduced severity of symptoms from the coronavirus if infection were to occur.
[1121] Before the subjects are administered a vaccine, a baseline titer of coronavirus -specific nAbs will be determined for each subject using the testing devices described herein. The subjects will then be administered a vaccine against the coronavirus and tested again to determine a titer of nAbs. Once the vaccine is administered, the subjects will then be tested regularly in monthly intervals to determine a titer of nAbs present in the subjects’ system as time passes. For each test, the subjects will be asked to submit biological samples comprising capillary blood. Blood cards will be distributed to the subjects for collection of the biological samples. The subjects, or healthcare workers, will perform a finger-prick to drop the capillary blood into each slot of the dried blood spot card. After collection of biological samples, the dried blood spot cardswill then be sent by mail to a centralized processing and testing facility, where the blood cards are processed, as describedin Example 132.
[1122] The data in the study will show the efficacy of the vaccine to protect subjects, with a high risk of exposure, against the coronavirus. The data will show a minimum nAbs titer necessary to protect subjects in such congregate living and high -population-density work environments.
[1123] The level of nAbs is also correlated to negative PCR test results. The study will show that a high nAbs titer in the subjects is indicative of a negative PCR test result.
Example 17. Performance of Neutralizing Antibody Test
[1124] In this example, the neutralizing antibody test is a time-resolved fluorescence energy transfer (TR-FRET) based homogenous assay to functionally measure neutralizing antibody activity for SARS-CoV-2 in serum obtained from individuals. The interaction between the virus Spike S1 protein with the host cell ACE2 receptor is mimicked in vitro with FRET pairs consisting of (1) an ACE2 receptor protein conjugated to a FRET donor (Europium) and (2) the SARS-CoV-2 Spike S1 subunit protein complexed to a FRET acceptor. When the FRET pairs are bound, excitation of the FRET donor results in energy transfer to and subsequent fluorescence of the FRET acceptor. In the presence of SARS-CoV-2 neutralizing antibodies, the binding of Spike S1 protein with ACE2 receptor is inhibited and results in decreased acceptor fluorescence.
[1125] Non-neutralizing antibodies that bind the Spike S1 protein without inhibiting the binding of the Spike S1 subunit with the ACE2 receptor will not decrease acceptor fluorescence. The neutralizing antibody test is a specific and direct measure of SARS-CoV-2 neutralizing antibodies. The neutralizing antibody test procedure consists of adding a single mixture of assay reagents with either patient specimen or with assay standards and/or external controls onto a 384-well plate. The reagent mixture contains (1) ACE2 receptor bound with Europium (EU) FRET donor and (2) SARS-CoV-2 Spike S1 subunit bound to a FRET acceptor. Equal volumes of the patient specimen (or assay standards/controls) and mixture of assay reagents (lOuL each) are incubated for 2 hours at room temperature, after which, the assay plate is read on a fluorescence microplate reader capable of measuring Time Resolve- Fluorescence Resonance Energy Transfer (TR-FRET). The neutralizing antibody test is formatted to be run either manually with single- -channel and multi-channel pipettes or it can be run on an automated high throughput platform.
[1126] The FRET reaction is activated at 337nm, and emission at 620nm and 665nm are determined. Light at 337nm is absorbed by the donor (Europium) and will transfer that energy to FRET acceptor when in close enough proximity to the donor (i.e. bound), and the acceptor will fluoresce at 665nm. If the FRET acceptor is not in near proximity (i.e. not bound), the energy on the donor is not transferred to the acceptor and resolves with the fluorescence of the donor at 620nm. The assay signal (665nm/620nm) will be greatest in the absence of binding inhibition when neutralizing antibody levels are low, and assay signal will be least in the presence of binding inhibition by neutralizing antibodies. In this way, the neutralization strength of any individual sera can be assessed by measuring its impact on the binding of the ACE2/Spike FRET pairs. The test signal is the ratio of light emission at 665nm/620nm and the neutralizing antibody test assay result is reported as % Neutralization (%N). The ratio equivalent to 0% Neutralization is defined by using Standard 1 , which does not contain any neutralizing antibody. The test signal equivalentto 100% neutralization is defined by using Standard 2, which contains a high level of NAb 414 monoclonal that completely blocks the Spike S-1 binding to ACE2 receptor. The % Neutralization activity of an individual specimen is calculated as follows: % Neutralization = 1- ((Ratio Specimen - RatioStd 2)/(RatioStd 1- RatioStd 2)) X 100%. [1127] In this example, the TR-FRET assay was used to determine whether the neutralizing antibody test described herein can measure neutralizing antibodies against SARS-CoV-2 accurately, and bindinginhibition was measured usingthe neutralizing antibody test and results were compared to a sample having a known IC50. The neutralizing antibody test was performed by performing a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction by the following steps:
[1128] Equal volumes (5 μl) of the ACE2 receptor protein complexed with EU, the SARS- CoV-2 Spike protein S1 subunit complexed with a FRET -acceptor for EU, and inhibitor were mixed, and were incubated for 1 hour at room temperature. In this example, the inhibitor is any solution containing neutralizing antibodies that inhibit the binding of the Spike protein S1 subunit to the ACE2 receptor protein. Specifically, multiple dilutions of a solution containing a known neutralizing antibody with an IC50 of 4nM was used as inhibitor. Following incubation, the fluorescent signal of the test mixture was then determined as shown in Table 3, and IC50 calculated. Read TR-FRET signal in a microtiter-plate readerunder settings described below in Table 2 (settings may need optimization depending on the instrument).
Table 2. Settings for TR-FRET Imaging
Figure imgf000204_0001
[1129] As shown in FIG. 15, the above protocol was performed on a 384 well plate (left) and a 1536 well plate (right), and in each case, the neutralizing antibody test accurately calculated the IC50 for the known neutralizing antibody. This demonstrates that the neutralizing antibody test described herein can measure inhibition of binding between the SARS-CoV-2 S protein and human ACE2 with accuracy. The signal to baseline (“S/B”) ratio of 4.70 and 4.54 demonstrate over 4 logs of separation between the baseline and maximum signal, indicating a large dynamic range for the neutralizing antibody test. The range in percent coefficient of variation (CV%) of 3.16-4.37 and 5.33-6.01 for the 384 well plate (left) and a 1536 well plate (right), respectively, are significantly lower than industry standard for a “good” test with respectto repeatability and reproducibility. Moreover, the Z’ score of0.81 and 0.74 forthe 384 well plate (left) and a 1536 well plate (right), respectively, indicate that the neutralizing antibody test is highly reproduceable. Together, these results show that the neutralizing antibody test described herein is accurate and highly reproduceable. In addition, the neutralizing antibody test described herein is scalable at least because of the small reagent requirements (<20 μl) and because the assay does not require a “wash” step.
Example 18. The Neutralizing Antibody Test Can Detect Varying Neutralizing Antibody Responses in Human Serum Samples
[1130] Vaccines against SARS-CoV-2 have been shown elicit a strong neutralizing antibody response in some, but not all, individuals, including vaccines that are licensed for commercial use. Measuring the binding inhibition between the SARS-CoV-2 Spike (S) protein and human ACE2 is a surrogate for measuring neutralizing antibody titers in a human subject, as shown in, as for example, Tan et al., A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediatedblockage of ACE2 -spike protein-protein interaction, NatBiotechnol. 2020 Sep; 38(9): 1073-1078, which is hereby incorporated by reference in its entirety. Since neutralizing antibody titers correlate with vaccine efficacy, measuring binding inhibition between the SARS-CoV-2 S protein and human ACE2 can evaluate vaccine efficacy for individuals in a given population. Earle, et al. Evidence for antibody as a protective correlate for COVID-19 vaccines, medRxiv preprint doi: 10.1101.2021.03.17.20200246 (March 20, 2021). The following experimental design is aimed at confirmingthatthe neutralizing antibody test described herein can quantitatively measure binding inhibition between SARS-CoV-2 S protein and human ACE2 as a surrogate for neutralizing antibody titers in human samples obtained from vaccinated subjects.
[1131] Three human blood serum samples are obtained that do not contain neutralizing antibodies against SARS-CoV-2. The samples are spiked with different known quantities of neutralizing antibodies against SARS-CoV-2 S protein, thereby mimicking human serum samples obtained from individuals in a given population vaccinated against SARS-CoV-2 having varying neutralizing antibody responses. The positive control that will be used contains a buffer with a known quantity of neutralizing antibodies against SARS-CoV-2 S protein. The negative control that will be used contains the buffer without any neutralizing antibodies against SARS-CoV-2 S protein.
[1132] Equal volumes of the ACE2 receptor protein complexed with EU, the SARS-CoV-2 Spike protein S1 subunit complexed with a FRET-acceptor for EU, and inhibitor are mixed, and are incubated for 1 hour at room temperature. In this example, the inhibitor is any solution containing neutralizing antibodies that inhibit the binding of the Spike protein S1 subunitto the ACE2 receptor protein. Specifically, multiple dilutions of a solution containing a known neutralizing antibody with an IC50 of 4nM is used as inhibitor. Following incubation, the fluorescent signal of the test mixture was then determined as shown in Table 2, and IC50 calculated. The above protocol is performed in a 384 well plate and a 1536 well plate. In each case, the results expected to be achieved are as follows:
[1133] It is expected that the neutralizing antibody test will accurately calculate the IC50 for the known neutralizing antibody forthose samples. The CV%, Z’ score, and S/B are expected to be similar to the results described in Example 17. Together, the results are expected to show that the neutralizing antibody test described herein is not only accurate, highly reproduceable, and scalable, but can measure binding inhibition between SARS-CoV-2 S protein and human ACE2 as a surrogate for neutralizing antibody titers in complex human serum samples that mimic samples obtained from vaccinated human subjects.
Example 19: Range of Neutralization Against a Coronavirus Vaccine
[1134] The neutralizing antibody response in serum samples obtained from 7 human subjects who had received complete doses of a vaccine against severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) was evaluated using the following protocol. 5 microliters (μl) of sera was added to 5 μl of ace2 conjugated to the FRET donor pair and 10 μl of spike S1 subunit conjugated to the FRET acceptor pair. The FRET reaction was performed in a 384 well microtiter plate in quadruplicate. Following a brief centrifugation to mix the reaction components and incubation at room -temperature, the plate was read in Synergy Neo 2 dual fluorescent reader, using excitation at 337nm and emission at 620nm and 665nm. The reaction signal is the 665nm/620nm ratio. Percent neutralization is determined by comparing the signal of each individual sera against known controls representing 0% neutralization and 100% neutralization, and is calculated as follows: Percent Neutralization = 1- (Signal of test sera - Signal 100% neutralization)/(Signal of 0% neutralization - Signal of 100% neutralization)* 100
[1135] There was a wide range of neutralization from 27% to 81% against a coronavirus vaccine formulated for human administration in human subj ects that were vaccinated with the coronavirus vaccine, as shown in FIG. 16. This data indicates that the elicitation of a neutralizing antibodies in response to vaccination is widely variable. This is surprising at least because, while the SARS-CoV-2 vaccine is highly effective when considering the population as a whole, this data show that on an individual level, the elicitation of neutralizing antibody response to the SARS-CoV-2 vaccine in humans varies widely. Measuring neutralizing antibody response to the SARS-CoV-2 vaccine may identify subjects as being at greater risk for infection and more severe clinical outcomes. The information gleaned from the neutralizing antibody test disclosed herein allows such subjects to make a more informed choice with respectto protective behavior, includingthe use of masks (indoors and outdoors), booster shots, social distancing, participating in high density events (e.g. air travel, airports, restaurants).
Example 20: Accuracy of the Neutralizing Antibody Test
[1136] The neutralizing antibody test disclosed herein have shown repeatable accurate results in serum samples with minimal matrix effects.
[1137] The methodology described in Example 17 above was repeated multiple times to demonstrate the robustness of the neutralizing antibody test disclosed herein. To assess accuracy, control solutions contained known amounts of neutralizing antibody were used as reference, and compared against negative sera containing known amounts of neutralizing antibody.
[1138] As shown in FIG. 17, the measurement of the IC50 value of Nab 414 (known IC50 of 8.0) is highly repeatable. Table 3 shows the results of the three runs.
Table 3. Confidence Intervals and IC50 of Known Neutralizing Antibody
Figure imgf000207_0001
[1139] Table 4 shows that accuracy in serum is greater than 90% across wide analyte concentration range (from 0.25uM to 0.00024uM NAb). Negative sera (sera without neutralizing antibodies) was used to generate different levels of neutralizing antibody. Buffer was also used to generate the same levels of neutralizing antibody. The observed (serum) was compared to the expected (buffer) as a measure of accuracy. At all levels tested, the accuracy was greater than 90%. These data indicate that the neutralizing antibody test is very accurate. Table 4. Accuracy of Neutralizing Antibody Test is Minimally Affected by Neutralizing
Antibody Concentrations
Figure imgf000208_0001
[1140] Table 5 illustrates that inter-assay repeatability is high (e.g., CV’s below 10%). Solutions with known levels of neutralizing antibody were prepared and tested over multiple runs to provide an estimate of inter-assay repeatability. These levels range from 0.5 nM down to 0.00617 nM. Note that the two lowest levels of neutralizing antibodies are near zero where variability is expected to be highest. For the 0.5, 0.1167 and 0.0556 nM level solutions, the variability (CV) was 1.7%, 2.1% and 3.8%, respectively. Even for the solutions containing neutralizing antibody at levels near zero, the variability was well below 10%. These data indicate the inter-assay repeatability is very high for the neutralizing antibody test.
Table 5. The Inter-Assay Repeatability is High for the Neutralizing Antibody Test
Figure imgf000208_0002
[1141] Table 6 illustrates that the measured inhibition does not change in varied dilutions of sera. Inhibition is not affected by sera. Twelve different negative sera were each diluted 40X, 80X, 160X and 320X, and then spiked with a single concentration of neutralizing antibody. Each sera was tested in quadruplicate according to the method described in Example 17 above. The assay signal is converted to % Neutralization by the formula below (please note that % Neutralization and % Inhibition are used interchangeably:
[1142] % Inhibition = (Signal of test sera - Signal 100% neutralization)/(Signal of 0% neutralization - Signal of 100% neutralization)* 100
[1143] The 0% neutralization signal is that signal obtained in the absence of neutralizing antibodies. For this level, the test reagents are mixed with buffer in place of patient sample. The 100% neutralization signal is that signal obtained in the presence of excess levels of neutralizing antibodies. To estimate 100% neutralization, 500nM of a potent neutralizing antibody (clone 414-1 obtained from BPS Bioscience) is used in place of patient sample. The data in Table 6 demonstrate that the measurement of percent inhibition is not affected by the serum matrix. Neither the 12 different sera used nor the concentration of sera used in these experiments affected the measurement of percent inhibition. These data demonstrate that the claimed neutralizing antibody test is not affected by differences in the sera amongst different human subjects.
Table 6. Inhibition Measured Using the Neutralizing Antibody Test Is Independent of Sera Dilutions
Figure imgf000209_0001
[1144] Table 7 illustrates that the measurement of the analyte is minimally impacted by the presence of serum. Similar to Table 6, twelve different sera were pre-diluted prior to the addition of a single level of a neutralizing antibody. Different from Table 6, the signal of the assay (and not percent inhibition) was directly compared. The variability of the signal was compared to understand the impact of different sera on assay signal. As can be seen by the data, the signal variability is reasonably low, ranging from 4.6% to 7.0%. It is low variability of assay signal, when evaluated against the 0% neutralization and 100% neutralization assay controls, that provides the low variability of the test result as seen in Table 6.
Table 7A. Percent Inhibition in 12 Sera Samples is Independent of Sera Dilutions
Figure imgf000210_0001
Table 7B. Average, Standard Deviation and Coefficient of Variation for 12 Sera Samples
Figure imgf000210_0002
[1145] Table 8 illustrates that the spike/recovery of Nab 414-1 is not affected by serum matrix. Similar to Table 6, twelve different sera were pre-diluted prior to the addition of a single level neutralizing antibody. In this analysis, the measurement of the target analyte is compared with that serum to understand the impact of individual serum dilution on assay signal. Of the twelve different sera evaluated in this manner, only one serum demonstrated a dilution -dependent variability greater than 10% . These data indicated thatthe dilution of serum utilized in the assay can be variable and will not unduly impact the assay result from the claimed neutralizing antibody test.
Table 8. Sera Matrix Does Not Affect Detection of NAb 414-1 with Known IC50
Figure imgf000210_0003
Figure imgf000211_0001
[1146] The neutralizing antibody test described herein has been shown to detect neutralizing antibodies against the SARS-CoV-2 vaccine in human sera with a high degree of accuracy and repeatability. The neutralizing antibody test accurately calculated the IC50 for the known neutralizing antibody in human sera, as shown in FIG. 17. This demonstrates that the neutralizing antibody test described herein can measure inhibition of binding between the SARS-CoV-2 S protein and human ACE2 with accuracy in human sera. Further, this accuracy was validated across a wide analyte concentration range (from 0.25uM to 0.00024uM NAb), as shown in Table 4. The range in percent coefficient of variation (CV%) for the neutralizing antibody testwas well below 10%, as shown in Table 5, indicatingthatthe claimed neutralizing antibody testis not only accurate, but precise. Together, these results show that the disclosed neutralizing antibody test is accurate and highly reproducible. Lastly, the percent inhibition measured using the claimed neutralizing antibody test was not affected by sera (presence/concentration) or analyte concentration, as shown in Tables 5-8.
Example 21. Screening for a Population at Risk of Infection
[1147] A population comprising a plurality of subjects is at risk of an influenza infection due to an outbreak. Accordingly, immunity of the population is determined by utilizingthe systems, devices, compositions, and methods described herein. Blood cards are distributed to the subjects for collection of biological sample comprising capillary blood. The healthcare workers perform finger-prick to drop the capillary blood into each slot of the dried blood spot card. After collection of biological samples, the dried blood spot cards are sent by mail to a centralized processing and testing facility, where the blood cards are processedby automated robotics. The blood samples are tested for presence or quantity of neutralizing antibody against at least one antigen of the influenza strain. Most of the subjects are determined to have sufficient quantity ofneutralizing antibody, which indicates that most the subjects can carry on with normal daily activities. Some of the subjects have absence or insufficient quantity of neutralizing antibody. These subjects are asked to take shelter -in-place measure and will be vaccinated with any one of the vaccines described herein to induce neutralizing antibody. [1148] In addition to determining the immunity due to the influenza outbreak, the population is routinely screened at a time interval of once every three months. Such routine screening of the population identifies the percentage of the subjects within the population with sufficient quantity of neutralizing antibody against influenza. If the percentage of the subjects with sufficient quantity of neutralizing antibody is above the percentage of the herd immunity (e.g over 70%), then the subjects with absence or insufficient quantity of neutralizing antibody do not need to be vaccinated.
[1149] The routine screening also identifies if any of the subjects are infected. The infected subjects can be asymptomatic but contagious. The routine screening is also conducted after the subjects are exposed to the influenza strain. Those exposed subjects with an initial absence of neutralizing antibody against the influenza strain are retested at least one week, at least one month, or at least one year after the last exposure. Screening of the subjects for neutralizing antibody for the influenza strain identifies the subjects with the neutralizing antibody who should undergo an evaluation forassessingthe symptoms or severity of the influenza infection.
Example 22. Accuracy of the Neutralizing Antibody Test: repeatability measures
[1150] In this example the TR-FRET assay was used to determine the accuracy and repeatability of the neutralizing antibody test. To perform the TR-FRET assay, a panel of sera samples obtained from human subjectsthat were vaccinated against SARS-CoV-2 (“vaccinated sera”) and samples obtained from human subjects that had recovered from an infection by SARS-CoV-2 (“convalescent sera”) were used to determine whether the neutralizing antibody test described herein can measure neutralizing antibodies against SARS-CoV-2 accurately in these two sample types, as measured by comparing binding inhibition of the neutralizing antibody test with sera samples spiked with a reference neutralizing antibodies having a known IC50 (NAb 414-1 with Known IC50 of 8.0). The TR-FRET assay was replicated three times (also referenced as Runs 1, 2 and 3) using different reagent lots and performed on difference days to show that there were not batch effects. For each run, TR-FRET reagents were added to the individual test samples (e.g., convalescent sera or vaccinated sera) or controls (e.g., assay standards and/or external control) as a single mixture into a 384-well plate. The reagent mixture contains (1) ACE2 receptor bound with Europium (EU) FRET donor and (2) SARS-CoV-2 Spike S1 subunit bound to a FRET acceptor. Equal volumes of the test samples (or assay standards/controls) and mixture of assay reagents (10 uL each) were incubated for 2 hours at room temperature, after which, the assay plate was read on a fluorescence microplate reader capable of measuring the TR-FRET (details in Example 17). In this example, the neutralizing antibody test was formatted to be run manually with single-channel pipettes, but it can be run with multi-channel pipettes, or on an automated high throughput platform. In this example, the negative control was a buffer solution that did not contain neutralizing antibodies, and the positive control was a buffer solution also negative for neutralizing antibodies against SARS- CoV-2 that were spiked with a known concentration of a neutralizing antibody with a known IC50 of 8.0.
[1151] The results of these triplicate runs are provided in FIG. 18A-C. As shownby correlation coefficient values obtained in paired comparisons shown in FIG. 18A-AC, the percent (%) neutralization results obtained when Run 1 versus Run 2 (FIG. 18A) or Run 1 versus Run 3 (FIG. 18B) were tightly comparable (R2 of 98%). The linear trend demonstrates that the neutralizing antibody test (runs 1, 2 and 3), which were performed on different days (FIG. 18A-B) or performed twice using different specimens and different reagent lots (FIG. 18C) were comparable across the different runs. These data shown in FIGS. 18A-18C demonstrate that any assay performed using the neutralizing antibody test reagents are consistently repeatable and are not impacted by batch effects.
Example 23. Neutralizing Antibody Test Correlates Strongly with Cell-based Neutralization Assay
[1152] A subset of the sera from Example 22 were also assayed using the conventional cellbased assay described in this example to validate that the neutralizing antibody test described here can be used as a surrogate for the conventional cell-based assay. Briefly, the cell based assay, also referred to as the Pseudovirion Neutralization Assay (PNA), was performed against Wuhan (WT) variant. On day 1, Vero E6 cells expressing the ACE-2 receptor were seeded in 96-well white plates at 20 000 cells/well to reach a cell confluence of 80% next day. On day 2, serum samples and controls were serially diluted. In parallel, SARS-CoV-2 pseudovirus were diluted as to reach the desired concentration (according to pre -determined TU/mL). Pseudovirus were added to diluted serum samples and pre-incubated for 1 hour at 37°C with 5% CO2. The mixture was added to the pre-seeded Vero E6 cell layers and plates will be incubated for 18-20 hours at 37°C with 5% CO2. On day 3, following incubation and removal of media, ONE-Glo EX Luciferase Assay Substrate will be added to cells and incubated for 3 minutes at RT with shaking. Luminescence will be measured using a SpectraMax iD3 microplate reader and SoftMax Pro v7.0.1. Luminescence results for each dilution were used to generate a titration curve using a 4 -parameter logistic regression (4PL). The titers were defined as the reciprocal dilution of the sample for which the luminescence is equal to a pre-determined cut-point of 50, corresponding to 50% neutralization. This cut-point is established using linear regression using 50% flanking points. Samples were analyzed using an array of acceptance criteria and will be repeated if that acceptance criteria are not met.
[1153] A comparison of the cell-based neutralization test and neutralizing antibody test of the present disclosure shows a strong agreement or correlation between the two test assays, as evidenced by the linearity of the titers in the linear plot shown in FIG. 19. This figure indicates these titers were co-linear at least up until about IC50 of 1,000 neutralizing antibody titers. These data strongly demonstrate that the titers obtained by the neutralizing antibody test disclosed herein and titers from the cell-based assay were very closely similar up to a titer of IC50 of about 1,000 (FIG. 19). The y-axis shows neutralizing titers measured by the neutralizing antib ody test expressed as neutralization inhibition; the x-axis showsthe cell-b ased results grouped by neutralizing antibody titer titrated or diluted between < 10, <10, 10 to 20, 201 to 400, 401 to 600, 601 to 800, 801 to 1000, and >1000. Therefore, the neutralizing antibody test disclosed herein can be used as a surrogate for the conventional cell-based assay. [1154] While preferred embodiments of the present inventive concept(s) have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the inventive concept(s). It should be understood that various alternatives to the embodiments of the inventive concept(s) described herein may be employed in practicing the inventive concept(s). It is intended that the following claims define the scope of the inventive concept(s) and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED:
1. A method of assessing adaptive immunity against a coronavirus in subject, the method comprising:
(a) bringing a biological sample of the subject into contact with (i) a firstpeptide orprotein comprising an Angiotensin-converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from the coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein;
(b) measuring percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample when the percent inhibition is calculated using a formula comprising: [(ST - SN)/(S0 - SN)* 100], wherein
ST is a signal from the binding complex,
SN is a signal expected for complete neutralization of binding between the first peptide or protein and the second peptide or protein, and
S0 is a signal expected for zero neutralization of binding between the firstpeptide or protein and the second peptide or protein; and
(c) comparing the percent inhibition of the binding measured in (b) to a percent inhibition of binding of the first peptide or protein to the second peptide or protein of in a control sample or index, wherein the control or the index is derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered fromthe infectionby the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus.
2. The method of claim 1, wherein the biological sample has an average volume of less than 1 milliliter when obtained from the subject.
3. The method of claim 1 or claim 2, wherein the biological sample is not a liquid when it is provided in (a).
4. The method of any one of claims 1 -3 , wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
5. The method of any one of claims 1 -4, wherein the one or more control subjects received a vaccine against the coronavirus within one month of comparing in (d).
6. The method of any one of claim 1-5, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
7. The method of any one of claim 1-6, wherein the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus.
8. The method of claim 7, wherein the SARS virus is SARS-CoV-2 or a variant thereof .
9. The method of any one of claim 1-8, wherein the subject is a human subject that has an age comprising more than or equal to about 50 years of age.
10. The method of any one of claim 1 -9, further comprising eluting the at least the portion of the biological sample from a dried blood spot card.
11. The method of any one of claim 1-10, wherein the subjectis unvaccinated against the coronavirus.
12. A method comprising: identifying a subject as having adaptive immunity against a coronavirus relative to a control subject that is vaccinated against the coronavims, wherein the identifying comprises:
(a) bringing a biological sample obtained from the subject into contact with (1) a first peptide or protein comprising an Angiotensin-converting enzyme2 (ACE2) polypeptide or a portion thereof, and (2) a second peptide or protein comprising a spike protein or a portion thereof derived from the coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein;
(b) measuring a percent inhibition of binding of the first peptide or protein to the second peptide or protein within the biological sample; and
(c) comparing the percent inhibition of the binding to a percent inhibition of binding of the first peptide or protein to the second peptide or protein in a control or an index, wherein the control or the index is derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received at least one booster vaccine against the coronavirus.
13. The method of claim 12, wherein the biological sample has an average volume of less than 1 milliliter when obtained from the subject.
14. The method of claim 12 or claim 13, wherein the biological sample comprises capillary blood.
15. The method of any one of claims 12-14, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
16. The method of any one of claims 12-15, wherein the control subject received a vaccine against the coronavirus within one month of comparing in (c).
17. The method of any one of claims 12-16, further comprising:
(d) identifying the adaptive immunity against the coronavirus of the subject as being lower than the adaptive immunity of the control or the index; and
(e) administering a vaccine against the coronavirus to the subject.
18. The method of any one of claims 12-17, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
19. The method of any one of claims 12-18, wherein the coronavirus is a Severe acute respiratory syndrome-related (SARS) virus.
20. The method of claim 19, wherein the SARS virus is SARS-CoV-2, or a variant thereof.
21. The method of any one of claims 12-20, wherein the human subject has an age comprising greater than or equal to about 50 years of age.
22. The method of any one of claims 12-21, wherein the first peptide or protein or the second peptide or protein is immobilized on a substrate
23. The method of any one of claims 12-22, further comprising detecting the binding complex using a lateral flow assay.
24. The method of claim 23, wherein detecting the binding complex in comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device.
25. The method of any one of claims 12-24, further comprising elutingthe biological sample from a dried blood spot card.
26. The method of any one of claims 12-25, wherein the subject is unvaccinated against the coronavirus.
27. A method of analyzing a biological sample, the method comprising:
(a) bringing a biological sample obtained from a subject into contact with (i) a first peptide orprotein comprisingan Angiotensin-converting enzyme2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein;
(b) measuring a percentinhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an accuracy of at least about 80%.
28. The method of claim 27, wherein the subject is vaccinated against the coronavirus.
29. The method of claim 27, wherein the subject is unvaccinated against the coronavirus.
30. The method of any one of claims 27-29, wherein the coronavirus comprises SARS- CoV-2 or a variant thereof.
31. The method of any one of claims 27-30, wherein the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide.
32. The method of any one of claims 27-31, wherein the second protein or peptide comprises the SI subunit of the spike protein.
33. The method of any one of claims 27-32, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
34. The method of any one of claims 27-33, wherein the biological sample comprises capillary blood.
35. The method of any one of claims 27-34, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
36. The method of claim 35, further comprising administering a vaccine against the coronavirus to the subject based on measuring in (b).
37. The method of claim 35, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
38. The method of any one of claims 27-37, wherein the subject is an age comprising greater than or equal to about 50 years of age.
39. The method of any one of claims 27-38, wherein the first peptide or protein or the second peptide or protein is immobilized on a substrate
40. The method of any one of claims 27-39, further comprising detecting the binding complex using a lateral flow assay.
41. The method of claim 40, wherein detecting the binding complex in (b) comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device.
42. The method of any one of claims 27-41, further comprising eluting the at least the portion of the biological sample from a dried blood spot card.
43. The method of any one of claim 27-42, wherein the accuracy is measured by:
(a) introducing a reference sample with (i) the first peptide or protein; and (ii) the second peptide or protein, wherein the reference sample comprises a known quantity of a reference neutralizing antibody, wherein the reference neutralizing antibody has a known IC50; and
(b) detecting a binding complex between the first peptide or protein and the second peptide or protein; and
(c) determiningan IC50 forthe neutralizing antibody, wherein the IC50is approximately the known IC50.
44. A method of analyzing a biological sample, the method comprising:
(a) bringing a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficient to form a binding complex between the first peptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein;
(b) measuring a percent inhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with an sensitivity of at least about 80%.
45. The method of claim 44, wherein the subject is vaccinated against the coronavirus.
46. The method of claim 44, wherein the subject is unvaccinated against the coronavirus.
47. The method of any one of claim 44-46, wherein the coronaviruscomprises SARS-CoV- 2 or a variant thereof.
48. The method of any one of claims 44-47, wherein the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof.
49. The method of any one of claims 44-48, wherein the second protein or peptide comprises the S1 subunit of the spike protein.
50. The method of any one of claims 44-49, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
51. The method of any one of claims 44-50, wherein the biological sample comprises capillary blood.
52. The method of any one of claims 44-51, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
53. The method of claim 44, further comprising administering a vaccine against the coronavirus to the subject based on measuring in (b).
54. The method of claim 44, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
55. The method of any one of claims 44-54, wherein the subject is an age comprising greater than or equal to about 50 years of age.
56. The method of any one of claims 44-55, further comprising eluting the at least the portion of the biological sample from a dried blood spot card.
57. The method of any one of claims 44-56, wherein the first peptide or protein or the second peptide or protein is immobilized on a substrate
58. The method of any one of claims 44-57, further comprising detecting the binding complex using a lateral flow assay.
59. The method of claim 58, wherein detecting the binding complex in (b) comprises visualizing a detectable moiety coupled to the first peptide or protein or the second peptide or protein of the binding complex using a naked eye or an imaging device.
60. A method of analyzing a biological sample, the method comprising:
(a) bringing at least a portion of a biological sample obtained from a subject into contact with (i) a first peptide or protein comprising an Angiotensin -converting enzyme 2 (ACE2) polypeptide or a portion thereof, and (ii) a second peptide or protein comprising a spike protein or a portion thereof derived from a coronavirus under conditions sufficientto form abinding complex between the firstpeptide or protein and the second peptide or protein in absence of neutralizing antibodies that block binding between the ACE2 polypeptide and the spike protein;
(b) measuring a percentinhibition of binding between the first peptide or protein and the second peptide or protein within the biological sample with percent coefficient of variation of less than 10%.
61. The method of claim 60, wherein the subject is vaccinated against the coronavirus.
62. The method of claim 60, wherein the subject is unvaccinated against the coronavirus.
63. The method of any one of claims 60-62, wherein the coronavirus comprises SARS- CoV-2 or a variant thereof.
64. The method of any one of claim 60-63, wherein the first peptide or protein comprises an extracellular portion of the ACE2 polypeptide or a portion thereof.
65. The method of any one of claims 60-64, wherein the second protein or peptide comprises the SI subunit of the spike protein.
66. The method of any one of claims 60-65, wherein the biological sample has an average volume of less than 1 milliliter when obtained.
67. The method of any one of claims 60-66, wherein the biological sample comprises capillary blood.
68. The method of any one of claims 60-67, wherein the method does not consist of utilizing an immortalized cell or immortalized cell culture.
69. The method of any one of claims 60-68, further comprising administering a vaccine against the coronavirus to the subject based on measuring in (b).
70. The method of any one of claims 60-69, wherein the spike protein of the coronavirus comprises a receptor binding domain specific for binding with ACE2 in vivo.
71. The method of any one of claims 60-70, wherein the subject is an age comprising greater than or equal to about 50 years of age.
72. The method of any one of claims 60-71, further comprising eluting the biological sample from a dried blood spot card.
73. The method of any of claims 1 -72, wherein the percent inhibition is predictive of a clinical endpoint when the subject received a pre-approved or licensed vaccine against the coronavirus.
74. The method of claim 73, wherein the pre-approved or licensed vaccine comprises Ad26.COV.S, mRNA-1273 orBNT162b2, or any combination thereof.
75. The method of any of claims 1 -74, wherein the percent inhibition is predictive of individual protection against another infection by the coronavirus.
76. The method of any of claims 1 -75, further comprising administering to the subject a vaccine booster against the coronavirus or a second dose of a vaccine against the coronavirus, when the percent inhibition is below low relative to a control of the index derived from one or more control subjects that (i) recovered from an infection by the coronavirus, (ii) is vaccinated against the coronavirus, (iii) has recovered from the infection by the coronavirus and is vaccinated against the coronavirus, or (iv) is vaccinated against the coronavirus and has received atleast one booster vaccine against the coronavirus.
77. The method of any of claims 1-76, wherein the subject has an underlying immune- compromising condition.
78. The method of claim 77, wherein the underlying immune-comprising condition comprises a pregnancy, a previous transplant, HIV/AIDS, cancer, receipt of a chemotherapy, receipt of radiotherapy, receipt of a proton treatment, or any combination thereof.
79. The method of any one of claims 1 -78, wherein the subject is breastfeeding.
80. The method of any one of claims 1 -79, wherein further comprising detecting one or more signals from one or more detectable moieties coupled to the binding complex.
81. The method of claim 80, further comprising detecting the binding complex using a lateral flow assay.
82. The method of claim 81, wherein the one or detectable moieties comprises a nanoparticle, a fluorescent metal, a fluorescent dye, an enzymatic label, or a colorimetric label, or any combination thereof.
83. The method of any one of claims 1 -82, further comprising determining that the biological sample comprises about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell-based pseudovirion neutralization assay (PNA), when the percent neutralization measured is about 62%.
84. The method of claim 83, further comprising determining that the subject is adequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of about 62%.
85. The method of any one of claims 1 -84, further comprising determining that the biological sample comprises less than about 400 reciprocal titers of the neutralizing antibodies in the biological sample as measured using a cell-based pseudovirion neutralization assay (PNA), when the percentneutralization measuredis less than about 62%.
86. The method of claim 85, further comprising determiningthatthe subjectis inadequately protected against an infection by the coronavirus, based at least in part, on the percent neutralization of less than about 62%.
87. The method of any one of claims 1 -86, wherein the method is performed without a wash step to wash away the first peptide or protein or the second peptide or protein that are unbound following the bringing in (a).
88. The method of any one of claims 1 -87, wherein the bringing the biological sample into contact with the first peptide or protein and the second peptide or protein is performed in a container.
89. The method of claim 88, wherein the container is optically clear.
90. The method of claim 88 or 89, wherein the container is a disposable cartridge.
91. The method of any one of claims 1 -90, further comprising repeating the method on a plurality of biological samples obtained from the subject, wherein at least two biological samples of the plurality of biological samples are obtained at different time points.
92. The method of any one of claims 1 -91, wherein the subject is receiving an immunosuppressant therapy.
93. The method of claim 92, further comprising:
(a) identifying the subject as needing a dose of a vaccine against the coronavirus, provided the percent neutralization is lower than 62%; and
(b) suspending a treatment regimen comprising the immunosuppressant therapy for a period of time during which period of time, the subject is administered the dose of the vaccine.
94. The method of any one of claims 1 -93, wherein the subject is a pregnant subject.
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Citations (3)

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WO2021168305A1 (en) * 2020-02-19 2021-08-26 Ubi Ip Holdings Designer peptides and proteins for the detection, prevention and treatment of coronavirus disease, 2019 (covid-19)
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WO2021168305A1 (en) * 2020-02-19 2021-08-26 Ubi Ip Holdings Designer peptides and proteins for the detection, prevention and treatment of coronavirus disease, 2019 (covid-19)
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