EP4649097A2

EP4649097A2 - Activation-induced marker assays

Info

Publication number: EP4649097A2
Application number: EP24742152.2A
Authority: EP
Inventors: Genevieve VAN DE BITTNER; Tatiana PAREDES SANTOS; Israel STEINFELD
Original assignee: Agilent Technologies Inc
Current assignee: Agilent Technologies Inc
Priority date: 2023-01-13
Filing date: 2024-01-16
Publication date: 2025-11-19
Also published as: CN120500499A; WO2024152056A9; WO2024152056A2; JP2026502483A; WO2024152056A3

Abstract

The present disclosure relates to the field of biology. In particular, the present disclosure relates to T cell activation induced marker assays that may be used, e.g., to determine whether a subject was previously exposed to an antigen of interest, e.g., via infection with a pathogen, and kits and compositions related thereto.

Description

AFS Ref. No.039062.00150 ACTIVATION-INDUCED MARKER ASSAYS Cross-Reference to Related Application [0001] This patent application claims priority to U.S. Provisional Patent Application No. 63/479,902, filed on Jan.13, 2023, which is herein incorporated by reference in its entirety. Technical Field [0002] The present disclosure relates to the field of biology. In particular, the present disclosure relates to T cell activation induced marker assays that may be used, e.g., to determine whether a subject was previously exposed to an antigen of interest, e.g., via infection with a pathogen, and kits related thereto. Background [0003] Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an ongoing and substantial threat to public health. The presence of neutralizing antibodies against SARS-CoV-2 is an indicator of protective immunity acquired as a result of past infection (or vaccination). In addition to humoral immune responses, T cells also play a pivotal role in coordinating the adaptive immune responses and as effectors against viral infection. Prior studies of acute and convalescent COVID-19 patients have reported that T cell responses are associated with reduced disease, suggesting that SARS-CoV-2–specific CD4+ T cell and CD8+ T cell responses are involved in the resolution of primary SARS-CoV-2 infection. See Liao et al. (2020); Moderbacher et al. (2020); Zhou et al. (2020). Furthermore, prior studies report that SARS-CoV-2-specific CD4+ and CD8+ T cells were detected in 100% and ~70% of convalescent individuals a short time after resolution. See Grifoni et al., 2020. [0004] In order to fully understand the immunological response elicited by SARS-CoV-2 infection, various components of the immune response, including B cells, CD4+ T cells, and AFS Ref. No.039062.00150 CD8+ T cells, must be considered, as the response kinetics of these different components may be independent of each other. Various assays are conventionally used to measure the quantity and qualities of antigen-specific T cells in humans. See Ten Brinke et al. (2017). Enzyme- linked immunospot (“ELISpot”) and intracellular cytokine staining (“ICS”) assays, in particular, are frequently used for such studies. See, e.g., Saade et al. (2012); Slota et al. (2011); Smith et al. (2015). The ELISpot assay involves stimulating peripheral blood mononucleocyte cells (“PBMCs”) with antigen in multi-well plates with membranes coated in an anti-cytokine capture antibody. Cytokines produced by antigen-specific T cells will bind to the capture antibody and are thus detectable using an enzyme-conjugated secondary antibody and a chromogenic development substrate. The ELISpot assay is highly sensitive, e.g., cells producing fewer than 100 cytokine molecules may be detected. See Shirai et al. (1993). As such, the ELISpot assay has become one of the most frequently used and highly validated assays for the detection of antigen-specific T cell responses in clinical trials. See, e.g., Britten et al. (2008); Moodie et al. (2010). However, the ELISpot assay has several major disadvantages that limit its use as a tool to investigate the immunological response to infection, including a limit on the number of parameters that can be investigated, the lack of phenotypic information, and preferential detection of effector cells. In view of these limitations, the ELISpot assay does not fully elucidate and may underestimate the total antigen-specific T cell response. [0005] As an alternative, intracellular cytokine staining (ICS) assays may be used to provide further information on the quantity and quality of antigen-induced T cells. In short, antigen-stimulated PBMCs are stained with fluorescently labelled anti-cytokine antibodies and analyzed by flow cytometry. ICS assays allow for detailed phenotypic and functional analyses of antigen-specific T cell populations. However, ICS assays are also limited by the number of parameters that can be assessed. Accordingly, ICS assays may be biased towards the detection AFS Ref. No.039062.00150 of a particular type of T cell. For example, ICS assay panels used in clinical trials typically assay for IFNγ, IL2, and TNFα and thus detect Th1-biased responses. See Coughlan et al. (2015); Horton et al. (2007). T cell responses to infection (or vaccination) are often highly heterogeneous and, therefore, detection based on the expression of one or more cytokines may underestimate the frequency of antigen-specific cells. See De Rosa et al. (2004). [0006] In view of these limitations, more recent studies have popularized the use of activation-induced markers (AIM) assays that can be used to identify and measure antigen- specific T cell responses based on the upregulation of TCR-stimulated surface markers. AIM assays may be used to generate a broader picture of the overall antigen-specific T cell response. However, AIM assays have not been fully evaluated and compared with more conventional cytokine-based methods. Moreover, given the large number of potentially upregulated surface markers, it is impossible to predict which individual marker, or combinations of markers, may be used to accurately detect antigen-specific T cells, absent trial and error. Brief Summary of the Disclosure [0007] Despite notable advances in the development of assays for detecting humoral and cellular immune responses to infection, there exists an ongoing need for additional improvements in this space in view of the diversity of possible antigens, including those from pathogens, and the limitations of current assays. In particular, there exists a need at this time for assays and methods that are capable of accurately detecting and/or measuring the antigen- specific T cell response elicited by SARS-CoV-2 infection, and other viral infections, in a human or animal subject. Such assays and methods may be used, e.g., for the diagnosis of past infection with this virus, as well as to provide guidance that may be used to determine when a subject would benefit from a booster vaccination. Accordingly, provided herein are T cell AIM assays, and kits and methods related thereto. In some aspects, these AIM assays, kits, and methods may be used for the detection of SARS-CoV-2-specific CD4+ and CD8+ T cells, AFS Ref. No.039062.00150 allowing for the detection and/or diagnosis of infection, and potential use as a tool to guide the scheduling of vaccination and treatment decisions. In others, such assays, kits, and methods may be used to for the detection CD4+ and CD8+ T cells specific for other clinically relevant viruses. [0008] In a first general aspect, the disclosure provides a kit comprising a plurality of antibodies, wherein each antibody is capable of specifically binding to a single cell surface marker, selected from a panel comprising CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and CD274. [0009] In some aspects, the plurality of antibodies comprises antibodies that collectively bind to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different cell surface markers selected from the panel. For example, a kit may comprise antibodies specific for any pair of cell surface markers described herein (e.g., CD69 and OX40, or CD25 and 41BB). In other aspects, the kit may comprise antibodies that are each specific for one biomarker selected from any combination of cell surface markers described herein. For example, in some aspects the plurality of antibodies comprises antibodies that specifically bind to any combination of CD25, CD69, OX40, 41BB, CD40L, CD107, CD38, and/or CD274 (e.g., to monitor cellular activation in the presence of or following treatment with an antigen stimulus). In some aspects the plurality of antibodies comprises antibodies that specifically bind to CD3, CD4, CD8, CD14, and/or CD19 (e.g., to isolate CD4+ and/or CD8+ cells); and/or to LAG3 (e.g., to identify exhausted cells). In some aspects, kits may comprise antibodies specific for all 14 of the cell surface biomarkers described in the preceding passage. It is understood that the kits set forth herein may utilize monoclonal or polyclonal antibodies, and/or antibody fragments, as a binding agent to detect the cell surface markers described herein. AFS Ref. No.039062.00150 [0010] In some aspects, the plurality of antibodies comprises antibodies that specifically bind to: a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; d) CD25 and OX40; e) CD274 and 41BB; and/or f) CD274 and CD69. [0011] In some aspects, the kits may comprise fluorophore-conjugated antibodies (e.g., to allow detection of the antigen-bound antibody using a flow cytometer or other instrument). In some aspects, a different fluorophore may be used for each cell surface marker. For example, an exemplary panel may comprise any or all of the following fluorophore-antibody conjugates: FITC/CD8, PE/CD25, PERCPCy5.5/CD3, BV421/CD137, BV510/CD4, BV605/LAG3, BV650/CD274, BV711/CD134 (OX40), BV785/CD38, APC/CD69, PE-Cy7/CD154, PE- Dazzle/CD107, Alexa 700/CD14, and/or Alexa 700/CD19). It is understood that any fluorophore disclosed herein may be used as a conjugate for any cell surface marker (or any other antigen) described herein. In some aspects, a kit may comprise a plurality of fluorophore- conjugated antibodies, wherein each of the fluorophore-conjugated antibodies is specific for a different cell surface marker and wherein a different fluorophore is used for each cell surface marker. In some aspects, the plurality of fluorophore-conjugated antibodies is provided as a single mixture, optionally comprising optimized concentrations or ratios of some or all of the fluorophore-conjugated antibodies. In some aspects, a kit according to the disclosure may comprise one or more alternative fluorophore-conjugated binding agents (e.g., an antibody fragment, an aptamer, or any other binding agent capable of specifically binding to a cell surface marker described herein) in place of any of the antibodies described in any of the various exemplary aspects described herein. [0012] The plurality of antibodies included as part of the kits described herein may be provided in dry (e.g., lyophilized) or liquid form. In some aspects, the plurality of antibodies may be provided as a single mixture, whereas in others the plurality of antibodies may be provided in separate compartments or containers (e.g., antibodies specific for each cell surface AFS Ref. No.039062.00150 marker, or for any subset of the cell surface markers, may be provided in a different compartment or container). In some aspects, kits may comprise a buffer or other solvent that may be used to prepare an antibody solution (e.g., to perform the assays described herein) using the plurality of antibodies. [0013] In some aspects, the kit further comprises a cell viability marker, wherein the cell viability marker comprises a binding agent or dye that selectively binds to and/or stains either live or dead cells. For example, in some aspects the cell viability marker comprises Live/Dead® Fixable Near-IR stain (Invitrogen®), or any of the other Live/Dead® stains (e.g., Fixable Blue/ Violet/Lime/Aqua/Yellow/Green/Olive/Orange/Red/Fixable Far Red) marketed by Invitrogen®. DAPI (4′,6-diamidino-2-phenylindole), and other dyes known to be useful for distinguishing live and dead eukaryotic cells may also be used, e.g., 7-AAD (7- Aminoactinomycin D), Hoechst dyes, or propidium iodide. Such dyes are available from multiple manufacturers and typically function based on the reaction of a fluorescent reactive dye with cellular proteins (amines) or via a fluorescence increase or shift that occurs following DNA intercalation. When used at typical concentrations for staining purposes, many of these dyes cannot penetrate live cell membranes, so only cell surface proteins are available to react with the dye, resulting in dim staining of live cells. In contrast, the reactive dye can permeate the damaged membranes of dead cells and stain both the interior and exterior amines, and so dead cells are subject to intense staining. Similarly, some cell viability marker dyes (e.g., DNA intercalating dyes) may show limited permeability when applied to live cell membranes, and a greater ability to penetrate and stain dead cells, when applied at concentrations typically used for live/dead staining. [0014] In some aspects, the kit may further comprise one or more secondary binding agents, wherein each secondary binding agent is capable of specifically binding to a cytokine (e.g., to IFN-γ, TNF-α, IL-2, IL-4, IL-6, or IL-10). Each secondary binding agent may comprise AFS Ref. No.039062.00150 an antibody, an antibody fragment, an aptamer, or any other binding agent capable of specifically binding to a cytokine, or any epitope or fragment thereof. The secondary binding agent may be used in the assays described herein, e.g., to detect and measure cytokine expression levels, providing an additional set of parameters that may be used (alone or in combination with expression level data for the cell surface markers) to determine whether a subject was previously infected with the SARS-CoV-2 virus (or to make any other determination described herein). [0015] In some aspects, the kit may further comprise one or more antigens (e.g., one or more peptides) that are capable of activating a peripheral blood mononuclear cell (“PBMC”), such as a CD4+ T cell or a CD8+ T cell. In some aspects, the antigen comprises at least one fragment of a polypeptide sequence of a protein produced by a strain of the SARS-CoV-2 virus. The antigen may comprise a peptide having a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or a length within a range defined by any pair of integers between 2 and 20. In some aspects, the at least one fragment comprises a peptide having the polypeptide sequence of a portion of a spike protein, membrane protein, outer membrane protein, or nucleocapsid protein of a strain of the SARS-CoV-2 virus. [0016] In some aspects, the kit further comprises one or more Fluorescence Minus One (“FMO”) control samples, wherein each FMO control sample comprises antibodies specific for all but one of cell surface markers. [0017] In some aspects, the kit further comprises one or more solvents and/or buffer solutions. [0018] In a second general aspect, the disclosure provides a method of determining that a subject has been infected by the SARS-CoV-2 virus, comprising: a) generating a treated sample by contacting a plurality of CD4+ T cells and/or CD8+ T cells obtained from the subject with at least one antigen, wherein the antigen comprises a peptide fragment of a protein of a strain AFS Ref. No.039062.00150 of the SARS-CoV-2 virus; b) contacting CD4+ T cells and/or CD8+ T cells in the treated sample with a plurality of fluorophore-conjugated antibodies to produce a labeled sample, wherein each antibody is specific for a cell surface marker selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274; c) detecting CD4+ T cells and/or CD8+ T cells in the labeled sample that express a plurality of cell surface markers selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274, based on a fluorescence signal generated by the fluorophore-conjugated antibodies; and d) determining that the subject has been infected by the SARS-CoV-2 virus, based on the detected CD4+ T cells and/or CD8+ T cells. In some aspects, steps a), b), c), and/or d) are performed using a kit according to any exemplary aspect described herein. [0019] In some aspects, the detecting step c) further comprises detecting the CD4+ T cells and/or CD8+ T cells in the labeled sample that express a plurality of cell surface markers using at least 2, 3, 4, 5, 6, 7, or 8 replicate samples. [0020] In some aspects, the detecting step c) further comprises a step of normalizing the fluorescence signal generated by the fluorophore-conjugated antibodies. The normalizing step may, e.g., be performed using a negative control sample consisting of a peptide diluent, such as water, or comprising water, another solvent, and/or a buffer. For example, the negative control may comprise the same peptide diluent in the same volume as is used for test sample(s) that receive the SARS-CoV-2 virus antigen (comprising a peptide fragment or pool of peptide fragments of a protein of a strain of the SARS-CoV-2 virus). Alternatively, the negative control may comprise the same peptide diluent in the same volume as is used for test sample(s) that receive a viral antigen of interest (comprising a peptide fragment or pool of peptide fragments of a protein of a strain of any virus of interest). In some aspects, the peptide diluent may be, e.g., water, or comprise dimethyl sulfoxide (DMSO) and phosphate buffered saline. In some AFS Ref. No.039062.00150 aspects, a positive control sample comprising a peptide pool containing a plurality of viral antigens may also be used, alone or in combination with a negative control. In some aspects, the peptide pool comprises a set of peptides comprising MHC class II restricted T cell epitopes from human Cytomegalovirus (CMV), Epstein Barr virus, Influenza virus, Tetanus toxin, and Adenovirus 5 (CEFTA). [0021] In some aspects, the normalizing step comprises: i) testing whether a fluorescence signal produced by fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample is higher or lower than a fluorescence signal obtained using the negative control sample; and ii) testing whether a fluorescence signal produced by fluorophore- conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample is higher or lower than a signal obtained using the positive control sample. In some aspects, the normalizing step further comprises subtracting the fluorescence signal obtained using the negative control sample from the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample, when the negative control fluorescence signal is lower. In some aspects, when the negative control fluorescence is higher, the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells may be set to zero. In some aspects, the normalizing step further comprises normalizing the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample, to the fluorescence signal obtained using the positive control sample, when the positive control fluorescence signal is higher. In some aspects, when the positive control fluorescence is lower, the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells may be set to a non-zero constant value, e.g., a value of one, two, or three, etc. The comparison of the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample, to the positive AFS Ref. No.039062.00150 and/or negative control, may be based on fluorescence signals obtained from a plurality of replicates. [0022] In some aspects, the determining step d) further comprises determining that the subject has been infected by the SARS-CoV-2 virus, based on an expression level of one or more cytokines by CD4+ T cells and/or CD8+ T cells in the treated sample. In some aspects, the determining step d) further comprises determining that the subject has been infected by the SARS-CoV-2 virus, based on a change in the amount of CD4+ T cells and/or CD8+ T cells in the labeled sample that express the plurality of cell surface markers, compared to a median or average amount determined using samples obtained from one or more SARS-CoV-2 naïve donors. The change in the amount of the plurality of cell surface markers may be measured, e.g., as: a) a percentage difference as compared to the median amount; b) fold-change difference as compared to the median amount; or b) a numerical difference as compared to the median amount. In other aspects, an analogous determination may be made for any other virus of interest being assayed for (e.g., based on a change in the amount of CD4+ T cells and/or CD8+ T cells in the labeled sample that express the plurality of cell surface markers, compared to a median or average amount determined using samples obtained from one or more naïve donors). [0023] In some aspects, a method of determining that a subject has been infected by the SARS-CoV-2 virus (or another virus of interest), or any other method described herein, may utilize plurality of fluorophore-conjugated antibodies used in step b) comprises antibodies specific for: a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; d) CD25 and OX40; e) CD274 and 41BB; and/or f) CD274 and CD69. For example, any combination of the aforementioned marker pairs may be used in the contacting or detecting steps of the methods described in the preceding passages. AFS Ref. No.039062.00150 [0024] In a third general aspect, the disclosure provides method of determining that a subject is in need of a SARS-CoV-2 virus vaccination (e.g., a booster vaccination), comprising: a) generating a treated sample by contacting a plurality of CD4+ T cells and/or CD8+ T cells obtained from the subject with at least one antigen, wherein the antigen comprises a peptide fragment of a protein of a strain of the SARS-CoV-2 virus; b) contacting CD4+ T cells and/or CD8+ T cells in the treated sample with a plurality of fluorophore-conjugated antibodies to produce a labeled sample, wherein each antibody is specific for a cell surface marker selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274; c) detecting CD4+ T cells and/or CD8+ T cells in the labeled sample that express a plurality of cell surface markers selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274, based on a fluorescence signal generated by the fluorophore-conjugated antibodies; and d) determining that the subject is in need of a SARS-CoV-2 virus vaccination, based on the detected CD4+ T cells and/or CD8+ T cells. In other aspects, an analogous determination may be made regarding the need for vaccination against any other virus of interest (e.g., by using an antigen comprising a peptide fragment of a protein of a strain of the virus of interest, and detecting CD4+ T cells and/or CD8+ T cells as described above). [0025] It is understood that in some aspects, a method of determining that a subject is in need of a SARS-CoV-2 virus vaccination, or a vaccination for another virus of interest, may further include any of the steps, components or parameters, of the methods of determining that a subject has been infected by the SARS-CoV-2 virus, or other virus of interest, described herein. [0026] To the accomplishment of the foregoing and related ends, the one or more aspects comprise the features hereinafter fully described and particularly pointed out in the claims. The following description and the annexed drawings set forth in detail certain illustrative features AFS Ref. No.039062.00150 of the one or more aspects. These features are indicative, however, of but a few of the various ways in which the principles of various aspects may be employed, and this description is intended to include all such aspects and their equivalents. Description of the Figures [0027] The drawings set forth herein illustrate and describe exemplary aspects of the disclosure and are not meant to limit the scope of the invention as defined by the claims. [0028] FIGs. 1-2 are cartoon representations illustrating an exemplary AIM assay workflow that may be used to determine whether a subject has been infected by the SARS- CoV-2 virus (or another virus of interest), as described herein. [0029] FIG. 3 summarizes the flow cytometry analysis and gating strategy for an exemplary AIM assay according to the disclosure. [0030] FIG.4 shows a series of plots summarizing the flow cytometry analysis and gating strategy for the cell surface marker pair CD25 and 41BB. [0031] FIG. 5 is a table showing exemplary cell surface marker pair that may be used in the kits and/or methods described herein. [0032] FIG.6 is a graph showing the percentage of activated CD4+ T cells detected using the cell surface marker pair CD25 and 41BB, measured using a kit according to the disclosure in samples obtained from a population of subjects that are convalescent for COVID-19 versus in samples obtained from a population of COVID-19 naïve donors. [0033] FIGs. 7-8 summarize data obtained from an ELISpot assay confirming T Cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. [0034] FIGs. 9A-9C are graphs summarizing cytokine release data confirming T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. AFS Ref. No.039062.00150 [0035] FIGs.10A-10B are graphs showing the percentage of activated CD4+ T cells (FIG. 10A), and activated CD8+ T cells (FIG.10B) detected using the cell surface marker pair CD25 and 41BB following AIM assays performed using a kit according to the disclosure, with samples obtained from SARS-CoV-2 naïve donors, versus COVID-19 convalescent donors that were serum positive or serum negative for SARS-CoV-2 antibodies. [0036] FIGs. 11-14 are graphs showing the fold change (FIGs. 11, 13) and difference (FIGs. 12, 14) in the CD4+ and CD8+ T cell response of COVID-19 convalescent donors, evaluated using an AIM assay according to the disclosure. [0037] FIGs.15-16 show two graphs that illustrate the normalization of three cell surface marker pairs (CD25 and OX40, CD25 and CD38, and CD25 and 41BB) using water (FIG.15) and a CEFTA pool (FIG.16). [0038] FIG. 17 is a hierarchical clustering diagram that shows the clustering of various CD4 T cell and CD8 T cell surface marker pairs tested with AIM assays according to the disclosure using samples from a COVID-19 naïve donors and COVID-19 convalescent donors. [0039] FIG. 18 is a graph showing the receiver operator characteristic (ROC) curve of a random forest model trained to determine whether a sample is from a COVID-19 convalescent or COVID-19 naïve donor using AIM data for a set of the cell surface markers described herein. [0040] FIG. 19 is a graph comparing random forest model importance score vs. the negative log of the Mann-Whitney U test p-value. This graph is annotated to highlight several cell surface marker pairs that stand out as relatively important features. [0041] FIG. 20 is a hierarchical clustering diagram that shows the clustering of the six features denoted by the annotation in FIG.19. [0042] FIG.21 is a table summarizing available data to support various cell surface marker pairs described herein. AFS Ref. No.039062.00150 [0043] FIG. 22 is chart showing the raw data used to generate the graph shown in FIG. 11. [0044] FIG. 23 is chart showing the raw data used to generate the graph shown in FIG. 12. [0045] FIG. 24 is chart showing the raw data used to generate the graph shown in FIG. 13. [0046] FIG. 25 is chart showing the raw data used to generate the graph shown in FIG. 14. [0047] FIG. 26 is chart showing the raw data used to generate the graph shown in FIG. 19. [0048] FIG. 27 is chart showing the raw data used to generate the graph shown in FIG. 20. [0049] FIGs. 28-30 are graphs showing the response (CD4+ cells) of donor samples observed during a study which evaluated the AIM assays described herein for the detection and/or diagnosis of CMV infection. FIG. 28 shows the response of CMV+ donor samples, whereas FIGs. 29-30 show the response of naïve donor samples (FIG. 30 being a magnified version of FIG.29). [0050] FIGs. 31-33 are graphs showing the response (CD8+ cells) of donor samples observed during a study which evaluated the AIM assays described herein for the detection and/or diagnosis of CMV infection. FIG. 31 shows the response of CMV+ donor samples, whereas FIGs. 32-33 show the response of naïve donor samples (FIG. 33 being a magnified version of FIG.32). [0051] FIG.34 is a graph showing the percentage of activated CD4+ T cells detected using the cell surface marker pair CD25 and 41BB following an AIM assay for the detection and/or diagnosis of CMV infection performed using a kit according to the disclosure. AFS Ref. No.039062.00150 Detailed Description [0052] The detailed description set forth below in connection with the appended drawings is intended as a description of various configurations and is not intended to represent the only configurations in which the concepts described herein may be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of various concepts. However, it will be apparent to those skilled in the art that these concepts may be practiced without these specific details. [0053] The investigation of cellular immune response offers a robust way to understand previous exposure to pathogens, or antigens from other sources. Post-infection, the cellular immune response to a pathogen is long-lived, as compared to the antibody production response, which tends to reduce over time for some pathogens. In the case of the global COVID-19 pandemic, caused by the SARS-CoV-2 virus, the production of antiviral serum antibodies is often undetectable after approximately 6 months. The T-cell-mediated immune response provides long-term protection against serious illness, and early studies indicate this response is detectable longer-term. See Sette et al., (2021). The current commercially available gold standard for evaluation of T cell response is the ELISpot test. Although reliable, the method cannot subtype the antigen-responsive T cells, and it has limited ability to measure T cell activation profiles. [0054] Marker Panels for AIM Assays [0055] The present disclosure addresses these and other shortcomings by providing diagnostic kits and methods based on a flow cytometry marker panel that can be used to accurately assess the CD4+ and/or CD8+ T cell immune response to SARS-CoV-2 peptides in COVID-19 convalescent donors (or viral peptides associated with any other virus of interest). In some aspects, this panel comprises one or more cell surface markers of CD4+ and CD8+ T cells (e.g., CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, AFS Ref. No.039062.00150 CD107, and/or CD274), and optionally at least one cell viability marker (e.g., a binding agent such as an antibody, dye, or stain that can be used to selectively distinguish live versus dead cells). In some aspects, the panel may comprise one or more cell surface markers that are useful for immunophenotyping and/or identifying exhausted cells (e.g., CD3, CD4, CD8, CD14, CD19 and/or LAG3), and one or more cell surface markers that are activation inducible following exposure to an antigen stimulus, (e.g., CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274). In some aspects, the panel may comprise any combination of the foregoing cell surface and/or cell viability markers (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the markers listed in this passage, or otherwise disclosed herein). As described in further detail below, it has been found that certain combinations of the cell surface markers described herein (e.g., CD69 and OX40; CD25 and 41BB; CD25 and CD38; CD25 and OX40; CD274 and 41BB; and/or CD274 and CD69) are particularly useful for detecting and evaluating the response of CD4+ and/or CD8+ cells following exposure to a SARS-CoV-2 virus antigen (or antigens), or antigens associated with an alternative virus of interest, allowing for an efficient and accurate determination as to whether a subject has, or was previously infected with the virus. [0056] FIGs. 1-2 summarize a flow cytometry analysis and gating strategy for an exemplary AIM assay using the marker panels and kits described herein. As illustrated by this, PBMCs from COVID-19 naïve and COVID-19 convalescent subjects may be collected and treated with one or more peptide fragments of one or more SARS-CoV-2 virus proteins as an antigen stimulus. The treated cells may then be separated from the supernatant liquid in the cell culture for subsequent processing. In this example, the treated cells are subjected to the AIM assay using a plurality of fluorophore-conjugated antibodies to detect the expression of one or more activation-induced cell surface markers, one or more cell surface markers useful for immunophenotyping and/or identifying exhausted cells, and at least one binding agent, dye, or AFS Ref. No.039062.00150 stain that may be used to distinguish live versus dead cells. In parallel, the supernatant may be collected and labeled to measure the expression of various cytokines (e.g., IFN-γ, TNF-α, IL- 2, IL-4, IL-6, and/or IL-10). In this case, a fluorescent bead-based assay was used. The labeled cells and supernatant may then be subjected to analysis by a flow cytometer. [0057] In this example, the flow cytometer is used to detect and measure fluorescent signals produced by the labeled cells and supernatant, e.g., to measure the expression level of various cytokines, and to evaluate the immune response of the PBMCs in response to the antigen stimulus. For example, the flow cytometer may be used to detect the number, percentage, or proportion of CD4+ and/or CD8+ T cells in the sample that express any one (or any combination) of the cell surface markers described herein. As demonstrated by the examples provided below, various combinations of the cell surface markers described herein may be used to accurately detect and measure the immune response of CD4+ and/or CD8+ T cells following treatment with one or more peptide fragments of one or more SARS-CoV-2 virus proteins. In this exemplary workflow, a portion of the originally collected sample (or a replicate) was collected and subjected to an ELISpot IFN-γ assay in parallel, to validate the accuracy of the AIM assay. [0058] FIG. 3 summarizes the flow cytometry analysis and gating strategy for an exemplary AIM assay according to the disclosure. As illustrated by this example, an exemplary gating strategy for the flow cytometer may comprise: 1) gating singlets (i.e., single cells) based on light scattering characteristics (side scattering, in this case); gating lymphocytes based on light scattering characteristics (side and forward scattering, in this case); 3) gating live cells (e.g., using a cell viability marker described here); gating T cells (e.g., based on the phenotype CD3+, CD14-, Cd19-); gating CD4+ and CD8+ T cells (e.g., using one or more markers described herein); and then gating CD4+ and/or CD8+ T cells that express one or more cell surface markers described herein. In this example, the final gating identified CD4+ T cells that AFS Ref. No.039062.00150 express CD40L and OX40, and CD8+ T cells that express CD25 and 41BB. The number of cells found to express the selected one or more cell surface markers may then be analyzed, e.g., to determine whether the sample donor has been infected with the SARS-CoV-2 virus. This determination may be used to guide treatment or vaccination decisions, e.g., by a medical professional. For example, a weak response may be used as an indicator that the sample donor requires a booster vaccination. [0059] FIGs.4-10 provide data illustrating the use of AIM assays based on the cell surface markers described herein. For example, FIGs.4-6 provide data for an AIM assay according to the disclosure, which evaluated CD4+ and CD8+ T cell activation following treatment with a SARS-CoV-2 virus antigen, using a pair of the cell surface markers described herein (CD25 and 41BB). Data for a negative control (water) and a positive control (using a CEFTA peptide pool) is also provided. As shown by this data (e.g., the FIG.4 plots), the percentage of CD4+ T cells found to be CD25+ and 41BB+ was significantly higher in the COVID-19 convalescent group (treated with a SARS-CoV-2 virus antigen obtained from Miltenyi Biotec or Mabtech). In some aspects, the SARS-CoV-2 virus antigen may comprise all, or any combination of, the peptides found in the “PepTivator®SARS-CoV-2 Select – premium grade,” sold by Miltenyi Biotec, or in “PepPool: SARS-CoV-2 (SNMO), human,” available from Mabtech. [0060] This AIM assay was repeated using various pairs of the cell surface markers described herein in order to evaluate the response of CD4+ and CD8+ cells. The various pairs of cell surface markers that were evaluated are summarized by FIG. 5. For each treatment condition (e.g., water or peptide pool), five replicates were evaluated, and all marker pairs were available for gating in each treatment condition. The initial results were averaged and normalized by background subtraction. FIG.6 is an exemplary plot of the results for one such assay, illustrating the increase in CD4+ T cells found to express the selected cell surface marker AFS Ref. No.039062.00150 pair in samples from COVID-19 convalescent donors compared to samples from COVID-19 naïve donors. [0061] FIGs. 7-9 provide additional data validating the use of AIM assays based on the cell surface markers described herein. The ELISpot assay is a conventional assay that may be used to detect and measure T cell activation. As shown by FIG. 7-8, an ELISpot assay confirmed T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS- CoV-2 peptide stimulation with the Mabtech peptide pool (when “Conv Ser Pos” samples and “Conv Ser Neg” samples were combined). As shown by FIG. 9, increased secretion of the cytokines: IL-2, IFN-γ and TNF-α was observed in COVID-19 convalescent donors (when “Conv Ser Pos” samples and “Conv Ser Neg” samples were combined), whereas the secretion of cytokines IL-4, Il-6 and IL-10 was not consistent among the donors. [0062] Diagnostic Methods [0063] In some aspects, the disclosure provides method of using the marker panels described herein, e.g., to determine whether a subject has been infected by the SARS-CoV-2 virus. Such methods may be performed using any of the various marker panels and/or kits described herein. In some aspects, such methods may involve the generation of a treated sample by contacting a plurality of CD4+ T cells and/or CD8+ T cells obtained from the subject with at least one antigen, wherein the antigen comprises a peptide fragment of a protein of a strain of the SARS-CoV-2 virus. The PBMCs (e.g., comprising CD4+ T cells and/or CD8+ T cells) obtained from the subject may optionally be aliquoted into separate portions prior to treatment (e.g., to create one or more replicates). One or more of the CD4+ T cells and/or CD8+ T cells in the treated sample may then be contacted with a plurality of fluorophore-conjugated antibodies to produce a labeled sample, wherein each antibody is specific for a cell surface marker selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274. In some aspects, the treated sample, or one or more AFS Ref. No.039062.00150 portions of the cells contained therein, may be separated into different replicates. The labeled sample (or one or more replicates derived thereof) may be analyzed using a flow cytometer to detect CD4+ T cells and/or CD8+ T cells in the labeled sample that express, or do not express, a plurality of cell surface markers selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274, based on a fluorescence signal generated by the fluorophore-conjugated antibodies. A determination may then be made as to whether the donor of the test sample has been infected by the SARS-CoV-2 virus, based on the detected CD4+ T cells and/or CD8+ T cells. As illustrated by the experiments shown in the examples provided herein, sensitive detection of T cell responses across a population with varying immune states often requires the measurement of multiple marker proteins. Therefore, it is useful to determine a combination of cell surface markers that enables robust detection of an infection or immune status, without requiring the measurement of every potential cell surface marker. The complexity of the immune system dictates that many cell surface markers might be important for a specific disease, but as more potential markers that are tested, the more possible combinations there are, and the greater the chance of increasing background noise of the measurements. Accordingly, in some aspects data normalization, alone or in combination with machine learning models, may be used to enable the selection of sets of cell surface markers specific for SARS-CoV-2 infection. While this section describes diagnostic methods directed to SARS-CoV-2 infection, such methods may be configured for the diagnosis of other viral infections (by using an antigen comprising a peptide fragment of a protein of a strain of the virus of interest). [0064] As noted above, various combinations of the cell surface markers described herein may be used to determine whether a subject’s CD4+ and/or CD8+ T cells display a response following stimulation with a SARS-CoV-2 antigen (or other viral antigen of interest). For example, in some aspects a marker panel may comprise any combination of the cell surface AFS Ref. No.039062.00150 marker pairs: CD25 and OX40; CD25 and 41BB; CD25 and CD38; CD25 and CD40L; CD69 and OX40; and/or CD69 and CD38. As illustrated by the data provided herein, these pairs may be particularly useful for detecting a response in CD4+ T cells. Similarly, a panel may comprise (alone or in addition to one or more of the preceding pairs), any combination of the cell surface marker pairs: CD274 and CD69; CD274 and CD38; CD25 and CD274; CD274 and 41BB; and/or CD25 and 41BB. As illustrated by the data provided herein, these pairs may be particularly useful for detecting a response in CD8+ T cells. FIG. 10 illustrates this cell-type specificity using CD25 and 41BB as a representative marker pair. As illustrated by FIG.10A and FIG.10B, this marker pair performed equally well as a means for detecting a response by CD4+ cells and CD8+ cells from COVID-19 convalescent donors. [0065] FIGs.11-14 provide further data regarding the selectivity of different marker pairs. In particular, these graphs summarize the data observed when various marker pairs were evaluated using CD4+ and CD8+ T cells from different COVID-19 convalescent donors. Each data point represents a single COVID-19 convalescent donor’s response to a single SARS- CoV-2 peptide pool (Miltenyi or Mabtech), as compared to the median value for a set of COVID-19 naïve donors. Notably, these graphs exemplify the use of two different calculation methods that may be used to compare the level of activation of a test sample, i.e., a fold-change approach or a difference analysis. These calculations are summarized as follows: [0066] Step 1A. Determine the median signal value observed for a set of replicate test samples (e.g., 5 replicates) for a single donor, following stimulation with the SARS-CoV-2 antigen. [0067] Step 1B. Background Subtraction: For each donor, subtract the signal value observed for a negative control (e.g., water) from the median signal determined in Step 1A. AFS Ref. No.039062.00150 [0068] Step 1C. Determine Naïve Sample Background: Determine the median signal observed for a set of samples (e.g., 8 samples) obtained from COVID-19 naïve donors, following stimulation with the SARS-CoV-2 antigen. [0069] Step 2A. Fold-Change Analysis: If a fold-change analysis is desired, divide the background-subtracted signal value for each COVID-19 convalescent donor sample by the median of the naïve donors’ background subtracted data (i.e., divide the result of Step 1B by the result of Step 1C). [0070] Step 2B. Difference Analysis: If a difference analysis is desired, subtract the background-subtracted signal value from each COVID-19 convalescent donor by the median of the naïve donors’ background-subtracted data (i.e., subtract the result of Step 1C from the result of Step 1B). [0071] The raw data used to generate FIGs.11-14 is provided in FIGs.22-25, respectively. [0072] In the case of FIGs. 11-14, each data point on the graphs represents a single COVID-19 convalescent donor’s signal, calculated according to either the fold-change or difference analysis methods, plotted with respect to the median of the naïve donors’ signals. As illustrated by these figures, the marker pairs CD25 and 41BB; CD25 and CD38; CD25 and CD69; CD40L and CD25; CD40L and CD69; CD40L and OX40; CD69 and CD38; and OX40 41BB performed particularly well with respect to the analysis of CD4+ T cells. Similarly, the marker pairs CD25 and 41BB; CD25 and CD38; CD25 and OX40; CD69 and CD107; CD69 and OX40; CD107 and CD25; C107 and CD38; CD274 and CD25; CD274 and CD38; CD274 and CD69; CD274 and OX40; and OX40 and 41BB performed particularly well with respect to the analysis of CD8+ T cells. It is understood that the calculation methods described above are merely non-limiting examples. In some aspects, other calculation techniques known in the art may be used as part of the analysis of the fluorescence signals detected by the flow cytometer used to perform the AIM assays described herein. AFS Ref. No.039062.00150 [0073] In some aspects, it may be desirable to perform normalization of the detected signals when conducting AIM assays using the marker panels described herein. In particular, normalization is often desirable when performing a cross-sample analysis (e.g., to correct for cross-sample variability as well as for cross marker-pair variability). Accordingly, in some aspects an AIM assay using any combination of the cell surface markers (or of pairs thereof) may involve a normalization step using a positive and/or negative control. For example, in some aspects, water may be used as a negative control and CEFTA peptide pool may be used as a positive control. In some aspects, a normalization process may proceed as follows: [0074] Step 1. Test if the signal value obtained following stimulation of a test sample with the SARS-CoV-2 antigen is significantly higher than the signal value obtained when a negative control consisting of water is assayed. Use one or more replicates to determine the statistical significance of the signal value increase. [0075] Step 2. If the signal value associated with the test sample is determined to be significantly higher in Step 1, subtract the signal value obtained from the assay of the negative control from the signal value obtained from the test sample. Otherwise, set the test sample signal value to zero. [0076] Step 3. Test if the signal value obtained following stimulation of a test sample with the SARS-CoV-2 antigen is significantly higher than the signal value obtained when a positive control consisting of a CEFTA peptide pool is assayed. Use one or more replicates to determine the statistical significance of the signal value increase. [0077] Step 4. If the signal value associated with the test sample is determined to be significantly higher in Step 3, set the test sample signal value to one. Otherwise, normalize the signal obtained from the test sample to the signal obtained from the assay of the CEFTA peptide pool. AFS Ref. No.039062.00150 [0078] It is understood that the steps shown above may be reordered (e.g., the positive control normalization described in Steps 3 and 4 may be carried out first). Moreover, the normalization method described above is a non-limiting example. In some aspects, other normalization techniques known in the art may be used as part of the analysis of the fluorescence signals detected by the flow cytometer used to perform the AIM assays described herein. FIGs.15-16 provide representative examples of normalized signal values generated by normalization using a negative control (water, FIG. 15) and a positive control (a CEFTA peptide pool, FIG.16). [0079] FIG. 17 shows a hierarchical clustering of various cell surface marker pairs described herein. As demonstrated by this figure, multiple cell surface marker pairs are capable of accurately distinguishing between COVID-19 convalescent and COVID-19 naïve samples using the AIM assays described herein. [0080] In some aspects, the marker panels described herein may be evaluated using a computer-implemented classifier. The term “classifier,” as used herein, refers broadly to a machine learning algorithm such as support vector machine(s), AdaBoost classifier(s), penalized logistic regression, elastic nets, regression tree system(s), gradient tree boosting system(s), naive Bayes classifier(s), neural nets, Bayesian neural nets, k-nearest neighbor classifier(s), deep learning systems, and random forest classifiers. [0081] A classification tree is an easily interpretable classifier with built in feature selection. A classification tree recursively splits the data space in such a way so as to maximize the proportion of observations from one class in each subspace. Classification trees are typically noisy. Random forests attempt to reduce this noise by taking the average of many trees. The result is a classifier whose error has reduced variance compared to a classification tree. Methods of building a Random Forest classifier, including software, are known in the art. Prinzie & Poel (2007) “Random Multiclass Classification: Generalizing Random Forests to AFS Ref. No.039062.00150 Random MNL and Random NB.” Database and Expert Systems Applications. Lecture Notes in Computer Science. 4653; Denisko & Hoffman (2018) “Classification and interaction in random forests.” PNAS 115(8): 1690-1692, the contents of which are incorporated by reference in its entirety. [0082] To classify a new observation using the random forest, classify the new observation using each classification tree in the random forest. The class to which the new observation is classified most often amongst the classification trees is the class to which the random forest classifies the new observation. Random forests reduce many of the problems found in classification trees but at the tradeoff of interpretability. [0083] Tools for implementing random forests as discussed herein are available, by way of non-limiting example, for the statistical software computing language and environment, R. For example, the R package “random Forest,” version 4.6-2, includes tools for creating, processing and utilizing random forests. [0084] In some aspects, a random forest classifier may be used to evaluate one or more signals generated by test samples assayed with the AIM assays described herein. An exemplary random forest classifier was trained used a set of 30 samples for which COVID-19 convalescence status was known, and evaluated using 10 test samples. As shown by the ROC data provided in FIG.18, this classifier was found to be 100% accurate in correctly detecting COVID-19 convalescent donors within the set of 10 samples. Additional random forest classifiers were generated using the same set of data (split between training and test groups), and various cell surface marker pairs described herein and found to display comparable results (not shown). FIG.19 is a graph comparing random forest model importance score vs. negative log of the Mann-Witney U test p-value. This graph is annotated to highlight six cell surface marker pairs that stand out as relatively important features (i.e., CD274 and CD69; CD25 and CD38; CD274 and 441B; CD25 and OX40; CD25 and 41BB; and CD69 and OX40). FIG.20 AFS Ref. No.039062.00150 shows a hierarchical clustering of these six cell surface marker pairs. As demonstrated by this figure, all six pairs are capable of accurately distinguishing between COVID-19 convalescent and COVID-19 naïve samples using the AIM assays described herein. In some aspects, the kits and methods described herein may assay for any of these six cell surface marker pairs, alone or in combination. [0085] FIG.21 is a table that lists several sets of cell surface marker pairs that may be used in the panels described herein, as well as exemplary evidence supporting the use of such marker panels. It is understood that any kit or method described herein may utilize any combination of the cell surface markers described shown in this figure or otherwise disclosed herein and that the pairs shown in this table are exemplary and non-limiting. [0086] FIGs. 22-25 provide charts containing the raw data used to generate the graphs shown in FIGs.11-14, respectively. Similarly, FIGs.26-27 provide charts containing the raw data used to generate the graphs shown in FIGs.19-20, respectively. [0087] FIGs.28-33 provide graphs showing the results of a study which evaluated the AIM assays described herein for the detection and/or diagnosis of CMV infection. FIG. 28 shows the response (CD4+ cells) of CMV+ donor samples, whereas FIGs.29-30 show the response of naïve donor samples (FIG.30 is a magnified version of FIG.29). Similarly, FIG.31 shows the response (CD8+ cells) of CMV+ donor samples, with FIGs. 32-33 showing the response of naïve donor samples (FIG. 33 is a magnified version of FIG. 32). FIG. 34 is a graph showing the percentage of activated CD4+ T cells detected using the exemplary cell surface marker pair CD25 and 41BB following this AIM assay. As illustrated by these figures, the present methods are not limited for use in the detection and/or diagnosis of SARS-CoV-2 infection, and may be used for the detection and/or diagnosis of any other virus of interest. Thus, it is understood that any of the methods, assays, and kits described herein may be used to detect and/or diagnose infections caused by other viruses (e.g., by using an antigen of the AFS Ref. No.039062.00150 virus of interest in such methods, assays, and/or kits, in place of a SARS-CoV-2 antigen). In some aspects, the virus may be for example an influenza virus (e.g., an influenza A or B-type virus), an adenovirus, a respiratory syncytial virus (RSV), a parainfluenza virus (e.g., type 1, 2, or 3), an enterovirus, a hepatitis virus, a herpesvirus, a flavivirus, a coronavirus, a human immunodeficiency virus, an infectious peritonitis virus, or any other type of virus. [0088] In some aspects, the methods and kits disclosed herein may also be used to determine whether a subject has been successfully vaccinated. Some individuals, especially those with compromised immune systems, may have difficulty mounting an immune response post-vaccination. The present methods provide a means to assess the CD4+ and/or CD8+ response of a subject, and consequently provide information that can be used to determine whether such individuals require further vaccination (e.g., an additional booster, or a new vaccination (e.g., using a different antigen stimulus). In some aspects, it may be desirable to measure the response of an individual at least 1, 2, 3, or 4 times per year, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after a prior vaccination was administered, or according to any other schedule (e.g., as directed by a medical professional). A determination that a subject has been successfully vaccinated may be made, based upon the detected response being above a predetermined threshold (e.g., measured as an actual, percentage, or fold-change increase compared to a baseline value and/or values obtained using a naïve control). The foregoing methods may similarly be used to evaluate vaccine candidates or to compare the efficacy of different vaccines (e.g., by measuring the response that the elicit in a population of test subjects). [0089] AIM Assay Kits [0090] In some aspects, the disclosed provides kits that may be used to conduct an AIM assay (e.g., using a flow cytometer), in order to identify and/or measure the response of CD4+ and/or CD8+ cells following exposure to an antigen (e.g., a viral antigen associated with any AFS Ref. No.039062.00150 virus of interest). Such kits may comprise a plurality of binding agents capable of specifically binding to a panel comprising one or more of the cell surface markers described herein (e.g., an antibody, antibody fragment, aptamer, or other binding agent), and optionally at least one binding agent, dye or stain that provides a signal that can be used to distinguish live versus dead cells. For example, monoclonal and polyclonal antibodies for the cell surface markers described herein are commercially available from ThermoFisher Scientific, Abcam plc, Bio- Rad Laboratories, Inc., and various other commercial vendors. One may practice the kits, assays, and methods described herein using commercial antibodies, and/or monoclonal or polyclonal antibodies generated by the user via conventional techniques. See, e.g., Hendriksen et al. (2002). In some aspects, the binding agents are combined into a mixture with other binding agents. In some aspects, the individual concentrations and conjugated fluorophores of the binding agents in the mixture are selected to allow sensitive, independent detection of each of the cell surface markers targeted by the panel, without requiring further titration. In some aspects, the kit may further comprise one or more buffers, solvents, or other reagents useful in connection with an AIM assay (e.g., a buffer to resuspend the binding agent, a solvent for dilution, positive and/or negative control samples). [0091] The term “antibody fragment,” as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with and bind to a cell surface marker epitope. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989)), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by AFS Ref. No.039062.00150 separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as “single-chain Fv” (scFv) antibodies. See e.g., Bird et al., (1988); and Huston et al., (1988). In addition, some animals such as camelids naturally make single-chain antibodies that can be used in biochemical assays. Such single-chain antibodies are also intended to be encompassed within the term “antibody fragment.” These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. [0092] Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv. See, e.g., Hollinger and Hudson (2005). Antibody fragments can also be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3). See, e.g., U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies. Antibody fragments can also be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1- VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. See Zapata et al. (1995). In addition to antibody fragments, marker proteins may be detected by other binding agents such as aptamers. Aptamers are nucleic acid-derived binding agents that can be composed or DNA, RNA, or a mixture of DNA and RNA nucleotides, and can be designed or selected to specifically bind to a target protein, such as the surface marker proteins described here. In view of the foregoing, it should be understood that the kits and methods may use any antibodies or antigen-binding antibody fragments known in the art, including but not limited to the various examples set forth herein. [0093] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject AFS Ref. No.039062.00150 matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular compound, composition, article, apparatus, methodology, protocol, and/or reagent, etc., described herein, unless expressly stated as such. In addition, those of ordinary skill in the art will recognize that certain changes, modifications, permutations, alterations, additions, subtractions and sub-combinations thereof can be made in accordance with the teachings herein without departing from the spirit of the present specification. [0094] Use of the terms “may” or “can” in reference to an embodiment or aspect of an embodiment also carries with it the alternative meaning of “may not” or “cannot.” As such, if the present specification discloses that an embodiment or an aspect of an embodiment may be or can be included as part of the inventive subject matter, then the negative limitation or exclusionary proviso is also explicitly meant, meaning that an embodiment or an aspect of an embodiment may not be or cannot be included as part of the inventive subject matter. In a similar manner, use of the term “optionally” in reference to an embodiment or aspect of an embodiment means that such embodiment or aspect of the embodiment may be included as part of the inventive subject matter or may not be included as part of the inventive subject matter. Whether such a negative limitation or exclusionary proviso applies will be based on whether the negative limitation or exclusionary proviso is recited in the claimed subject matter. [0095] Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual AFS Ref. No.039062.00150 value of a numerical range is incorporated into the present specification as if it were individually recited herein. [0096] The terms “a,” “an,” “the” and similar references used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Further, ordinal indicators—such as “first,” “second,” “third,” etc.—for identified elements are used to distinguish between the elements, and do not indicate or imply a required or limited number of such elements, and do not indicate a particular position or order of such elements unless otherwise specifically stated. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0097] When used in the claims, whether as filed or added per amendment, the open-ended transitional term “comprising” (and equivalent open-ended transitional phrases thereof like including, containing and having) encompasses all the expressly recited elements, limitations, steps and/or features alone or in combination with unrecited subject matter; the named elements, limitations and/or features are essential, but other unnamed elements, limitations and/or features may be added and still form a construct within the scope of the claim. Specific embodiments disclosed herein may be further limited in the claims using the closed-ended transitional phrases “consisting of” or “consisting essentially of” in lieu of or as an amended for “comprising.” When used in the claims, whether as filed or added per amendment, the closed-ended transitional phrase “consisting of” excludes any element, limitation, step, or AFS Ref. No.039062.00150 feature not expressly recited in the claims. The closed-ended transitional phrase “consisting essentially of” limits the scope of a claim to the expressly recited elements, limitations, steps and/or features and any other elements, limitations, steps and/or features that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. Thus, the meaning of the open-ended transitional phrase “comprising” is being defined as encompassing all the specifically recited elements, limitations, steps and/or features as well as any optional, additional unspecified ones. The meaning of the closed-ended transitional phrase “consisting of” is being defined as only including those elements, limitations, steps and/or features specifically recited in the claim whereas the meaning of the closed-ended transitional phrase “consisting essentially of” is being defined as only including those elements, limitations, steps and/or features specifically recited in the claim and those elements, limitations, steps and/or features that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. Therefore, the open-ended transitional phrase “comprising” (and equivalent open- ended transitional phrases thereof) includes within its meaning, as a limiting case, claimed subject matter specified by the closed-ended transitional phrases “consisting of” or “consisting essentially of.” As such embodiments described herein or so claimed with the phrase “comprising” are expressly or inherently unambiguously described, enabled and supported herein for the phrases “consisting essentially of” and “consisting of.” [0098] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any AFS Ref. No.039062.00150 other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. [0099] Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described. References 1. Sette, A., & Crotty, S. (2021). “Adaptive immunity to SARS-CoV-2 and COVID-19.” Cell, 184(4), 861-880. 2. Bowyer, G., Rampling, T., Powlson, J., Morter, R., Wright, D., Hill, A. V., & Ewer, K. J. (2018). “Activation-induced markers detect vaccine-specific CD4+ T cell responses not measured by assays conventionally used in clinical trials.” Vaccines, 6(3), 50. 3. Dan, J. M., Mateus, J., Kato, Y., Hastie, K. M., Yu, E. D., Faliti, C. E., ... & Crotty, S. (2021). “Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.” Science, 371(6529), eabf4063. 4. Jung, J. H., Rha, M. S., Sa, M., Choi, H. K., Jeon, J. H., Seok, H., ... & Shin, E. C. (2021). “SARS-CoV-2-specific T cell memory is sustained in COVID-19 convalescent patients for 10 months with successful development of stem cell-like memory T cells.” Nature Communications, 12(1), 1-12. 5. Grifoni, A., Weiskopf, D., Ramirez, S. I., Mateus, J., Dan, J. M., Moderbacher, C. R., ... & Sette, A. (2020). “Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals.” Cell, 181(7), 1489-1501. 6. Hendriksen, C, and Jann H. “Production of polyclonal and monoclonal antibodies.” Handbook of laboratory animal science 2 (2002): 391-411. 7. Le Bert, N., Tan, A. T., Kunasegaran, K., Tham, C. Y., Hafezi, M., Chia, A., ... & Bertoletti, A. (2020). “SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.” Nature, 584(7821), 457-462. 8. Peng, Y., Mentzer, A. J., Liu, G., Yao, X., Yin, Z., Dong, D., ... & Dong, T. (2020). “Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK AFS Ref. No.039062.00150 convalescent individuals following COVID-19.” Nature Immunology, 21(11), 1336- 1345. Moderbacher, C. R., Ramirez, S. I., Dan, J. M., Grifoni, A., Hastie, K. M., Weiskopf, D., ... & Crotty, S. (2020). “Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity.” Cell, 183(4), 996- 1012. Zhou, R., To, K. K. W., Wong, Y. C., Liu, L., Zhou, B., Li, X., ... & Chen, Z. (2020). “Acute SARS-CoV-2 infection impairs dendritic cell and T cell responses.” Immunity, 53(4), 864-877. Liao, M., Liu, Y., Yuan, J., Wen, Y., Xu, G., Zhao, J., ... & Zhang, Z. (2020). “Single- cell landscape of bronchoalveolar immune cells in patients with COVID-19.” Nature Medicine, 26(6), 842-844. Ten Brinke, A., Marek-Trzonkowska, N., Mansilla, M. J., Turksma, A. W., Piekarska, K., Iwaszkiewicz-Grześ, D., ... & Gregori, S. (2017). “Monitoring T-cell responses in translational studies: optimization of dye-based proliferation assay for evaluation of antigen-specific responses.” Frontiers in Immunology, 8, 1870. Saade, F., Gorski, S. A., & Petrovsky, N. 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(2008). “Toward the harmonization of immune monitoring in clinical trials: quo vadis?.” Cancer Immunology, Immunotherapy, 57(3), 285-288. Moodie, Z., Price, L., Gouttefangeas, C., Mander, A., Janetzki, S., Löwer, M., ... & Britten, C. M. (2010). “Response definition criteria for ELISPOT assays revisited.” Cancer Immunology, Immunotherapy, 59(10), 1489-1501. AFS Ref. No.039062.00150 Coughlan, L., & Lambe, T. (2015). “Measuring cellular immunity to influenza: methods of detection, applications and challenges.” Vaccines, 3(2), 293-319. Horton, H., Thomas, E. P., Stucky, J. A., Frank, I., Moodie, Z., Huang, Y., ... & De Rosa, S. C. (2007). “Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination.” J. Immunological Methods, 323(1), 39-54. De Rosa, S. C., Lu, F. X., Yu, J., Perfetto, S. P., Falloon, J., Moser, S., ... & Roederer, M. 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R.; Oppermann, H. “Protein engineering of antibody binding sites-recovery of specific activity in an anti-digoxin single-chain FV analog produced in Escherichia coli.” Proc. Natl. Acad. Sci. USA, 85, 5879-5883. Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., & Winter, G. (1989). “Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli.” Nature, 341(6242), 544-546.

Claims

AFS Ref. No.039062.00150 CLAIMS We claim 1. A kit, comprising: a plurality of antibodies, wherein each antibody is capable of specifically binding to a single cell surface marker selected from a panel comprising CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and CD274. 2. The kit of claim 1, wherein the plurality of antibodies comprises antibodies that collectively bind to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 different cell surface markers selected from the panel. 3. The kit of claims 1 or 2, wherein the kit further comprises a cell viability marker, wherein the cell viability marker comprises a binding agent or dye that selectively binds to or stains either live or dead cells. 4. The kit of any one of claims 1-3, wherein the plurality of antibodies comprises antibodies that specifically bind to CD25, CD69, OX40, 41BB, CD38, and/or CD274. 5. The kit of claim 4, wherein the plurality of antibodies further comprises antibodies that specifically bind to CD40L and/or CD107 6. The kit of any one of claims 1-5, wherein the plurality of antibodies comprises a) CD3, CD4, CD8, CD14, and CD19; b) LAG3; or c) a) and b). 7. The kit of any one of claims 1-6, wherein the plurality of antibodies comprises fluorophore- conjugated antibodies. 8. The kit of claim 7, wherein a different fluorophore is used for each cell surface marker selected from the panel. AFS Ref. No.039062.00150 9. The kit of any one of claim 8, wherein the fluorophore-conjugated antibodies are provided as a single mixture, and wherein each fluorophore-conjugated antibody is present in the mixture at a concentration that allows detection of each cell surface marker targeted by the mixture. 10. The kit of any one of claims 1-8, wherein the kit further comprises one or more antigens capable of activating a peripheral blood mononuclear cell (PBMC); optionally wherein the PBMC is a CD4+ T cell and/or a CD8+ T cell. 11. The kit of claims 9 or 10, wherein the one or more antigens comprise one or more peptides. 12. The kit of claim 11, wherein the one or more peptides comprise at least one fragment of a polypeptide sequence of a protein produced by a strain of the SARS-CoV-2 virus, wherein the at least one fragment has a length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, or a length within a range defined by any pair of integers between 2 and 20. 13. The kit of claim 12, wherein the at least one fragment comprises a fragment of a spike protein, a membrane protein, an outer membrane protein, or a nucleocapsid protein of the strain of the SARS-CoV-2 virus. 14. The kit of any one of claims 1-13, wherein the kit further comprises one or more Fluorescence Minus One (“FMO”) control samples, wherein each FMO control sample comprises antibodies specific for all but one of cell surface markers. 15. The kit of any one of claims 1-14, wherein the kit further comprises one or more solvents and/or buffer solutions. 16. The kit of any one of claims 1-15, wherein the kit further comprises one or more secondary binding agents, wherein each secondary binding agent is capable of specifically binding to a cytokine. 17. The kit of any one of claims 1-15, wherein the kit further comprises one or more secondary binding agents, wherein each secondary binding agent is capable of specifically binding to IFN- γ, TNF-α, IL-2, IL-4, IL-6, or IL-10. AFS Ref. No.039062.00150 18. The kit of any one of claims 1-17, wherein the plurality of antibodies comprises antibodies specific for: a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; and/or d) CD25 and OX40. 19. The kit of any one of claims 1-17, wherein the plurality of antibodies comprises antibodies specific for: a) CD274 and 41BB; and/or b) CD274 and CD69. 20. The kit of any one of claims 1-17, wherein the plurality of antibodies comprises antibodies specific for: a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; d) CD25 and OX40 e) CD274 and 41BB; and/or f) CD274 and CD69. 21. The kit of any one of claims 1-17, wherein the plurality of antibodies comprises one or more lyophilized antibodies. 22. A method of determining whether a subject has been infected by the SARS-CoV-2 virus, comprising: a) generating a treated sample by contacting a plurality of CD4+ T cells and/or CD8+ T cells obtained from the subject with at least one antigen, wherein the antigen comprises a peptide fragment of a protein of a strain of the SARS-CoV-2 virus; b) contacting CD4+ T cells and/or CD8+ T cells in the treated sample with a plurality of fluorophore-conjugated antibodies to produce a labeled sample, wherein each antibody is specific for a cell surface marker selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274; AFS Ref. No.039062.00150 c) detecting CD4+ T cells and/or CD8+ T cells in the labeled sample that express a plurality of cell surface markers selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274, based on a fluorescence signal generated by the fluorophore-conjugated antibodies; and d) determining that the subject has been infected by the SARS-CoV-2 virus, based on the detected CD4+ T cells and/or CD8+ T cells. 23. The method of claim 22, wherein steps a), b), c), and/or d) are performed using the kit of any one of claims 1-21. 24. The method of claims 22 or 23, wherein the detecting step c) further comprises detecting the CD4+ T cells and/or CD8+ T cells in the labeled sample that express a plurality of cell surface markers using at least 2, 3, 4, 5, 6, 7, or 8 replicate samples. 25. The method of any one of claims 22-24, wherein the detecting step c) further comprises a step of normalizing the fluorescence signal generated by the fluorophore-conjugated antibodies. 26. The method of claim 25, wherein the step of normalizing is performed using: a negative control sample, comprising at least one peptide diluent selected from water, another solvent, and/or a buffer; and/or a positive control sample, comprising a peptide pool. 27. The method of claim 26, wherein the peptide pool comprises a set of peptides comprising MHC class II restricted T cell epitopes from human Cytomegalovirus, Epstein Barr virus, Influenza virus, Tetanus toxin, and Adenovirus 5 (“CEFTA”). 28. The method of claim 26, wherein the normalizing step comprises: i) testing whether a fluorescence signal produced by fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample is higher than a fluorescence signal obtained using the negative control sample; and ii) testing whether a fluorescence signal produced by fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample is lower than a signal obtained using the positive control sample. AFS Ref. No.039062.00150 29. The method of claim 28, further comprising: subtracting the fluorescence signal obtained using the negative control sample from the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample, when the negative control fluorescence signal is lower. 30. The method of claim 28, further comprising: setting the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample to zero, when the negative control fluorescence signal is higher. 31. The method of any one of claims 28-30, further comprising: normalizing the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample, to the fluorescence signal obtained using the positive control sample, when the positive control fluorescence signal is higher. 32. The method of any one of claims 28-31, further comprising: setting the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample to a non-zero constant value, when the positive control fluorescence signal is lower. 33. The method of any one of claims 28-32, wherein the comparison of the fluorescence signal produced by the fluorophore-conjugated antibodies bound to CD4+ T cells and/or CD8+ T cells in the labeled sample, to the positive and/or negative control, is based on fluorescence signals obtained from a plurality of replicates. 34. The method of any one of claims 22-33, wherein the determining step d) further comprises determining that the subject has been infected by the SARS-CoV-2 virus, based on an expression level of one or more cytokines by CD4+ T cells and/or CD8+ T cells in the treated sample. AFS Ref. No.039062.00150 35. The method of any one of claims 22-33, wherein the determining step d) further comprises determining that the subject has been infected by the SARS-CoV-2 virus, based on a change in the amount of CD4+ T cells and/or CD8+ T cells in the labeled sample that express the plurality of cell surface markers, compared to a median or average amount determined using samples obtained from one or more SARS-CoV-2 naïve donors. 36. The method of claim 35, wherein the change in the amount of the plurality of cell surface markers is measured as: a) a percentage difference as compared to the median amount; b) fold-change difference as compared to the median amount; or b) a numerical difference as compared to the median amount. 37. The method of any one of claims 22-36, wherein the plurality of fluorophore-conjugated antibodies used in step b) comprises antibodies specific for: a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; d) CD25 and OX40; e) CD274 and 41BB; and/or f) CD274 and CD69. 38. The method of claim 37, wherein the plurality of cell surface markers used in step c) comprises: a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; d) CD25 and OX40 e) CD274 and 41BB; and/or f) CD274 and CD69. 39. A method of determining that a subject is in need of a virus vaccination, comprising: a) generating a treated sample by contacting a plurality of CD4+ T cells and/or CD8+ T cells obtained from the subject with at least one antigen, wherein the antigen comprises a peptide fragment of a protein of a strain of a virus; AFS Ref. No.039062.00150 b) contacting CD4+ T cells and/or CD8+ T cells in the treated sample with a plurality of fluorophore-conjugated antibodies to produce a labeled sample, wherein each antibody is specific for a cell surface marker selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274; c) detecting CD4+ T cells and/or CD8+ T cells in the labeled sample that express a plurality of cell surface markers selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and/or CD274, based on a fluorescence signal generated by the fluorophore-conjugated antibodies; and d) determining that the subject is in need of the virus vaccination, based on the detected CD4+ T cells and/or CD8+ T cells. 40. The method of claim 39, wherein step c) further comprises: normalizing the fluorescence signal generated by the fluorophore-conjugated antibodies to produce a normalized signal; and comparing the normalized signal to (1) a fluorescence signal generated by one or more naïve CD4+ T cells and/or CD8+ T cells; or (2) a median or average fluorescence signal generated by a plurality of COVID-19 naïve CD4+ T cells and/or CD8+ T cells. 41. The method of claims 39 or 40, wherein the virus is SARS-CoV-2.