Activation-induced marker assay

Abstract

The present disclosure relates to the field of biology.In particular, the present disclosure relates to the T cell activation-inducing marker assay, which can be used to determine whether subject has previously been exposed to the antigen of interest, for example, through infection with pathogen, and its related kit and composition.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 63 / 479,902, filed January 13, 2023, which is incorporated herein by reference.

[0002] The present disclosure relates to the field of biology.In particular, the present disclosure relates to the T cell activation-inducing marker assay and its related kit, which can be used to determine whether a subject has previously been exposed to an antigen of interest, for example, through infection with a pathogen. [Background technology]

[0003] Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses an ongoing and significant threat to public health. The presence of neutralizing antibodies against SARS-CoV-2 is an indicator of protective immunity resulting from previous infection (or vaccination). In addition to humoral immune responses, T cells also play a crucial role in regulating adaptive immune responses and as effectors against viral infection. Previous studies of acute and convalescent COVID-19 patients have reported that T cell responses are associated with disease progression, suggesting that SARS-CoV-2-specific CD4+ 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, previous studies have reported that SARS-CoV-2-specific CD4+ and CD8+ T cells were detected in 100% and approximately 70% of convalescent individuals immediately after resolution, respectively (see Grifoni et al., 2020).

[0004] 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 CD8+ T cells, must be considered, as the response kinetics of these various components may be independent of one another. Various assays have traditionally been used to measure the quantity and quality of antigen-specific T cells in humans. See Ten Brinke et al. (2017). In particular, enzyme-linked immunospot (ELISpot) and intracellular cytokine staining (ICS) assays are frequently used in such investigations. See, for example, Saade et al. (2012); Slota et al. (2011); Smith et al. (2015). ELISpot assays involve stimulating peripheral blood mononuclear cells (PBMCs) with antigen in multiwell plates with membranes coated with anti-cytokine capture antibodies. Cytokines produced by antigen-specific T cells are believed to bind to the capture antibody and can therefore be detected using an enzyme-conjugated secondary antibody and a chromogenic substrate. The ELISpot assay is extremely sensitive; for example, cells producing less than 100 cytokine molecules can be detected. See Shirai et al. (1993). As a result, the ELISpot assay has become one of the most frequently used and highly validated assays for detecting antigen-specific T cell responses in clinical trials. See, for example, Britten et al. (2008); Moody et al. (2010). However, the ELISpot assay has several major drawbacks that limit its use as a tool for examining immunological responses to infection, including a limited number of parameters that can be examined, a lack of phenotypic information, and preferential detection of effector cells. Given these limitations, the ELISpot assay may not fully reveal or underestimate the entire antigen-specific T cell response.

[0005] Alternatively, intracellular cytokine staining (ICS) assays can be used to provide further information about the quantity and quality of antigen-induced T cells. Briefly, antigen-stimulated PBMCs are stained with fluorescently labeled anti-cytokine antibodies and analyzed by flow cytometry. ICS assays allow for detailed phenotypic and functional analysis of antigen-specific T cell populations. However, ICS assays are also limited by the number of parameters that can be assessed. Therefore, ICS assays may be biased toward detecting specific types of T cells. For example, ICS assay panels used in clinical trials typically assay IFNγ, IL2, and TNFα, thus detecting 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] Given these limitations, more recent research has popularized the use of activation-induced marker (AIM) assays, which can be used to identify and measure antigen-specific T cell responses based on the upregulation of surface markers upon TCR stimulation. AIM assays can be used to generate a broader picture of the overall antigen-specific T cell response. However, AIM assays have not been fully evaluated or compared with more traditional cytokine-based methods. Furthermore, given the large number of potentially upregulated surface markers, it is impossible to predict, without trial and error, which individual markers or combinations of markers can be used to accurately detect antigen-specific T cells. Summary of the Invention

[0007] Despite significant progress in the development of assays for detecting humoral and cellular immune responses to infection, there is a continuing need for further improvement in this area, given the diversity of possible antigens, including those derived from pathogens, and the limitations of current assays. In particular, there is currently a need for assays and methods that can accurately detect and / or measure antigen-specific T cell immune responses elicited by SARS-CoV-2 infection and other viral infections in human or animal subjects. Such assays and methods can be used, for example, to diagnose past infection with this virus and to provide guidance that can be used to determine when a subject would benefit from booster vaccination. Accordingly, T cell AIM assays and related kits and methods are provided herein. In some embodiments, these AIM assays, kits, and methods can be used to detect SARS-CoV-2-specific CD4+ and CD8+ T cells, enabling their potential use as tools for detecting and / or diagnosing infection and guiding vaccination and treatment decision schedules. In other cases, such assays, kits, and methods can be used to detect CD4+ and CD8+ T cells specific for other clinically relevant viruses.

[0008] In a first general aspect, the present disclosure provides a kit comprising a plurality of antibodies, each antibody capable of specifically binding to a single cell surface marker selected from a panel including CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and CD274.

[0009] In some embodiments, 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, the kit may comprise antibodies specific to any pair of cell surface markers described herein (e.g., CD69 and OX40, or CD25 and 41BB). In other embodiments, the kit may comprise antibodies each specific to one biomarker selected from any combination of cell surface markers described herein. For example, in some embodiments, 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., for monitoring cell activation in the presence of or after treatment with antigenic stimulation). In some embodiments, the plurality of antibodies includes 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 embodiments, the kit may include antibodies specific for all 14 of the cell surface biomarkers described in the preceding paragraph. It is understood that the kits specified herein may utilize monoclonal or polyclonal antibodies, and / or antibody fragments, as binding agents for detecting the cell surface markers described herein.

[0010] In some embodiments, 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 embodiments, the kit may include a fluorophore-conjugated antibody (e.g., to enable detection of antigen-bound antibodies using a flow cytometer or other instrument). In some embodiments, a different fluorophore may be used for each cell surface marker. For example, an exemplary panel may include 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, Alexa700 / CD14, and / or Alexa700 / CD19. Any fluorophore disclosed herein may be used as a conjugate for any cell surface marker (or any other antigen) described herein. In some embodiments, the kit may include multiple fluorophore-conjugated antibodies, each specific for a different cell surface marker, with a different fluorophore being used for each cell surface marker. In some embodiments, the multiple fluorophore-conjugated antibodies are provided as a single mixture, optionally containing optimized concentrations or ratios of some or all of the fluorophore-conjugated antibodies. In some embodiments, the kit according to the present disclosure may include one or more alternative fluorophore-conjugated binding agents (e.g., antibody fragments, aptamers, or any other binding agents capable of specifically binding to the cell surface markers described herein) in place of any of the antibodies described in any of the various exemplary embodiments described herein.

[0012] The multiple antibodies included as part of the kits described herein can be provided in dried (e.g., lyophilized) or liquid form. In some embodiments, the multiple antibodies can be provided as a single mixture, while in others, the multiple antibodies can be provided in separate compartments or containers (e.g., antibodies specific for each cell surface marker or for any subset of cell surface markers can be provided in different compartments or containers). In some embodiments, the kits can include buffers or other solvents that can be used to prepare antibody solutions (e.g., for performing the assays described herein) using the multiple antibodies.

[0013] In some embodiments, the kit further includes a cell viability marker, which comprises a binder or dye that selectively binds to and / or stains either live or dead cells. For example, in some embodiments, the cell viability marker comprises Live / Dead® Fixable Near-IR Stain (Invitrogen®) or any of the other Live / Dead® stains sold by Invitrogen® (e.g., Fixable Blue / Violet / Lime / Aqua / Yellow / Green / Olive / Orange / Red / Fixable Far Red). DAPI (4',6-diamidino-2-phenylindole) and other dyes known to be useful for distinguishing between live and dead eukaryotic cells, such as 7-AAD (7-aminoactinomycin D), Hoechst dye, or propidium iodide, may also be used. Such dyes are available from several manufacturers and typically function based on the reaction of fluorescently reactive dyes with cellular proteins (amines) or through a fluorescence increase or shift that occurs after DNA intercalation. When used at typical concentrations for staining purposes, many of these dyes cannot penetrate viable cell membranes, thereby limiting the availability of cell surface proteins that react with the dye, resulting in dim staining of viable cells. In contrast, reactive dyes can penetrate the damaged membranes of dead cells and stain both internal and external amines, resulting in intense staining of dead cells. Similarly, some cell viability marker dyes (e.g., DNA intercalation dyes) may exhibit limited permeability when applied to viable cell membranes but a greater ability to penetrate and stain dead cells when applied at concentrations typically used for live / dead staining.

[0014] In some embodiments, the kit may further include one or more secondary binding agents, each 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 an antibody, antibody fragment, aptamer, or any other binding agent capable of specifically binding to a cytokine, or any epitope or fragment thereof. Secondary binding agents may be used in the assays described herein, for example, 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 cell surface markers) to determine whether a subject has previously been infected with the SARS-CoV-2 virus (or to make any other determination described herein).

[0015] In some embodiments, the kit may further include one or more antigens (e.g., one or more peptides) capable of activating peripheral blood mononuclear cells ("PBMCs"), such as CD4+ T cells or CD8+ T cells. In some embodiments, the antigen comprises at least a fragment of a polypeptide sequence of a protein produced by a SARS-CoV-2 viral strain. 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 embodiments, the at least one fragment comprises a peptide having a polypeptide sequence of a portion of a spike protein, membrane protein, envelope protein, or nucleocapsid protein of a SARS-CoV-2 viral strain.

[0016] In some embodiments, the kit further comprises one or more Fluorescence Minus One (“FMO”) control samples, each FMO control sample comprising antibodies specific for all but one of the cell surface markers.

[0017] In some embodiments, the kit further comprises one or more solvents and / or buffer solutions.

[0018] In a second general aspect, the disclosure provides a method for detecting a SARS-CoV-2 virus strain, comprising the steps of: a) contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from a subject with at least one antigen to generate a treated sample, wherein the antigen comprises a peptide fragment of a protein of the SARS-CoV-2 virus strain; b) contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from the subject with at least one antigen to generate a treated sample; contacting T cells 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 the fluorescent signals generated by the fluorophore-conjugated antibodies; and d) detecting the detected CD4+ T cells and / or CD8+ T cells. Provided are methods for determining whether a subject is infected with the SARS-CoV-2 virus, comprising determining whether the subject is infected with the SARS-CoV-2 virus based on T cells. In some embodiments, steps a), b), c), and / or d) are performed using a kit according to any exemplary embodiment described herein.

[0019] In some embodiments, the detecting step c) further comprises detecting 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 embodiments, the detecting step c) further comprises normalizing the fluorescent signal generated by the fluorophore-conjugated antibody. The normalizing step can be performed, for example, using a negative control sample consisting of a peptide diluent such as water, or containing water, another solvent, and / or a buffer. For example, the negative control can contain the same volume of peptide diluent used in the test sample receiving the SARS-CoV-2 viral antigen (including a peptide fragment or pool of peptide fragments of a protein of the SARS-CoV-2 viral lineage). Alternatively, the negative control can contain the same volume of peptide diluent used in the test sample receiving the viral antigen of interest (including a peptide fragment or pool of peptide fragments of a protein of any viral lineage of interest). In some embodiments, the peptide diluent can be, for example, water, or can include dimethyl sulfoxide (DMSO) and phosphate-buffered saline. In some embodiments, a positive control sample containing a peptide pool containing multiple viral antigens can also be used, alone or in combination with the negative control. In some embodiments, the peptide pool comprises a set of peptides comprising MHC class II-restricted T cell epitopes derived from human cytomegalovirus (CMV), Epstein-Barr virus, influenza virus, tetanus toxoid, and adenovirus 5 (CEFTA).

[0021] In some embodiments, the normalizing step includes: i) testing whether the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample is higher or lower than the fluorescent signal obtained using a negative control sample; and ii) testing whether the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample is higher or lower than the signal obtained using a positive control sample. In some embodiments, the normalizing step further includes subtracting the fluorescent signal obtained using the negative control sample from the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample if the negative control fluorescent signal is lower. In some embodiments, if the negative control fluorescent signal is higher, the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells can be set to zero. In some embodiments, the normalizing step further comprises normalizing the fluorescent signal produced by the fluorophore-conjugated antibody bound to the CD4+ T cells and / or CD8+ T cells in the labeled sample to the fluorescent signal obtained using the positive control sample if the positive control fluorescent signal is higher. In some embodiments, if the positive control fluorescent signal is lower, the fluorescent signal produced by the fluorophore-conjugated antibody bound to the CD4+ T cells and / or CD8+ T cells can be set to a constant value other than zero, such as 1, 2, or 3. The comparison of the fluorescent signal produced by the fluorophore-conjugated antibody bound to the CD4+ T cells and / or CD8+ T cells in the labeled sample to the positive and / or negative controls can be based on fluorescent signals obtained from multiple replicates.

[0022] In some embodiments, determining step d) further comprises determining that the subject is infected with the SARS-CoV-2 virus based on the expression levels of one or more cytokines by CD4+ T cells and / or CD8+ T cells in the treated sample. In some embodiments, determining step d) further comprises determining that the subject is infected with 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 a plurality of cell surface markers compared to a median or average amount determined using samples obtained from one or more SARS-CoV-2 naive donors. The change in the amount of a plurality of cell surface markers can be measured, for example, as a) a percentage difference compared to the median amount; b) a difference in fold change compared to the median amount; or b) a numerical difference compared to the median amount. In other embodiments, similar determinations can be made for any other virus of interest that is assayed (e.g., based on changes in the amount of CD4+ T cells and / or CD8+ T cells in a labeled sample that express multiple cell surface markers compared to a median or mean amount determined using samples obtained from one or more naive donors).

[0023] In some embodiments, the method of determining whether a subject is infected with the SARS-CoV-2 virus (or another virus of interest), or any other method described herein, may utilize multiple fluorophore-conjugated antibodies used in step b), including 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 paragraph.

[0024] In a third general aspect, the disclosure provides a method for detecting a SARS-CoV-2 virus strain protein comprising the steps of: a) contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from a subject with at least one antigen to generate a treated sample, wherein the antigen comprises a peptide fragment of a protein of a SARS-CoV-2 virus strain; b) contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from a subject with at least one antigen to generate a treated sample; contacting T cells 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 the fluorescent signals generated by the fluorophore-conjugated antibodies; and d) detecting the detected CD4+ T cells and / or CD8+ T cells. Methods for determining whether a subject is in need of SARS-CoV-2 virus vaccination (e.g., a booster vaccination) are provided, comprising determining whether the subject is in need of SARS-CoV-2 virus vaccination based on T cells. In other embodiments, a similar determination can be made regarding the need for vaccination against any other virus of interest (e.g., by detecting CD4+ T cells and / or CD8+ T cells using an antigen comprising a peptide fragment of a protein of the virus strain of interest, as described above).

[0025] It is understood that in some embodiments, the methods of determining that a subject is in need of SARS-CoV-2 virus vaccination or vaccination against another virus of interest may further include any of the steps, components, or parameters of the methods of determining that a subject is infected with SARS-CoV-2 virus or another 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 of the one or more aspects. These features are indicative, however, of but a few of the various ways in which the principles of the various aspects may be employed, and the description is intended to include all such aspects and their equivalents.

[0027] The drawings specified herein illustrate and describe exemplary aspects of the present disclosure and are not intended to limit the scope of the invention as defined by the claims. [Brief explanation of the drawings]

[0028] [Figure 1] 1 is a cartoon representation illustrating an exemplary AIM assay workflow that may be used to determine whether a subject is infected with the SARS-CoV-2 virus (or another virus of interest), as described herein. [Figure 2] 1 is a cartoon representation illustrating an exemplary AIM assay workflow that may be used to determine whether a subject is infected with the SARS-CoV-2 virus (or another virus of interest), as described herein. [Figure 3] FIG. 1 summarizes flow cytometry analysis and gating strategy for an exemplary AIM assay according to the present disclosure. [Figure 4]1 is a series of plots summarizing flow cytometry analysis and gating strategy for the cell surface marker pair CD25 and 41BB. [Figure 5] 1 is a table showing exemplary cell surface marker pairs that may be used in the kits and / or methods described herein. [Figure 6] 1 is a graph showing the percentage of activated CD4+ T cells detected using the cell surface marker pair CD25 and 41BB in samples obtained from a population of COVID-19 convalescent subjects versus samples obtained from a population of COVID-19 naive donors, as measured using a kit according to the present disclosure. [Figure 7] Figure summarizes data obtained from ELISpot assays confirming T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. [Figure 8] Figure summarizes data obtained from ELISpot assays confirming T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. [Figure 9A] Graph summarizing cytokine release data confirming T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. [Figure 9B] Graph summarizing cytokine release data confirming T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. [Figure 9C] Graph summarizing cytokine release data confirming T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation. [Figure 10A]1 is a graph showing the percentage of activated CD4+ T cells detected using the cell surface marker pair CD25 and 41BB after an AIM assay performed using a kit according to the present disclosure for samples obtained from SARS-CoV-2 naive donors versus COVID-19 convalescent donors who were seropositive or seronegative for SARS-CoV-2 antibodies. [Figure 10B] 1 is a graph showing the percentage of activated CD8+ T cells detected using the cell surface marker pair CD25 and 41BB after an AIM assay performed using a kit according to the present disclosure for samples obtained from SARS-CoV-2 naive donors versus COVID-19 convalescent donors who were seropositive or seronegative for SARS-CoV-2 antibodies. [Figure 11] 1 is a graph showing fold change in CD4+ and CD8+ T cell responses in COVID-19 convalescent donors assessed using the AIM assay according to the present disclosure. [Figure 12] 1 is a graph showing the difference in CD4+ and CD8+ T cell responses in COVID-19 convalescent donors assessed using the AIM assay according to the present disclosure. [Figure 13] 1 is a graph showing fold change in CD4+ and CD8+ T cell responses in COVID-19 convalescent donors assessed using the AIM assay according to the present disclosure. [Figure 14] 1 is a graph showing the difference in CD4+ and CD8+ T cell responses in COVID-19 convalescent donors assessed using the AIM assay according to the present disclosure. [Figure 15] 1 is a graph illustrating the normalization of three cell surface marker pairs (CD25 and OX40, CD25 and CD38, and CD25 and 41BB) using water. [Figure 16] 1 is a graph illustrating the normalization of three cell surface marker pairs (CD25 and OX40, CD25 and CD38, and CD25 and 41BB) using the CEFTA pool. [Figure 17]FIG. 1 is a hierarchical clustering diagram showing the clustering of various CD4 and CD8 T cell surface marker pairs tested with the AIM assay according to the present disclosure using samples from COVID-19 naive and COVID-19 convalescent donors. [Figure 18] 1 is a graph showing the receiver operating characteristic (ROC) curve of a random forest model trained to determine whether a sample is from a COVID-19 convalescent or COVID-19 naive donor using AIM data for a set of cell surface markers described herein. [Figure 19] Figure 1 shows a graph comparing random forest model importance scores versus the negative logarithm of the Mann-Whitney U test p-value. The graph is annotated to highlight several cell surface marker pairs that stand out as relatively significant. [Figure 20] FIG. 20 is a hierarchical clustering diagram showing the clustering of the six traits represented by the annotations in FIG. 19. [Figure 21] 1 is a table summarizing the available data supporting the various cell surface marker pairs described herein. [Figure 22] FIG. 12 is a chart showing the raw data used to generate the graph shown in FIG. 11. [Figure 23] FIG. 13 is a chart showing the raw data used to generate the graph shown in FIG. 12. [Figure 24] FIG. 14 is a chart showing the raw data used to generate the graph shown in FIG. 13. [Figure 25] FIG. 15 is a chart showing the raw data used to generate the graph shown in FIG. 14. [Figure 26] FIG. 20 is a chart showing the raw data used to generate the graph shown in FIG. 19. [Figure 27] FIG. 21 is a chart showing the raw data used to generate the graph shown in FIG. 20. [Figure 28]1 is a graph showing donor sample responses (CD4+ cells) observed during a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection, showing the response of a CMV+ donor sample. [Figure 29] 1 is a graph showing donor sample responses (CD4+ cells) observed during a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection, showing the response of naive donor samples. [Figure 30] 29A and 29B are graphs showing donor sample responses (CD4+ cells) observed during a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection, showing responses from naive donor samples and an expanded version of FIG. 29. [Figure 31] 1 is a graph showing donor sample responses (CD8+ cells) observed during a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection, showing the response of CMV+ donor samples. [Figure 32] 1 is a graph showing donor sample responses (CD8+ cells) observed during a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection, showing the response of naive donor samples. [Figure 33] 32A and 32B are graphs showing donor sample responses (CD8+ cells) observed during a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection, showing responses from naive donor samples; [Figure 34] 1 is a graph showing the percentage of activated CD4+ T cells detected using the cell surface marker pair CD25 and 41BB after an AIM assay for the detection and / or diagnosis of CMV infection performed using a kit according to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0029] The detailed description set forth below in connection with the accompanying 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 the various concepts. However, it will be apparent to those skilled in the art that these concepts may be practiced without these specific details.

[0030] Examination of cellular immune responses provides a robust means for understanding previous exposure to pathogens or antigens from other sources. After infection, cellular immune responses to pathogens are long-lived compared to antibody responses, which tend to decline 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 six months. T cell-mediated immune responses provide long-term protection against severe disease, and early studies indicate that this response is detectable for a longer period of time. See Sette et al. (2021). The current commercially available gold standard for assessing T cell responses is the ELISpot test. Although reliable, this method cannot subtype antigen-responsive T cells and is limited in its ability to measure T cell activation profiles.

[0031] Marker panel for AIM assay The present disclosure addresses these and other shortcomings by providing diagnostic kits and methods based on flow cytometry marker panels that can be used to accurately assess CD4+ and / or CD8+ T cell immune responses to SARS-CoV-2 peptides (or viral peptides associated with any other virus of interest) in COVID-19 convalescent donors. In some embodiments, the panels include 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, 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 between live and dead cells). In some embodiments, the panel may include 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-induced after exposure to antigenic stimulation (e.g., CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and / or CD274). In some embodiments, the panel may include 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 section or otherwise disclosed herein). As described in more detail below, certain combinations of 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) have been found to be particularly useful in detecting and assessing CD4+ and / or CD8+ cell responses following exposure to a SARS-CoV-2 viral antigen (or antigens), or an antigen associated with a surrogate virus of interest, and allow for efficient and accurate determination of whether a subject is or has previously been infected with the virus.

[0032] Figures 1-2 summarize the flow cytometry analysis and gating strategy for an exemplary AIM assay using the marker panel and kit described herein. As illustrated here, PBMCs from COVID-19 naive and COVID-19 convalescent subjects can be collected and treated with one or more peptide fragments of one or more SARS-CoV-2 viral proteins as an antigenic stimulus. The treated cells can then be separated from the cell culture supernatant for subsequent processing steps. In this example, the treated cells are subjected to an AIM assay using multiple 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 binder, dye, or stain that can be used to distinguish live versus dead cells. In parallel, supernatants can 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 supernatants can then be subjected to analysis by flow cytometry.

[0033] In this example, a flow cytometer is used to detect and measure fluorescent signals produced by labeled cells and supernatants, e.g., to measure the expression levels of various cytokines, and evaluate the immune response of PBMCs in response to antigen stimulation. For example, a flow cytometer can be used to detect the number, percentage, or proportion of CD4+ and / or CD8+ T cells in a 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 can be used to accurately detect and measure the immune response of CD4+ and / or CD8+ T cells after treatment with one or more peptide fragments of one or more SARS-CoV-2 viral proteins. In this exemplary workflow, a portion (or replicate) of the initially collected sample was collected in parallel and subjected to an ELISpot IFN-γ assay to verify the accuracy of the AIM assay.

[0034] Figure 3 summarizes the flow cytometry analysis and gating strategy for an exemplary AIM assay according to the present disclosure. As illustrated by this example, an exemplary gating strategy for a flow cytometer can include: 1) gating singlets (i.e., single cells) based on light scatter characteristics (in this case, side scatter); gating lymphocytes based on light scatter characteristics (in this case, side and forward scatter); 3) gating live cells (e.g., using cell viability markers described herein); gating T cells (e.g., based on the phenotypes 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 expressing one or more cell surface markers described herein. In this example, the final gating step identified CD4+ T cells expressing CD40L and OX40, and CD8+ T cells expressing CD25 and 41BB. The number of cells found to express the selected cell surface marker(s) can then be analyzed to determine, for example, whether the sample donor is infected with the SARS-CoV-2 virus. This determination can be used, for example, by a medical professional, to guide treatment or vaccination decisions. For example, a weak response can be used as an indicator that the sample donor requires a booster vaccination.

[0035] Figures 4-10 provide data illustrating the use of AIM assays based on the cell surface markers described herein. For example, Figures 4-6 provide data for AIM assays according to the present disclosure that used a pair of cell surface markers (CD25 and 41BB) described herein to assess CD4+ and CD8+ T cell activation after treatment with SARS-CoV-2 viral antigens. Data for a negative control (water) and a positive control (using a CEFTA peptide pool) are also provided. As shown by this data (e.g., the plot in Figure 4), the percentage of CD4+ T cells found to be CD25+ and 41BB+ was significantly higher in COVID-19 convalescent groups (treated with SARS-CoV-2 viral antigens obtained from Miltenyi Biotec or Mabtech). In some embodiments, the SARS-CoV-2 viral antigens may include all or any combination of peptides found in "PepTivator® SARS-CoV-2 Select-premium grade" sold by Miltenyi Biotec or in "PepPool: SARS-CoV-2 (SNMO), human" available from Mabtech.

[0036] This AIM assay was repeated using various pairs of cell surface markers described herein to evaluate CD4+ and CD8+ cell responses. The various pairs of cell surface markers evaluated are summarized in Figure 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. Initial results were averaged and normalized by background subtraction. Figure 6 is an exemplary plot of results for one such assay, showing an increase in CD4+ T cells found to express selected cell surface marker pairs in samples from COVID-19 convalescent donors compared to samples from COVID-19 naive donors.

[0037] Figures 7-9 provide additional data validating the use of the cell surface marker-based AIM assay described herein. The ELISpot assay is a conventional assay that can be used to detect and measure T cell activation. As shown by Figures 7-8, the ELISpot assay confirmed T cell activation and IFN-γ release by COVID-19 convalescent donors after SARS-CoV-2 peptide stimulation with Mabtech peptide pools (when "Convalescent Ser Pos" and "Convalescent Ser Neg" samples were combined). As shown by Figure 9, increased secretion of the cytokines IL-2, IFN-γ, and TNF-α was observed in COVID-19 convalescent donors (when "Convalescent Ser Pos" and "Convalescent Ser Neg" samples were combined), while secretion of the cytokines IL-4, IL-6, and IL-10 was inconsistent between donors.

[0038] Diagnostic methods In some embodiments, the present disclosure provides methods using the marker panels described herein, for example, to determine whether a subject is infected with the SARS-CoV-2 virus. Such methods can be performed using any of the various marker panels and / or kits described herein. In some embodiments, such methods can involve generating a processing sample by contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from a subject with at least one antigen, where the antigen comprises a peptide fragment of a protein of a strain of the SARS-CoV-2 virus. PBMCs obtained from a subject (e.g., comprising CD4+ T cells and / or CD8+ T cells) can optionally be aliquoted into separate portions (e.g., to generate one or more replicates) prior to processing. One or more CD4+ T cells and / or CD8+ T cells in the processed sample can then be contacted with multiple fluorophore-conjugated antibodies to produce a labeled sample, each antibody 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 embodiments, the processed sample, or one or more portions of the cells contained therein, can be separated into different replicates. The labeled sample (or one or more replicates 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 multiple cell surface markers selected from CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and / or CD274 based on the fluorescent signals generated by the fluorophore-conjugated antibodies. A determination may then be made as to whether the donor of the test sample is infected with 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 populations with various immune states often requires measurement of multiple marker proteins. Therefore, it is useful to determine a combination of cell surface markers that allows for robust detection of infection or immune status without requiring measurement of every potential cell surface marker. The complexity of the immune system dictates that, although many cell surface markers may be important for a specific disease, the more potential markers tested, the greater the number of possible combinations and the greater the opportunity for increasing background noise in the measurement. Thus, in some embodiments, data normalization, alone or in combination with a machine learning model, may be used to enable the selection of a set of cell surface markers specific to SARS-CoV-2 infection. While this section describes diagnostic methods directed at SARS-CoV-2 infection, such methods may be configured for the diagnosis of other viral infections (by using antigens containing peptide fragments of proteins from the viral strain of interest).

[0039] As noted above, various combinations of the cell surface markers described herein can be used to determine whether a subject's CD4+ and / or CD8+ T cells exhibit a response after stimulation with SARS-CoV-2 antigens (or other viral antigens of interest).For example, in some embodiments, the marker panel can include any combination of cell surface 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 can be particularly useful for detecting responses in CD4+ T cells.Similarly, the panel can include (alone or in addition to one or more of the aforementioned pairs) any combination of cell surface markers: 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 responses in CD8+ T cells. Figure 10 illustrates this cell type specificity using CD25 and 41BB as a representative marker pair. As illustrated by Figures 10A and 10B, this marker pair performed equally well as a means of detecting responses by CD4+ and CD8+ cells from COVID-19 convalescent donors.

[0040] Figures 11-14 provide further data regarding the selectivity of various marker pairs. In particular, these graphs summarize the data observed when various marker pairs were evaluated using CD4+ and CD8+ T cells from various COVID-19 convalescent donors. Each data point represents the response of a single COVID-19 convalescent donor to a single SARS-CoV-2 peptide pool (Miltenyi or Mabtech) compared to the median for a set of COVID-19 naive donors. Notably, these graphs illustrate the use of two different calculation methods that can be used to compare the levels of activation of test samples: the fold-change approach or differential analysis. These calculations are summarized as follows:

[0041] Step 1A. After stimulation with SARS-CoV-2 antigen, determine the median signal value observed for a set of replicate test samples (e.g., 5 replicates) for a single donor.

[0042] Step 1B. Background subtraction: For each donor, subtract the signal value observed for the negative control (eg, water) from the median signal determined in step 1A.

[0043] Step 1C. Determine naive sample background: Determine the median signal observed for a set of samples (e.g., 8 samples) obtained from COVID-19 naive donors after stimulation with SARS-CoV-2 antigen.

[0044] Step 2A. Fold Change Analysis: If fold change analysis is desired, divide the background-subtracted signal value for each COVID-19 convalescent donor sample by the median of the background-subtracted data from naive donors (i.e., divide the results from step 1B by the results from step 1C).

[0045] Step 2B. Differential Analysis: If differential analysis is desired, subtract the background-subtracted signal value from each COVID-19 convalescent donor by the median of the background-subtracted data from naive donors (i.e., subtract the results of step 1C from the results of step 1B).

[0046] The raw data used to generate Figures 11-14 are provided in Figures 22-25, respectively.

[0047] For Figures 11-14, each data point on the graph represents the signal of a single COVID-19 convalescent donor, calculated according to either fold change or differential analysis, and plotted against the median signal of naive donors. 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 for analyzing CD4+ T cells. Similarly, the marker pair CD25 and 41BB; CD25 and CD38; CD25 and OX40; CD69 and CD107; CD69 and OX40; CD107 and CD25; CD107 and CD38; CD274 and CD25; CD274 and CD38; CD274 and CD69; CD274 and OX40; and OX40 and 41BB have shown particularly excellent performance for analyzing CD8+ T cells.It is understood that the calculation method described above is merely a non-limiting example.In some embodiments, other calculation techniques known in the art can be used as part of the analysis of the fluorescent signal detected by the flow cytometer that is used to carry out the AIM assay described herein.

[0048] In some embodiments, when performing an AIM assay using the marker panel described herein, it may be desirable to normalize the detected signal. In particular, normalization is often desirable when performing inter-sample analysis (for example, to correct for inter-sample variation and inter-marker pair variation). Thus, in some embodiments, an AIM assay using any combination of cell surface markers (or pairs thereof) may involve a normalization step using positive and / or negative controls. For example, in some embodiments, water may be used as a negative control, and a CEFTA peptide pool may be used as a positive control. In some embodiments, the normalization process may proceed as follows:

[0049] Step 1. Test whether the signal value obtained after stimulation of the test sample with SARS-CoV-2 antigen is significantly higher than the signal value obtained when assaying a negative control consisting of water. One or more replicates are used to determine the statistical significance of the increased signal value.

[0050] 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 negative control assay from the signal value obtained from the test sample. Otherwise, set the test sample signal value to zero.

[0051] Step 3. Test whether the signal value obtained after stimulation of the test sample with SARS-CoV-2 antigen is significantly higher than the signal value obtained when assaying a positive control consisting of a CEFTA peptide pool. One or more replicates are used to determine the statistical significance of the increased signal value.

[0052] 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 1. Otherwise, normalize the signal obtained from the test sample to the signal obtained from assaying the CEFTA peptide pool.

[0053] 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 performed first). Furthermore, the normalization methods described above are non-limiting examples. In some embodiments, other normalization techniques known in the art may be used as part of the analysis of the fluorescent signals detected by the flow cytometer used to perform the AIM assays described herein. Figures 15-16 provide representative examples of normalized signal values ​​generated by normalization using a negative control (water, Figure 15) and a positive control (CEFTA peptide pool, Figure 16).

[0054] Figure 17 shows hierarchical clustering of various cell surface marker pairs described herein. As demonstrated by this figure, multiple cell surface marker pairs can accurately distinguish between COVID-19 convalescent and COVID-19 naive samples using the AIM assay described herein.

[0055] In some embodiments, the marker panel described herein can be evaluated using computer-implemented classifier.The term " classifier " used herein broadly refers to machine learning algorithms such as support vector machine, AdaBoost classifier, penalized logistic regression, elastic net, regression tree system, gradient tree boosting system, naive Bayes classifier, neural net, Bayesian neural net, k nearest neighbor classifier, deep learning system and random forest classifier.

[0056] Classification trees are easily interpretable classifiers that incorporate feature selection. Classification trees recursively partition the data space 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 averaging over many trees. The result is a classifier with reduced error variance compared to classification trees. Methods for constructing random forest classifiers, including software, are known in the art. Prinzie & Poel (2007) "Random Multiclass Classification: Generalizing Random Forests to 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 their entireties.

[0057] To classify a new observation using a random forest, each classification tree in the random forest is used to classify the new observation. The class into which the new observation is most frequently classified in a classification tree is the class into which the random forest classifies the new observation. Random forests reduce many of the problems found in classification trees, but at the expense of interpretability.

[0058] 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 Forests" version 4.6-2 includes tools for creating, processing, and utilizing random forests.

[0059] In some embodiments, a random forest classifier may be used to evaluate one or more signals generated by test samples assayed using the AIM assay described herein. An exemplary random forest classifier was trained using a set of 30 samples with known COVID-19 recovery status and evaluated using 10 test samples. As shown by the ROC data provided in Figure 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 into training and test groups) and various cell surface marker pairs described herein and were found to exhibit comparable results (not shown). Figure 19 is a graph comparing the random forest model importance score versus the negative logarithm of the Mann-Whitney U test p-value. The graph is annotated to highlight six cell surface marker pairs (i.e., CD274 and CD69; CD25 and CD38; CD274 and 441B; CD25 and OX40; CD25 and 41BB; and CD69 and OX40) that stand out as relatively important traits. Figure 20 shows hierarchical clustering of these six cell surface marker pairs. As demonstrated by this figure, all six pairs can accurately distinguish between COVID-19 convalescent and COVID-19 naive samples using the AIM assay described herein. In some embodiments, the kits and methods described herein can assay any of these six cell surface marker pairs alone or in combination.

[0060] Figure 21 is a table listing 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 cell surface markers shown in this figure or otherwise disclosed herein, and that the pairs shown in this table are exemplary and non-limiting.

[0061] Figures 22-25 provide charts containing the raw data used to generate the graphs shown in Figures 11-14, respectively. Similarly, Figures 26-27 provide charts containing the raw data used to generate the graphs shown in Figures 19-20, respectively.

[0062] Figures 28-33 provide graphs showing the results of a study evaluating the AIM assay described herein for the detection and / or diagnosis of CMV infection. Figure 28 shows the response (CD4+ cells) of a CMV+ donor sample, while Figures 29-30 show the response of a naive donor sample (Figure 30 is an expanded version of Figure 29). Similarly, Figure 31 shows the response (CD8+ cells) of a CMV+ donor sample, and Figures 32-33 show the response of a naive donor sample (Figure 33 is an expanded version of Figure 32). Figure 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 method is not limited to use in the detection and / or diagnosis of SARS-CoV-2 infection, but 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 can be used to detect and / or diagnose infections caused by other viruses (e.g., by using an antigen of the virus of interest in such a method, assay, and / or kit instead of a SARS-CoV-2 antigen). In some embodiments, the virus can be, for example, an influenza virus (e.g., influenza A or B virus), adenovirus, respiratory syncytial virus (RSV), parainfluenza virus (e.g., types 1, 2, or 3), enterovirus, hepatitis virus, herpesvirus, flavivirus, coronavirus, human immunodeficiency virus, infectious peritonitis virus, or any other type of virus.

[0063] In some embodiments, the methods and kits disclosed herein can also be used to determine whether a subject has been successfully vaccinated. Some individuals, particularly those with a compromised immune system, may have difficulty mounting an immune response after vaccination. The present method provides a means for assessing a subject's CD4+ and / or CD8+ response, thereby providing information that can be used to determine whether such individuals require additional vaccinations (e.g., additional boosters) or new vaccinations (e.g., using different antigen stimulation). In some embodiments, it may be desirable to measure the individual's response at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the administration of the previous vaccination, at least 1, 2, 3, or 4 times per year, or according to any other schedule (e.g., as instructed by a medical professional). The determination that a subject has been successfully vaccinated can be based on the detected response exceeding a predetermined threshold (e.g., measured as an actual increase, percentage increase, or fold increase change compared to baseline values ​​and / or values ​​obtained using naive controls). The methods described above may similarly be used to evaluate vaccine candidates or to compare the efficacy of various vaccines (eg, by measuring the response elicited in a population of test subjects).

[0064] AIM Assay Kit In some embodiments, the present disclosure provides kits that can be used to perform AIM assays (e.g., using a flow cytometer) to identify and / or measure the response of CD4+ and / or CD8+ cells after exposure to an antigen (e.g., a viral antigen associated with any virus of interest). Such kits can include multiple binding agents (e.g., antibodies, antibody fragments, aptamers, or other binding agents) that can specifically bind to a panel including one or more of the cell surface markers described herein, and optionally at least one binding agent, dye, or stain that provides a signal that can be used to distinguish between live and dead cells. For example, monoclonal and polyclonal antibodies against the cell surface markers described herein are commercially available from ThermoFisher Scientific, Abcam plc, Bio-Rad Laboratories, Inc., and various other commercial suppliers. Commercial antibodies and / or monoclonal or polyclonal antibodies generated by users using conventional techniques can be used to practice the kits, assays, and methods described herein. See, for example, Hendriksen et al. (2002). In some embodiments, the binding agent is combined into a mixture with other binding agents. In some embodiments, the individual concentrations of the binding agents in the mixture and the conjugated fluorophores are selected to enable the highly sensitive and independent detection of each of the cell surface markers targeted by the panel without the need for further titration. In some embodiments, the kit may further comprise one or more buffers, solvents, or other reagents useful in connection with AIM assay (e.g., buffers for resuspending binding agents, solvents for dilution, positive and / or negative control samples).

[0065] The term "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact and bind to a cell surface marker epitope. Examples of binding fragments include, but are not limited to, Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bridges at the hinge region; Fd fragments, which are composed of VH and CH1 domains; Fv fragments, which are composed of the VL and VH domains of a single antibody arm; dAb fragments (Ward et al., (1989)); and isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of Fv fragments, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods to produce a single protein chain, in which the VL and VH domains pair to form a monovalent molecule known as a "single-chain Fv" (scFv) antibody. See, e.g., Bird et al., (1988); and Huston et al., (1988). In addition, some animals, such as camelids, naturally make single-chain antibodies, which 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 skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0066] Antibody fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. See, e.g., Hollinger and Hudson (2005). Antibody fragments can also be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3). See, e.g., U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies. Antibody fragments can also be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions with complementary light chain polypeptides. See, Zapata et al. (1995). In addition to antibody fragments, marker proteins can be detected by other binding agents, such as aptamers. Aptamers are nucleic acid-derived binding agents that may be composed of 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 herein. In light of the foregoing, it should be understood that the kits and methods can use any antibody or antigen-binding antibody fragment known in the art, including but not limited to the various examples set forth herein.

[0067] Finally, while aspects of the specification have been emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. It should be understood, therefore, that the disclosed subject matter is in no way limited to the specific compounds, compositions, articles, devices, methodologies, protocols, and / or reagents, etc., described herein, unless expressly stated as such. Additionally, those skilled in the art will recognize that certain changes, modifications, permutations, alterations, additions, subtractions, and subcombinations thereof can be made in accordance with the teachings herein without departing from the spirit of the specification.

[0068] The use of the terms "may" or "can" with respect to an embodiment or aspect of an embodiment also carries the alternative meaning of "may not" or "cannot." Thus, where the specification discloses that an embodiment or aspect of an embodiment may or may be included as part of the inventive subject matter, any negative limitation or exclusionary condition is also expressly intended, meaning that the embodiment or aspect of an embodiment may not or cannot be included as part of the inventive subject matter. In a similar manner, the use of the term "optionally" with respect to an embodiment or aspect of an embodiment means that such embodiment or aspect of an embodiment may or may not be included as part of the inventive subject matter. Whether such a negative limitation or exclusionary condition applies will depend on whether the negative limitation or exclusionary condition is stated in the claimed subject matter.

[0069] Although 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. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated herein as if it were individually set forth herein.

[0070] As used in the context of describing the present invention (particularly in the context of the claims that follow), the terms "a," "an," "the," and similar references should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Furthermore, ordinal designations such as "first," "second," and "third" referring to specified elements are used to distinguish elements, unless specifically stated otherwise, and do not indicate or imply a required or limited number of such elements, nor do they indicate a particular location or order of such elements. 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., "etc.") provided herein is intended merely to further clarify the invention and does not impose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0071] When used in the claims, whether as filed or added by amendment, the open transitional term "comprising" (and its equivalent open transitional phrases such as including, containing, and having) includes all explicitly recited elements, limitations, steps, and / or characteristics, either alone or in combination with unrecited subject matter; while the named elements, limitations, and / or characteristics are essential, other unnamed elements, limitations, and / or characteristics may be added and still form a structure within the scope of the claim. Specific embodiments disclosed herein may be further limited in the claims using the closed transitional phrase "consisting of" or "consisting essentially of" in place of or as an amendment to "comprising." When used in the claims, whether as filed or added by amendment, the closed transitional phrase "consisting of" excludes any element, limitation, step, or feature not expressly recited in the claim. The closed transitional phrase "consisting essentially of" limits the scope of a claim to the explicitly recited elements, limitations, steps, and / or characteristics, as well as any other elements, limitations, steps, and / or characteristics that do not materially affect the basic and novel characteristics of the claimed subject matter. Thus, the meaning of the open transitional phrase "comprising" is defined to include all specifically recited elements, limitations, steps, and / or characteristics, as well as any optional additional unspecified ones. The meaning of the closed transitional phrase "consisting of" is defined to include only those elements, limitations, steps, and / or characteristics specifically recited in the claim, while the meaning of the closed transitional phrase "consisting essentially of" is defined to include only those elements, limitations, steps, and / or characteristics specifically recited in the claim, as well as those elements, limitations, steps, and / or characteristics that do not materially affect the basic and novel characteristics of the claimed subject matter.Thus, the open transitional phrase "comprising" (and equivalent open transitional phrases) includes within its meaning, in limiting cases, the claimed subject matter specified by the closed transitional phrases "consisting of" or "consisting essentially of." As such, the embodiments described herein or claimed using the phrase "comprising" are expressly or inherently clearly described, enabled, and supported herein by the phrases "consisting essentially of" and "consisting of."

[0072] All patents, patent publications, and other publications referenced and identified in this 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 may 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 taken as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0073] Finally, 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. Thus, the present invention is not limited to that precisely as shown and described.

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Claims

1. A kit comprising a plurality of antibodies, each antibody capable of specifically binding to a single cell surface marker selected from a panel including CD3, CD4, CD8, CD14, CD19, LAG3, CD25, CD69, OX40, 41BB, CD38, CD40L, CD107, and CD274.

2. 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. 3. The kit of claim 1 or 2, further comprising a cell viability marker, wherein the cell viability marker comprises a binder or dye that selectively binds to or stains either live or dead cells.

4. The kit of claim 1 , 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 an antibody that specifically binds to CD40L and / or CD107.

6. the plurality of antibodies a) CD3, CD4, CD8, CD14, and CD19; b) LAG3; or c) a) and b) 6. The kit of claim 1, comprising:

7. 7. The kit of claim 1, wherein the plurality of antibodies comprises fluorophore-conjugated antibodies.

8. 8. The kit of claim 7, wherein a different fluorophore is used for each cell surface marker selected from the panel.

9. 9. The kit of claim 8, wherein the fluorophore-conjugated antibodies are provided as a single mixture, and each fluorophore-conjugated antibody is present in the mixture at a concentration that allows for detection of each cell surface marker targeted by the mixture.

10. 9. The kit of any one of claims 1 to 8, further comprising one or more antigens capable of activating peripheral blood mononuclear cells (PBMCs), optionally wherein the PBMCs are CD4+ T cells and / or CD8+ T cells.

11. 11. The kit of claim 9 or 10, wherein the one or more antigens comprise one or more peptides.

12. the one or more peptides comprise at least a fragment of a polypeptide sequence of a protein produced by a strain of the SARS-CoV-2 virus; 12. The kit of claim 11, 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. 13. The kit of claim 12, wherein the at least one fragment comprises a fragment of a spike protein, a membrane protein, an envelope protein, or a nucleocapsid protein of the SARS-CoV-2 virus strain.

14. 14. The kit of any one of claims 1 to 13, further comprising one or more Fluorescence Minus One ("FMO") control samples, each FMO control sample comprising antibodies specific for all but one of the cell surface markers.

15. 15. The kit of any one of claims 1 to 14, further comprising one or more solvents and / or buffer solutions.

16. 16. The kit of any one of claims 1 to 15, further comprising one or more secondary binding agents, each secondary binding agent capable of specifically binding to a cytokine.

17. 16. The kit of any one of claims 1 to 15, further comprising one or more secondary binding agents, each secondary binding agent capable of specifically binding to IFN-γ, TNF-α, IL-2, IL-4, IL-6, or IL-10.

18. the plurality of antibodies a) CD69 and OX40; b) CD25 and 41BB; c) CD25 and CD38; and / or d) CD25 and OX40 18. The kit of claim 1, comprising an antibody specific for

19. the plurality of antibodies a) CD274 and 41BB; and / or b) CD274 and CD69 18. The kit of claim 1, comprising an antibody specific for

20. the plurality of antibodies 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 18. The kit of claim 1, comprising an antibody specific for

21. 18. The kit of any one of claims 1 to 17, wherein the plurality of antibodies comprises one or more lyophilized antibodies.

22. a) generating a treated sample by contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from a 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+ 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+ 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 the fluorescent signal generated by the fluorophore-conjugated antibody; and d) determining that the subject is infected with the SARS-CoV-2 virus based on the detected CD4+ T cells and / or CD8+ T cells. A method for determining whether a subject is infected with the SARS-CoV-2 virus, comprising:

23. 23. The method of claim 22, wherein steps a), b), c) and / or d) are performed using a kit according to any one of claims 1 to 21.

24. 24. The method of claim 22 or 23, wherein said detecting step c) further comprises detecting the CD4+ 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. 25. The method of any one of claims 22 to 24, wherein the detecting step c) further comprises normalizing the fluorescent signal produced by the fluorophore-conjugated antibody.

26. The normalizing step comprises: a negative control sample comprising at least one peptide diluent selected from water, another solvent, and / or a buffer; and / or Positive control sample containing a peptide pool 26. The method of claim 25, wherein the method is performed using

27. 27. The method of claim 26, wherein the peptide pool comprises a set of peptides comprising MHC class II-restricted T cell epitopes derived from human cytomegalovirus, Epstein-Barr virus, influenza virus, tetanus toxoid, and adenovirus 5 ("CEFTA").

28. The normalizing step comprises: i) testing whether the fluorescent signal produced by the fluorophore-conjugated antibody bound to the CD4+ T cells and / or CD8+ T cells in the labeled sample is higher than the fluorescent signal obtained using the negative control sample; and ii) testing whether the fluorescent signal produced by the fluorophore-conjugated antibodies bound to the CD4+ T cells and / or CD8+ T cells in the labeled sample is lower than the signal obtained using the positive control sample.

27. The method of claim 26, comprising:

29. if the negative control fluorescent signal is lower, subtracting the fluorescent signal obtained using the negative control sample from the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample.

30. The method of claim 28, further comprising:

30. setting the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample to zero if the fluorescent signal of the negative control is higher.

30. The method of claim 28, further comprising:

31. If the positive control fluorescent signal is higher, normalizing the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample to the fluorescent signal obtained using the positive control sample.

31. The method of any one of claims 28 to 30, further comprising:

32. If the fluorescent signal of the positive control is lower, setting the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample to a constant value other than zero.

32. The method of any one of claims 28 to 31, further comprising:

33. 33. The method of any one of claims 28 to 32, wherein the comparison of the fluorescent signal produced by the fluorophore-conjugated antibody bound to CD4+ T cells and / or CD8+ T cells in the labeled sample with the positive and / or negative control is based on fluorescent signals obtained from multiple replicates.

34. 34. The method of any one of claims 22 to 33, wherein said determining step d) further comprises determining that the subject is infected with the SARS-CoV-2 virus based on the expression level of one or more cytokines by CD4+ T cells and / or CD8+ T cells in the treated sample.

35. 34. The method of any one of claims 22 to 33, wherein said determining step d) further comprises determining that the subject is infected with the SARS-CoV-2 virus based on a change in the amount of CD4+ and / or CD8+ T cells in the labeled sample that express the plurality of cell surface markers compared to a median or mean amount determined using samples obtained from one or more SARS-CoV-2 naive donors.

36. the change in the amount of the plurality of cell surface markers is a) the percentage difference compared to the median amount; b) the difference in fold change compared to the median amount; or b) the numerical difference compared to the median amount 36. The method of claim 35, wherein the measured value is:

37. the plurality of fluorophore-conjugated antibodies used in step b) 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 37. The method of any one of claims 22 to 36, comprising an antibody specific for

38. The plurality of cell surface markers used in step c) are 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, comprising:

39. a) generating a treated sample by contacting a plurality of CD4+ T cells and / or CD8+ T cells obtained from a subject with at least one antigen, wherein the antigen comprises a peptide fragment of a protein of a viral strain; b) contacting CD4+ 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+ 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 the fluorescent signal generated by the fluorophore-conjugated antibody; and d) determining that the subject is in need of viral vaccination based on the detected CD4+ T cells and / or CD8+ T cells. A method for determining whether a subject is in need of a viral vaccination, comprising:

40. Step c) is normalizing the fluorescent signal produced by the fluorophore-conjugated antibody to produce a normalized signal; and comparing the normalized signal to (1) the fluorescence signal generated by one or more naive CD4+ T cells and / or CD8+ T cells; or (2) the median or mean fluorescence signal generated by a plurality of COVID-19 naive CD4+ T cells and / or CD8+ T cells.

40. The method of claim 39, further comprising:

41. 41. The method of claim 39 or 40, wherein the virus is SARS-CoV-2.

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