EP4616189A1 - Methods for assessing antigen specific t-cell responses - Google Patents

Abstract

The present disclosure provides methods and kits for determining antigen-specific T-cell responses in a subject based upon the use of a whole blood sample, rather than isolated, concentrated, and/or extracted peripheral blood mononuclear cells. The use of a whole blood sample to measure antigen-specific T-cell responses in a subject streamlines the method by decreasing sample preparation and consumption. The methods for determining antigen antigen-specific T-cell responses in a subject include, contacting a whole blood sample from the subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, and analyzing the mixture for antigen-specific T-cells.

Description

METHODS FOR ASSESSING ANTIGEN SPECIFIC T-CELL RESPONSES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is being filed on November 13, 2023, as a PCT International Patent application and claims the benefit of and priority to U.S. Provisional Patent Application No. 63/383,412, filed on November 11, 2022, the entire disclosure of which is hereby incorporated by reference in its entirety.

INTRODUCTION

[0002] The development of specific adaptive immune response is decisive to control and clear infections, including viral infections. T-cells are able to orchestrate and execute an immune response against different antigens and acquire a highly diversified set of functional properties, which provides the basis for immune protection. Accordingly, analysing and understanding specific T-cell responses is critical to better understand protective immunity in many infectious diseases.

[0003] The ability to analyse and characterize the build-up of cellular immunity is an important milestone for the development of vaccines and treatments across many fields, including virology, oncology, and immunology. The presence of antigen-specific T- cells can be evaluated in vitro based on the ability of T-cells to proliferate, and express activation markers and/or produce cytokines and transcription factors in the presence of an antigen of interest. Among the available methods, the Enzyme-Linked Immunospot (“ELISpof ’) assay is a technique to determine the frequency of antigen-specific immune cells secreting a cytokine upon recall antigen stimulation. The IFN-y ELISpot technique represents the most common approach in clinical settings to assess both T- CD4+ and T-CD8+ antigen specific responses. Based on the use of isolated, concentrated, and/or extracted peripheral blood mononuclear cells (“PBMC”), this technique enables the detection of low-frequency antigen-specific T-cells but doesn’t allow for differentiation of CD4+ and CD8+ driven responses.

[0004] Another method to measure and characterize specific T-cell responses is Intracellular Cytokine Staining (‘ICS”). Combined with multi-parameter flow cytometry, ICS allows the simultaneous detection of different markers, both extra- and intracellular, leading to the identification of subsets of responding cells based on cell phenotyping, with the functional readout of cytokine production. Generally, ICS makes it possible to explore the polarization of the cellular response by analysing cytokines such as IL-2, TNF and IFN-y. An isolated, concentrated, and/or extracted PBMC preparation is often perceived as a crucial prerequisite to ICS and therefore often represents the first step of an ICS workflow. PMBC preparation is time consuming and depends on technically competent individuals who are often already busy in a diagnostic lab. As a direct result, ICS is rarely considered in clinical routine.

[0005] Classical approaches for determining antigen-specific T-cell responses in a subject use isolated, concentrated, and/or extracted PBMCs. As discussed, examples of these approaches include ELISpot and ICS. Both ELISpot and ICS require the extraction of PMBCs from whole blood prior to determining antigen-specific T-cell responses. Current techniques, including ELISpot and ICS, suffer from a lack of resolution, lack of sensitivity, and/or from labor intensive and tedious workflows. Additional deficiencies with current techniques include that many current techniques only look at a single activation marker. These deficiencies with current techniques are compounded by the low frequency of antigen specific T-cells, which often include percentages as low as 1 cell per 100,000 T-cells.

BRIEF SUMMARY OF THE INVENTION

Methods for assessing an antigen-specific T-cell response in a subject.

[0006] The present disclosure provides methods and kits for determining antigenspecific T-cell responses in a subject based upon the use of a whole blood sample, rather than isolated, concentrated, and/or extracted PBMC. The use of a whole blood sample to measure antigen-specific T-cell responses in a subject streamlines the method by simplifying sample preparation and analysis, decreasing sample volume, and reducing sample consumption. The methods and kits disclosed herein may provide a loss-less and more realistic physiological environment for the stimulation of antigenspecific T -cells. The methods and kits may further simplify sample preparation and analysis through the use of dry reagent technology and/or the use of intact antigens rather than pre-processed peptides.

[0007] In its broadest aspect, the present disclosure provides methods and kits for determining antigen-specific T-cell responses in a subject, the method comprising contacting a whole blood sample from the subject with at least one antigen to form a mixture, contacting the mixture with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen, or a combination thereof, to form a mixture, and analyzing the mixture for T-cells responding to the antigen. In embodiments, the analyzing is by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL.

[0008] In an embodiment, at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof. In an embodiment, at least one reagent is added to the mixture formed by contacting the whole blood sample with at least one antigen.

[0009] In certain embodiments, the disclosed methods further comprise a step of incubating the mixture formed by contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining regent and/or after contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining regent. In certain embodiments, the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours.

[0010] The methods of the present disclosure may also include obtaining a cell concentrate from the mixture after the step of incubating the mixture, wherein the step of obtaining a cell concentrate from the mixture comprises one or more of the following steps: adding a fixation reagent to the mixture, adding a lysing reagent to the mixture, adding a permeabilization reagent to the mixture, staining the mixture with a staining reagent, and washing and concentrating the mixture to obtain the cell concentrate.

[0011] In certain embodiments, the staining reagent is a dry staining reagent and may be provided in at least one pre-filled reagent vessel. In embodiments, the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of extracellular and/or intracellular T-cell markers.

[0012] In certain embodiments, the at least one antigen is a dry reagent and may be provided in at least one pre-filled reagent vessel. In some embodiments, the at least one dry antigen reagent contained within a pre-filled dry reagent vessel further comprises and at least one dry staining reagent. [0013] Some examples of extracellular T-cell markers include, but are not limited to CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, and/or any combination thereof. Some examples of the intracellular T-cell markers include, but are not limited to IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGF (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, and/or any combination thereof.

[0014] In certain embodiments, the step of analyzing the mixture comprises analyzing the presence of activation markers of CD4+ T-cells, and/or CD8+ T-cells, and/or the presence of intracellular cytokines or transcription factors. In certain embodiments, the step of analyzing the mixture comprises analyzing the presence of markers indicating Thl, Th2, Thl7, or Treg specific T-cells in response to an allergen during immunotherapy. The activation markers of CD4+ T-cells may include, but are not limited to CD69, CD154, CD137 and CD107a. The activation markers of CD8+ T-cells may include, but are not limited to CD69, CD154, CD137 and CD107a.

[0015] In certain embodiments, the at least one antigen comprises at least one virusspecific antigen. The at least one virus-specific antigen may include, but is not limited to a full-length protein, protein derivative, peptide, or peptide pool derived from SARS- CoV-2, SARS-CoV, MERS, Cytomegalovirus (CMV), Respiratory Syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), Monkey Pox (MP), Human Immunodeficiency virus (HIV), or Human Papilloma virus (HPV). The full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV -2 may include, but is not limited to a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, and/or any combination thereof.

[0016] In certain embodiments, the at least one antigen may include at least one bacteria-specific antigen, at least one parasite-specific antigen, and/or at least one allergen-specific antigen. In certain embodiments, the at least one antigen is one or more proteins, variants, or fusion proteins isolated from a bacterial lysate derived from the infection of at least one bacterial strain. In certain embodiments, the at least one antigen is from tuberculosis, L. monocytogenes, E. coli, Borreha bacterium. In some embodiments, the at least one antigen is derived from one or more pollen or environmental allergen. A wide range of allergenic pollen species are known, including grasses, weeds, and trees, and are available through knowledge in the literature. In some embodiments, the at least one antigen is derived from one or more food allergen. A wide range of allergenic food species are known, including dairy', wheat and nuts, and are available through knowledge in the literature.

[0017] In certain embodiments, the methods exclude a step of isolation, concentration, and/or extraction of peripheral blood mononuclear cells (PBMCs).

[0018] In certain embodiments, methods for determining antigen-specific T-cell responses in a subject are used to analyze an antigen-specific T-cell response of a subject before and/or after vaccination. In certain embodiments, methods for determining antigen-specific T-cell responses in a subject are used for monitoring an immune status of a subject after an incident of viral infection. In certain embodiments, methods for determining antigen-specific T-cell responses in a subject are used monitoring an immune status of a subject prior to an incident of viral infection.

[0019] In certain embodiments, the present disclosure is directed to kits for determining antigen-specific T-cell responses in a subject comprise at least one antigen and at least one staining reagent. In embodiments, the at least one antigen and at least one staining reagent is compatible for a whole blood sample of about 10 pL to about 10 mL. In certain embodiments, the at least one antigen is a dry reagent and may be provided in at least one pre-filled reagent vessel. In some embodiments, the at least one dry antigen reagent is contained within a pre-filled dry reagent vessel. In other embodiments, the at least one staining reagent is a dry staining reagent and may be provided in at least one pre-filled reagent vessel. In other embodiments, a pre-filled dry reagent vessel may contain at least one dry staining reagent and at least one dry antigen reagent.

[0020] In some embodiments, kits can be prepared for determining antigen-specific T- cell responses based on cell types of interest or toward specific applications such as prediction of a response to a therapeutic agent, diagnosis and prognosis of various diseases or condition. In some embodiments, kits of the present disclosure may also be used to identify antigen-specific T-cell responses to determine the effectiveness of treatment for cancer, allergies, autoimmunity, bacterial infections, viral infections, and/or parasitic infections. Kits of the present disclosure may also be used to identify antigen-specific T-cell responses to determine immunity to and/or from cancer, allergies, bacterial infections, viral infections, and/or parasitic infections. In some embodiments, kits of the present disclosure may also be used to identify antigenspecific T-cell responses to determine a subject’s recovery from treatment for cancer, allergies, bacterial infections, viral infections, and/or parasitic infections.

[0021] In certain embodiments, kits for determining an antigen-specific T-cell response may include staining reagents specific to detect extracellular markers of a T- cell response. In certain embodiments, kits for determining an antigen-specific T-cell response may include staining reagents specific to intracellular markers of a T-cell response, including cytokine-specific and transcription factor-specific markers.

Staining reagents may include antibody reagents for identification of extracellular T- cell markers, including but not limited to CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, and/or any combination thereof. Staining reagents may also include antibody reagents for identification of intracellular T-cell markers including but not limited to IL-2, IL-4, IL- 5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGF|3 (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, or any combination thereof.

[0022] In certain embodiments, kits for determining an antigen-specific T-cell response may include at least one pre-filled reagent vessel, a lysing reagent, a permeabilization reagent, a fixation reagent, or any combination thereof. In certain embodiments, the at least one antigen or the at least one staining reagent is a dry reagent. In an embodiment, at least one dry reagent is provided in at least one pre-filled reagent vessel. [0023] In certain embodiments, kits for determining an antigen-specific T-cell response may include at least one virus-specific antigen. The virus specific antigen can be, but is not limited to a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, Cytomegalovirus (CMV), Respiratory Syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), Monkey Pox (MP), or Human Papilloma virus (HPV). The virus specific antigen can be, but is not limited to a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprising a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof. [0024] In certain embodiments, kits for determining an antigen-specific T-cell response may include at least one parasite-specific antigen, at least one allergenspecific antigen, or at least one bacteria-specific antigen. In certain embodiments, kits for determining an antigen-specific T-cell response may be used for analyzing the antigen-specific T-cell response of a subject before and/or after vaccination, or for monitoring an immune status of a subject prior to or after an incident of viral infection. [0025] In certain embodiments, kits for determining an antigen-specific T-cell response may include staining reagents specific to characterize the phenotype of a variety of T-cell subsets, including but not limited to Naive T-cells, memory T-cells, regulatory T-cells, helper T-cells, cytotoxic T-cells, and/or natural killer T-cells.

[0026] In certain embodiments, the disclosed kits comprise a combination of T-cell activation markers to extracellular and intracellular markers. The disclosed kits can also comprise one or more additional vials, tubes, inhibitors, modulators, therapeutic agents, fixatives, buffers, physical devices and software for carrying out the analysis of the cells, such as by flow cytometry.

[0027] In one aspect, the present disclosure provides a method of determining an antivirus immune response to SARS-CoV-2 in a subject, the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen, or a combination thereof, to form a mixture. In embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL, or any volume in between 10 about 10 pL to about 10 mL. In certain embodiments, the whole blood sample is greater than about 10 pL but less than about 1 ml. In certain embodiments, the whole blood sample is between about 200 pL and about 1 ml.

[0028] In an embodiment, at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof. In an embodiment, at least one reagent is added to the mixture formed by contacting the whole blood sample with at least one antigen. In certain embodiments, analyzing the mixture includes identifying a T-cell response to contacting the whole blood sample with at least one antigen.

[0029] In certain embodiments, the disclosed methods further comprise a step of incubating the mixture formed by contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining regent and/or after contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining regent. In certain embodiments, the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours.

[0030] The methods of the present disclosure may also include obtaining a cell concentrate from the mixture after the step of incubating the mixture, wherein the step of obtaining a cell concentrate from the mixture comprises one or more of the following steps: adding a fixation reagent to the mixture, adding a lysing reagent to the mixture, adding a permeabilization reagent to the mixture, staining the mixture with a staining reagent, and washing and concentrating the mixture to obtain the cell concentrate.

[0031] In certain embodiments, the staining reagent is a dry staining reagent and may be provided in at least one pre-filled reagent vessel. In embodiments, the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of extracellular and/or intracellular T-cell markers.

[0032] Some examples of extracellular T-cell markers include, but are not limited to CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL). CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, and/or any combination thereof. Some examples of the intracellular T-cell markers include, but are not limited IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGFp (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, and/or any combination thereof.

[0033] In certain embodiments, the step of analyzing the mixture comprises analyzing the presence of activation markers of CD4+ T-cells, and/or CD8+ T-cells, and/or the presence of intracellular cytokines and/or transcription factors. The activation markers of CD4+ T-cells may include, but are not limited to CD69, CD 154, CD 137 and CD107a. The activation markers of CD8+ T-cells may include, but are not limited to CD69, CD154, CD137 and CD107a.

[0034] In certain embodiments, the at least one antigen comprises at least one virusspecific antigen. The at least one virus-specific antigen may include, but is not limited to a full-length protein, protein derivative, peptide, or peptide pool derived from SARS- CoV-2. The full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 may include, but is not limited to a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, and/or any combination thereof.

[0035] In certain embodiments, the methods exclude a step of isolation, concentration, and/or extraction of peripheral blood mononuclear cells (PBMCs).

[0036] In certain embodiments, methods for determining an anti-virus immune response to SARS-CoV-2 in a subject are used to analyze an antigen-specific T-cell response of a subject before and/or after vaccination. In certain embodiments, methods for determining an anti-virus immune response to SARS-CoV-2 in a subject are used for monitoring an immune status of a subject after an incident of viral infection. In certain embodiments, methods for determining an anti-virus immune response to SARS-CoV-2 in a subject are used for monitoring an immune status of a subject prior to an incident of viral infection.

BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1 shows exemplary workflows for Extracellular Staining Workflow (Figure 1A and IB), Intracellular Staining Workflow I (Figure 1C), and Intracellular Staining Workflow II (Figure ID) as discussed in Examples 1-6.

[0038] Figure 2 shows the data and information from a two blood donor samples using an embodiment of Extracellular Staining Workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and/or CD137. Figures 2A-2B shows the flow cytometry gating of the two donor samples. Figures 2C-2D shows the flow cytometry analysis of CD4+ T-cells of the two donor samples using a negative control (Neg), one activation condition for Cytomegalovirus (CMV), and one activation condition for CEFX. Figures 2E-2F shows the flow cytometry analysis of CD8+ T-cells of the two donor samples using a negative control (Neg), one activation condition for Cytomegalovirus (CMV), and one activation condition for CEFX.

[0039] Figure 3 shows the data and information from a whole blood sample and PBMC sample using an embodiment of Extracellular Staining Workflow. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and/or CD137. Figures 3A-3B shows the flow cytometry gating of the two samples. Figures 3C-3D shows the flow cytometry analysis of CD4+ T-cells of the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for Spike. Figures 3E-3F shows the flow cytometry analysis of CD8+ T-cells of the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for Spike. Figures 3G-3H shows the flow cytometry analysis of NK-cells of the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for Spike. Figures 3I-3J shows the flow cytometry analysis ofNKT-cells of the two samples using a negative control (Neg), one activation condition for CEFX, and one activation condition for Spike.

[0040] Figure 4 shows the data and information from a whole blood sample using an embodiment of Extracellular Staining Workflow generating antigen-specific T-cells to intact proteins versus peptides. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and/or CD154. Figure 4A shows the flow cytometry gating of the whole blood sample. Figure 4B shows the flow cytometry analysis of CD4+ T-cells of the whole blood sample using a negative control (Neg), one activation condition for Spike JPT, one activation condition for intact Spike (7.2 pmol concentration), one activation condition for intact Spike (14.4 pmol concentration), and one activation condition for intact Spike (28.8 pmol concentration). Figure 4C shows the flow cytometry analysis of CD8+ T-cells of the whole blood sample using a negative control (Neg), one activation condition for Spike JPT, one activation condition for intact Spike (7.2 pmol concentration), one activation condition for intact Spike (14.4 pmol concentration), and one activation condition for intact Spike (28.8 pmol concentration).

[0041] Figure 5 shows the data and information from a whole blood sample using an embodiment of Extracellular Staining Workflow generating antigen-specific T-cells to intact using antigens in either dried or liquid format. The cells were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and/or CD 154. Figure 5 A shows the flow cytometry gating of the whole blood sample. Figure 5B shows the flow cytometry analysis of CD4+ T-cells of the whole blood sample using a negative control (Neg), one activation condition for liquid Spike (Liq), one activation condition for dry Spike (Dry), one activation condition for liquid EMN (Liq) and one activation condition for dry EMN (Dry). Figure 5C shows the flow cytometry analysis of CD84+ T-cells of the whole blood sample using a negative control (Neg), one activation condition for liquid Spike (Liq), one activation condition for dry Spike (Dry), one activation condition for liquid EMN (Liq) and one activation condition for dry EMN (Dry).

[0042] Figure 6 shows the data and information from whole blood samples using an embodiment of Intracellular Staining Workflow using antigens in either dried or liquid format. The cells were stained for CD3, CD4, CD8, IFNy, CD45, CD154, IL-4, TNFa, and/or IL-17. Figure 6A shows the flow cytometry analysis of CD4+ T-cells of a sample from a patient with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for liquid antigen for P8A, one activation condition for liquid antigen for P8B, one activation condition for liquid antigen for PAD2, one activation condition for liquid antigen for PAD4, one activation condition for dry antigen for Cytomegalovirus (Dry CMV), one activation condition for dry antigen for Dry CEFX, and one activation condition for dry antigen for Dry PMA/ionomycin (PMA/Iono). Figure 6B shows the flow cytometry analysis of CD8+ T-cells of a sample from a patient with rheumatoid arthritis (RA) using a negative control (CTRL), one activation condition for liquid antigen for P8A, one activation condition for liquid antigen for P8B, one activation condition for liquid antigen for PAD2, one activation condition for liquid antigen for PAD4, one activation condition for dry antigen for Cytomegalovirus (Dry CMV), one activation condition for dry antigen for Dry CEFX, and one activation condition for dry antigen for Dry PMA/ionomycin (PMA/Iono). Figure 6C shows the flow cytometry analysis of CD4+ T-cells of a sample from a healthy patient using a negative control (CTRL), one activation condition for liquid antigen for P8A, one activation condition for liquid antigen for P8B, one activation condition for liquid antigen for PAD2, one activation condition for liquid antigen for PAD4, one activation condition for dry antigen for Cytomegalovirus (Dry CMV), one activation condition for dry antigen for Dry CEFX, and one activation condition for dry antigen for Dry PMA/ionomycin (PMA/Iono). Figure 6D shows the flow cytometry analysis of CD8+ T-cells of a sample from a healthy patient using a negative control (CTRL), one activation condition for liquid antigen for P8A, one activation condition for liquid antigen for P8B, one activation condition for liquid antigen for PAD2, one activation condition for liquid antigen for PAD4, one activation condition for dry antigen for Cytomegalovirus (Dry CMV), one activation condition for dry antigen for Dry CEFX, and one activation condition for dry antigen for Dry PMA/ionomycin (PMA/Iono). [0043] Figure 7 shows the data and information from two blood donor samples using an embodiment of Intracellular Staining Workflow II. The cells were stained for CD3, CD4, CD8, IFNy, CD 19, CD45, CD 154, IL-4, IL- 10, TNFa, and/or IL- 17. Figure 7A shows the flow cytometry analysis of CD4+ T-cells of a sample using a negative control (NEG), one activation condition for antigen for SARS-CoV-2-Spike Glycoprotein, one activation condition for antigen for CMV Global, one activation condition for antigen for CMV pp65, one activation condition for an Allergen Mix, and one activation condition for antigen for PMA/ionomycin (PMA/Iono). Figure 7B shows the flow cytometry analysis of CD8+ T-cells of a sample using a negative control (NEG), one activation condition for antigen for SARS-CoV-2-Spike Glycoprotein, one activation condition for antigen for CMV Global, one activation condition for antigen for CMV pp65, one activation condition for an Allergen Mix, and one activation condition for antigen for PMA/ionomycin (PMA/Iono). Figure 7C shows the flow cytometry analysis of CD 19+ B-cells-cells of a sample using a negative control (NEG), one activation condition for antigen for SARS-CoV-2-Spike Glycoprotein, one activation condition for antigen for CMV Global, one activation condition for antigen for CMV pp65, one activation condition for an Allergen Mix, and one activation condition for antigen for PMA/ionomycin (PMA/Iono). Figure 7D shows the flow cytometry analysis of NK-cells-cells of a sample using a negative control (NEG), one activation condition for antigen for SARS-CoV-2-Spike Glycoprotein, one activation condition for antigen for CMV Global, one activation condition for antigen for CMV pp65, one activation condition for an Allergen Mix, and one activation condition for antigen for PMA/ionomycin (PMA/Iono). Figures 7E-7H show the resulting aggregate average significant differences in marker expression across multiple whole blood samples by activator type. Figure 7E shows the significant differences for CD4+ cells, Figure 7F shows CD8+ cells, Figure 7G shows CD 19+ B-cells, and Figure 7H shows NK cells.

[0044] Figure 8 shows the data and information from three blood donor samples using an embodiment of Intracellular Staining Workflow II. The whole blood cells were stained for CD3, CD4, CD45RA, CD8, CD154, CD69, CD107a, and/or CD137 and not activated, activated with SPIKE JPT or activated with CEFX. Figure 8A shows the flow cytometry gating of the three donor samples. Figure 8B shows the flow cytometry analysis of CD4+ T-cells of the three whole blood samples using a negative control (Neg), one activation condition for SPIKE JPT, and one activation condition for CEFX. Figure 8C shows the flow cytometry analysis of CD4+ T-cells of the three whole blood samples using a negative control (Neg), one activation condition for SPIKE JPT, and one activation condition for CEFX.

DETAILED DESCRIPTION

[0045] While the concepts of the present disclosure are illustrated and described in detail in the figures and descriptions herein, results in the figures and their description are to be considered as examples and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. [0046] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure. [0047] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods described herein are readily apparent from the description of the disclosure contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the disclosure or any embodiment thereof. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the disclosure.

[0048] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are now described.

Definitions.

[0049] As used herein, “g” represents gram; “L” represents liter; “mg” represents “milligram (10-3 gram);” “mL” or “cc” represents milliliter (10-3 liter). One “pL” equals to one microliter (10-6 liter). The unit of temperature used herein is degree Celsius (°C).

[0050] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. Whether or not modified by the term “about,” the claims include equivalents to the quantities. [0051] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a method” includes having two or more methods that are either the same or different from each other. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0052] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By way of a hypothetical illustrative example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4- 5.

[0053] The term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0054] The term “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0055] The term “antigen” as used herein is defined as any substance or molecule (e.g. a polypeptide) capable of causing a specific immune response. An antigen can be or can be derived from or can be immunologically cross-reactive with an infectious pathogen, epitope, biomolecule, cell or tissue associated with an infection, cancer, autoimmune disease, allergy, or any other condition in which stimulation of an antigenspecific immune response is involved or would be desirable or beneficial. An antigen may be a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof.

[0056] The term “extracellular T-cell markers” as used herein encompasses any markers or molecules characteristic of the cytoplasmic membrane of a T-cell, or those partially or fully exposed on the outer surface of the cytoplasmic membrane and can be accessed without modulating cell permeability. Extracellular T-cell markers as used herein include those expressed in resting, activated, responding, and/or diseased cells, and include, but are not limited to CD2, CD3, CD4, CD8, CD1 la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, and/or any combination thereof.

[0057] The term “intracellular T-cell markers” as used herein encompasses any markers or molecules located inside a T-Ocell and include, but are not limited to IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGF (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, and/or any combination thereof. Intracellular T-cell markers may include cytokines and transcription factors. Intracellular T-cell markers and extracellular T-cell markers can be detected on the same cell. The detection of some intracellular T-cell markers may require disruption or other manipulation of the cell membrane integrity, for example, preparation and or/treatment of the cells with one or more permeabilizing reagents as described herein.

[0058] The term “full-length protein” as used herein encompasses any amino acid sequence that represents the complete amino acid sequence for a given nucleotide sequence. One of ordinary skill in the art will understand the term “full-length protein” as used herein. The National Institute of Health’s sequence database GenBank® may be used as a reference to identify full-length protein sequences and is available here https://www.ncbi.nlm.nih.gov/genbank/.

[0059] The term “peptide pool” as used herein encompasses combinations of mixed peptides of the same or different lengths, often from about 2 amino acids to about 50 amino acid residues. The peptides within the peptide pool may include synthetic and/or naturally occurring peptides. Peptide pools may include overlapping peptides from an antigen. Peptide pools are the standard for stimulation of antigen-specific T-cells in functional T-cell assays such as ELISpot and ICS, and may be used with the present disclosure.

[0060] The term “protein derivative” as used herein encompasses nonisomorphous derivative proteins.

[0061] The term “dry reagent” as used herein encompasses regents provided in dry rather than liquid form, and often do not require refrigeration and are more stable. [0062] The term “dry staining reagent” as used herein encompasses staining regents that allow the identification of a target, e g., a monoclonal antibody that allows identification of a T-cell based on its interaction with a specific target marker. Dry staining reagents are provided in dry rather than liquid form, and often do not require refrigeration and are more stable. An example of dry staining reagents are the DURA Innovations reagents available from Beckman Coulter (https://www.beckman.com/resources/technologies/dura-innovations).

[0063] The term “Allergen Mix” as used herein encompasses raw allergenic extract, recombinant or purified allergens, whether it is in the form of native, entire, undigested proteins or denatured, entire, denatured proteins or denatured, enzyme digested proteins or synthetic peptides of allergen proteins.

[0064] The term “spike” as used herein encompasses purified or recombinant SARS- CoV-2 protein or synthetic peptides, either as individual peptides or pools of several peptides.

[0065] The term “spike JPT” as used herein encompasses a synthetic peptide pool of the entire or specific domains of the SARS-CoV-2 protein. An example of spike JPT is sold by JPT Technologies (JPT Peptide Technologies GmbH, Volmerstrasse, 12489 Berlin, Germany.)

[0066] The term “CEFX” as used herein encompasses a mixture of 176 synthetic peptides epitopes from a diverse viral and bacterial organisms. An example of CEFX is sold by JPT Technologies.

General Description

[0067] The present disclosure overcomes the deficiencies of classical approaches to analyzing antigen-specific T-cell responses by replacing the use of isolated, concentrated, and/or extracted PBMCs with whole blood and intact antigens rather than pre-processed peptides. In an embodiment, the methods for determining antigenspecific T-cell responses in a subject avoids the isolation, concentration, and/or extraction of PBMC by contacting a whole blood sample from a subject with at least one antigen to form a mixture. Contacting the whole blood sample with at least one staining reagent, e.g., a dry staining reagent contained within a reagent vessel, wherein the dry staining reagent comprises monoclonal antibody reagents that allow identification of T-cell markers. Incubating the mixture, obtaining a cell concentrate from the mixture, and then analyzing the mixture to identify a T-cell response to the antigen. In embodiments, the analyzing is by flow cytometry.

[0068] Methods and kits of the present disclosure can be applied in a variety of fields, including determining antigen-specific T-cell responses to infectious diseases, autoimmune disorders, cancer, allergies, and aging. In addition, methods and kits of the present disclosure can be used to determine the need for treatment, e.g., a vaccine, a subject’s response to treatment, and/or the necessity for, or lack thereof, additional treatment.

[0069] Some embodiments of the methods of the present disclosure overcome limitations of prior methods through the simplified use of intact or whole proteins as antigens. Manufacturing of synthetic peptides is labor intensive and can be a challenge to be exacting, in particular if the intent is to cover a full protein sequence. Further, the use of peptide pools may bias a T-cell response toward a non-naturally responding set of T-cells; for example, short peptides could bias a response toward CD8+ T-cells. Use of non-modified, intact or larger proteins could include hundreds of 8- to 12-mer overlapping peptides for a single antigen. These methods can also open the possibility to test for antigen-specific T-cell responses in a subject sample mixed with a potentially antigenic mixture of proteins, without any a priori toward one or another protein.

[0070] Disclosed herein are streamlined methods and kits that utilize whole blood, rather than isolated, extracted, or concentrated PBMCs, for determining antigenspecific T-cell responses in a subject, and are based on the use of extracellular activation markers and/or intracellular activation markers. This was developed and further compared to more conventional techniques such as ELISpot and flow cytometry-based ICS. The methods of the present disclosure rely on the direct detection of rare antigen-specific lymphocytes by identifying membrane markers of T-cell activation on whole blood samples, referred to as the Extracellular Staining Protocol or by identifying intracellular markers of T-cell activation on whole blood samples Intracellular Staining Protocol I and Intracellular Staining Protocol II. These simple, streamlined methods were developed that may be used in a wide variety of settings, including clinical settings. The simple streamlined methods are capable of individual stratification capabilities, e.g., using methods of the present disclosure, a combination of extracellular markers (CD154, CD137 and CD107a) exhibited a higher orthogonality than cytokines (IFN-y, TNF-a and IL-2) commonly considered in ICS.

[0071] The present disclosure provides methods and kits for determining antigenspecific T-cell responses in a subject, the method contacting a whole blood sample from the subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture for T-cells responding to the antigen contact.

[0072] In embodiments, the subject is a mammal, a domesticated pet (e.g., a dog or a cat), or a human. In certain embodiments, the subject is being screened for antigenspecific T-cell responses to determine the presence of cancer, allergies, autoimmunity , bacterial infections, viral infections, and/or parasitic infections. Methods of the present disclosure may also be used to identify antigen-specific T-cell responses to determine the effectiveness of treatment for cancer, allergies, autoimmunity, bacterial infections, viral infections, and/or parasitic infections. Methods of the present disclosure may also be used to identify antigen-specific T-cell responses to determine immunity to and/or from cancer, allergies, bacterial infections, viral infections, and/or parasitic infections. Methods of the present disclosure may also be used to identify antigen-specific T-cell responses to determine a subject’s recovery from treatment for cancer, allergies, bacterial infections, viral infections, and/or parasitic infections.

[0073] In certain embodiments, methods for determining antigen-specific T-cell responses in a subject are used to analyze an antigen-specific T-cell response of a subject before treatment and/or vaccination. Methods of the present disclosure may also be used for determining antigen-specific T-cell responses in a subject are used to analyze an antigen-specific T-cell response of a subject after treatment and/or vaccination. Methods of the present disclosure may also be used for determining antigen-specific T-cell responses in a subject are used for monitoring an immune status of a subject after an incident of infection, including a viral infection or a bacterial infection. Methods of the present disclosure may also be used for determining antigenspecific T-cell responses in a subject are used for monitoring an immune status of a subject prior to an incident of an infection, including a bacterial infection or a viral infection.

[0074] In certain embodiments, methods of the present disclosure are used for determining an anti-virus immune response to SARS-CoV-2 in a subject prior to infection, to determine the presence of infection, and/or after infection. Methods of the present disclosure may also be used for determining an anti-virus immune response to SARS-CoV-2 in a subject prior to vaccination, to determine the presence of vaccination, after vaccination, or some combination thereof. [0075] In certain embodiments, methods of the present disclosure may be used for determining an anti -tumor immune response in a subject prior to and after telomerasebased vaccination, to determine the presence of antigen-specific T-cells.

[0076] In certain embodiments, methods of the present disclosure may be used for determining an allergen-specific immune response in a subject prior to, during, and after receiving allergen immunotherapy.

[0077] In embodiments, the whole blood sample is about 10 pL to about 10 mL. In certain embodiments, the whole blood sample is about 10 pL to about 2 mL, about 10 pL to about 1 mL, 10 pL to about 750 pL, about 50 pL to about 500 pL, or about 100 pL to about 400 pL. In certain embodiments, the whole blood sample is greater than zero less than about 2 mL, less than about 1 mL, less than about 750 pL, less than about 500 pL, less than about 300 pL, less than about 250 pL, less than about 100 pL, or less than about 50 pL. In certain embodiments, the whole blood sample is greater than about 500 pL, greater than about 1 mL, greater than about 2 mL, greater than about 3 mL, or greater than about 4 mL.

[0078] In certain embodiments, methods for determining antigen-specific T-cell responses comprise at least one antigen. In an embodiment, the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof. The peptide pools may include peptides derived from CEFX or CMV or any other commercially available pool. The antigens, including whole proteins, peptides, peptide pools, and protein derivatives may be commercially available, may be naturally occurring, may be synthetically engineered, or some combination thereof. In an embodiment, the at least one antigen comprises an intact protein, as opposed to a processed protein. In an embodiment, the at least one antigen comprises a single epitope. In an embodiment, the at least one antigen comprises a partial proteome. In an embodiment, the at least one antigen comprises a full proteome containing hundreds of intact proteins. In such an embodiment, the at least one antigen may comprise a raw extract, for example, of an allergen. In certain embodiments, the at least one antigen is an allergen extract comprising a homogenous mixture of polypeptides and other biomolecules. [0079] The number of antigens added to a mixture of whole blood is not limited by the parameters of the disclosed methods and kit. In an embodiment, the number of antigen(s)in a single tube, well, sample or mixture is between 1 and 20, is between 1 and 15, is between 1 and 10, is between 3 and 8. In an embodiment, the number of antigen(s) is greater than 2, is greater than 3, is greater than 5, is greater than 7, is greater than 9, or is greater than 15. In an embodiment, the number of antigens is at least 1 but is less than 20, is less than 15, is less than 10, or is less than 5.

[0080] In certain embodiments, at least one reagent is added to the mixture formed by contacting the whole blood sample with at least one antigen. The at least one reagent can include one or more reagents commonly used for flow cytometry, cellular sample preparation, ELISpot or ICS, and know n to one of ordinary skill in the art. For example, the at least one reagent may include phosphate buffered saline, water, acids, bases, pH adjusting reagents, modulators, therapeutic agents, fixatives, pH stabilizing reagents, lysis buffers, wash buffers, and/or some combination thereof. The at least one reagent may include commercially available reagents, e.g., DURAclone flow cytometryreagents (Beckman Coulter) or Optilyse C (Beckman Coulter). The potential reagents may include, but are not limited to permeabilization, fixative, and/or lysis reagents commonly used for sample preparation for flow cytometry .

[0081] In certain embodiments, analyzing the mixture by flow cytometry includes identifying a T-cell response to contacting the whole blood sample with at least one antigen. The analyzing the mixture by flow⁷ cytometry can include one or more techniques, assays, algorithms, formulas, programs, or some combination thereof that are well known to one of ordinary skill in the art. For example, analyzing the mixture by flow cytometry may include using Kaluza Analysis Software version 2.1 (available from Beckman Coulter). The analyzing the mixture by flow cytometry may include, but is not limited to analyzing the presence or absence of markers of different compartments of cells to deliver maximum stratification and exhaustively assess the one or more phenotypes present in the blood sample. This may include compartments delineated as responding or non-responding cells, immature or mature cells, memory cells, or more specific phenotypes such as CD4, Thl, Th2, Th9, Thl7, Th22, Treg, NKT, CD8, Tel, Tc2, Tc9, TEFF, TCM, TSCM, TRM, TEM, yoT cells, and/or the presence of intracellular cytokines and transcription factors. [0082] In certain embodiments, the present disclosure further comprises a step of incubating the mixture formed by contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining regent and/or after contacting the whole blood sample with at least one antigen before contacting the whole blood with at least one staining regent. There may be no incubation steps, a single incubation step, at least two incubation steps, or more than three incubation steps. The incubation(s) step may comprise an agitation or mixing of the mixture via shaking, rocking, stirring, vortexing, and/or some combination thereof.

[0083] In certain embodiments, staining reagents (e.g., an antibodies) are added to a mixture before addition of an antigen, after addition of antigen, concurrent with the addition of an antigen, or any combination thereof. For example, in certain embodiments at least one staining reagent is added both before and after antigen stimulation.

[0084] In certain embodiments, the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours. In embodiments, an incubation of a mixture may be at room temperature, at about 37°C, at about 4°C, or some combination thereof. In certain embodiments, the incubation of a mixture may be for about 30 seconds to about 24 hours, for about 5 minutes to about 24 hours, for about 10 minutes to about 12 hours, or for about 10 minutes to about 30 minutes. In certain embodiments, the incubation will be greater than 0 seconds but less than about 24 hours, less than about 16 hours, less than about 12 hours, less than about 6 hours, less than about 2 hours, less than about 1 hour, less than about 30 minutes, less than about 15 minutes, or less than about 10 minutes. In certain embodiments, the incubation will include greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 1 hour, greater than about 12 hours, or greater than about 16 hours.

[0085] The methods of the present disclosure may also include obtaining a cell concentrate from the mixture after the step of incubating the mixture, wherein the step of obtaining a cell concentrate from the mixture comprises one or more of the following steps: adding a fixation reagent to the mixture, adding a lysing reagent to the mixture, adding a permeabilization reagent to the mixture, staining the mixture with a staining reagent, and washing and concentrating the mixture to obtain the cell concentrate. The methods of the present disclosure may include one or more of these steps in any order. Example lysing reagents include, but are not limited to OptiLyse (Beckman Coulter) and VersaLyse (Beckman Coulter). Example permeabilization and fixation reagents reagents include, but are not limited to, Intraprep Permeabilization Reagent (Beckman Coulter) and PerFix reagents (Beckman Coulter). Example staining reagents include, but are not limited to, monoclonal antibody reagents that allow identification of extracellular and/or intracellular T-cell markers. For example, conjugated antibodies from Beckman Coulter against CD3 (APC-Alexa750, clone UCHT1), CD4 (APC, clone 13B8.2), CD8 (Alexa700, clone B9. l l), CD154 (PE, clone TRAP-1), and CD69 (FITC, clone FN50) and intracellular cytokines with conjugated antibodies from BioLegend against TNF-a (PC7, clone Mabl l), IL-2 (BV605, clone MQ1-17H12), and IFN-y (BV650, clone 4S.B3).

[0086] In certain embodiments, the staining reagent is a dry staining reagent and may be provided in at least one pre-filled reagent vessel. The pre-filled reagent vessel may include single well plates, multi-well plates (e.g., 96 well plates), a test tube, or an Eppendorf tube. The dry staining reagent may be loose or affixed to the bottom of the reagent vessel, the side of the reagent vessel, or distributed throughout the reagent vessel. In embodiments, the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of extracellular and/or intracellular T-cell markers, including those monoclonal antibody reagents identified herein.

[0087] Some examples of extracellular T-cell markers include, but are not limited to CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, and/or any combination thereof.. Some examples of the intracellular T-cell markers include, but are not limited to IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGF|3 (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, and/or any combination thereof. [0088] In certain embodiments, the at least one antigen comprises at least one virusspecific antigen. The at least one virus-specific antigen may include, but is not limited to a full-length protein, protein derivative, peptide, or peptide pool derived from SARS- CoV-2, SARS-CoV, MERS, Cytomegalovirus (CMV), Respiratory Syncytial virus (RSV), Epstein-Barr vims (EBV), influenza (H1N1), Monkey Pox (MP), or Human Papilloma virus (HPV). The full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV -2 may include, but is not limited to, a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, and/or any combination thereof.

[0089] In certain embodiments, the at least one antigen may include at least one bacteria-specific antigen, at least one parasite-specific antigen, and/or at least one allergen-specific antigen.

[0090] In certain embodiments, the methods exclude a step of isolation, concentration, and/or extraction of PBMCs.

[0091] In one aspect, the present disclosure provides a method of determining an antivirus immune response to SARS-CoV -2 in a subject, the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL.

[0092] In one aspect, the present disclosure provides a method of determining a T-cell response to CPI in a subject, the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL. [0093] In one aspect, the present disclosure provides a method of determining a specific T-cell response in a subject receiving telomerase-based vaccination as part of a tumor therapy, the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL.

[0094] In one aspect, the present disclosure provides a method of determining an allergen-specific T-cell response to in a subject receiving allergen immunotherapy (AIT), the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL.

[0095] In one aspect, the present disclosure provides a method of determining a T-cell response in a subject with autoimmunity against Peptidyl Arginyl Deiminases (PAD), the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL.

[0096] In one aspect, the present disclosure provides a method of determining a T-cell response to CMV in a subject, the method comprising contacting a whole blood sample from a subject with at least one antigen to form a mixture, contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before, concurrent with, or after contacting the whole blood sample with at least one antigen to form a mixture, and analyzing the mixture. In some embodiments, the mixture is analyzed by flow cytometry. In embodiments, the whole blood sample is about 10 pL to about 10 mL.

EXAMPLES

[0097] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some expenmental errors and deviations should be accounted for. Unless indicated otherwise, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0098] Figure 1 depicts the workflows required for each of the assays considered herein. While both the IFN-y ELISpot and ICS assays require the initial preparation of PBMCs, the assays disclosed herein rely on the direct incubation of whole blood and antigenic compositions(s). Working with whole blood instead of isolated, concentrated, or extracted PBMCs provides a multitude of benefits, including a decrease in the necessary volume of blood samples, a decrease in the technical time required to complete the assay, a decrease in the difficulty of the assay, and an increase in the robustness of the assay.

Example 1

Extracellular Workflow Protocol

[0099] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T-cell activation on whole blood samples.

[00100] In this example (using the method as depicted in Figure 1A) for staining extracellular T-cell markers, 250 mL of whole blood was incubated for 16-20 hours at 37°C with antigens (CEFX or CMV) and antibodies specific for CD107a and CD1 4. At the end of the incubation, the samples were stained for 30 minutes at RT (room temperature) with the remaining antibodies. Next, 1.25 mL Optilyse C was added and incubated for 10 minutes. Next, samples were centrifuged at 300g, supernatant was removed, and the pellet was reconstituted in 250 mL of PBS for analysis by flow cytometry. [00101] As depicted in Figures 2A-2F, the whole blood samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD 154, CD69, CD 107a, and/or CD 137 and activated with CMV or CEFX. Figures 2A-2B shows the flow cytometry gating of the two donor samples. Figures 2B-2F show the resulting flow cy tometry analysis of T-cells according to the disclosed method. The analysis of the flow cy tometry data was performed using Kaluza Analysis Software version 2. 1 (Beckman Coulter). The populations of interest were manually gated.

Example 2

Extracellular Workflow Protocol: Whole Blood v. PBMC

[00102] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T-cell activation on whole blood samples.

[00103] In this example (using the method as depicted in Figures 1 A and IB) for staining extracellular T-cell markers, 250 mL of whole blood or isolated PBMC were incubated for 16-20 hours at 37°C with antigens (CEFX or Spike) and antibodies specific for CD 107a and CD 154. At the end of the incubation, a 30 minute staining step at RT with the remaining antibodies was performed. Next, 1.25 mL Optilyse C was added, incubated for 10 minutes. Next, samples were centrifuged at

300g, supernatant was removed, and the pellet was reconstituted in 250 mL of PBS for analysis by flow cytometry.

[00104] As depicted in Figures 3A-3J, the whole blood or PBMC samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD154, CD69, CD107a, and/or CD137 and activated with CMV or CEFX. Figures 3A-3B shows the flow cytometry gating of the samples. Figures 3B-3J show the resulting flow cytometry analysis of T-cells, NK cells, and NKT cells according to the disclosed method. The analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). The populations of interest were manually gated.

Example 3

Extracellular Workflow Protocol: Intact versus peptide of Spike protein at varying concentrations

[00105] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T-cell activation on whole blood samples responding to intact proteins versus peptides. [00106] In this example (using the method as depicted in Figures 1 A and IB) for staining extracellular T-cell markers, 250 mL of whole blood was incubated for 16- 20 hours at 37°C with antigens (Spike JPT, or intact Spike at concentrations from 7.2 pmol-28.8 pmol) and antibodies specific for CD107a and CD154. At the end of the incubation, a 30 minute staining step at RT with the remaining antibodies was performed. Next, 1.25 mL Optilyse C was added, incubated for 10 minutes. Next, samples were centrifuged at 300g, supernatant was removed, and the pellet was reconstituted in 250 mL of PBS for analysis by flow cytometry.

[00107] As depicted in Figure 4A-4C, the whole blood samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and/or CD 154 and activated with Spike JPT, or varying concentrations of intact Spike. Figure 4A shows the flow cytometry gating of the samples. Figures 4B-4C show the resulting flow cytometry analysis of T-cells according to the disclosed method. The analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2. 1 (Beckman Coulter). The populations of interest were manually gated.

Example 4

Extracellular Workflow Protocol: Liquid versus Dry Antigen

[00108] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T-cell activation on whole blood samples responding to intact proteins versus peptides. [00109] In this example (using the method as depicted in Figures 1 A and IB) for staining extracellular T-cell markers, 250 mL of whole blood or isolated PBMC were incubated for 16-20 hours at 37°C with antigens (Spike JPT, or intact Spike at concentrations from 7.2 pmol-28.8 pmol) and antibodies specific for CD107a and CD154. At the end of the incubation, a 30 minute staining step at RT with the remaining antibodies was performed. Next, 1.25 mL Optilyse C was added, incubated for 10 minutes. Next, samples were centrifuged at 300g, supernatant was removed and the pellet was reconstituted in 250 mL of PBS for analysis by flow cytometry.

[00110] As depicted in Figure 5A-5C, the whole blood samples were stained for CD3, CD4, CD45, CD45RA, CD8, CD56, CCR7, CD154, CD69, CD107a, CD137, and/or CD 154 and activated with liquid or dry Spike or EMN. Figure 5 A shows the flow cytometry gating of the samples. Figures 5B-5C show the resulting flow cytometry analysis of T-cells according to the disclosed method. The analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). The populations of interest were manually gated.

Example 5

Intracellular Workflow I Protocol: Liquid versus Dry Antigen

[00111] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound and intracellular markers of T-cell activation on whole blood samples using liquid versus dry antigens.

[00112] In this example (using the method as depicted in Figure 1C), for staining intracellular T-cell markers, 250 uL of whole blood was incubated with brefeldin (dried or liquid) and an antigen (dried or liquid) for 5 hours. Next, each sample received 1.25 mL of Intraprep Permeabilization Reagent 1 (Rl) (Beckman Coulter) followed by 15 minutes of incubation. Next, each sample received 1.5 mL of Intraprep Permeabilization Reagent 2 (R2) followed by 10 minutes of incubation. The samples were then centrifuged at 500g for 5 minutes and the supernatant was removed. Additional reconstituted staining markers were added (if dried, they were first reconstituted in 1.5 mL) on the cell pellet, followed by 45 minutes of incubation. Next, 3 mL of IntraPrep Permeabilization Reagent 3 (R3) at IX was added to the stained cells and the samples were centrifuged at 500g for 5 min and the supernatant was removed. Then, 250 mL of R3 at IX was added to reconstitute cell pellet. The samples were analyzed by flow cytometry.

[00113] As depicted in Figures 6A-6D, the whole blood samples were stained for CD3, CD4, CD8, IFNy, CD45, CD154, IL-4, TNFa, and/or IL-17 and activated with liquid or dry antigens (liquid: P8A, P8B, PAD2, PALM; dry: CMV, CEFX, PMA/Iono). Figure 6A shows the flow cytometry gating of the samples. Figures 6B-6D show the resulting flow cytometry analysis of T-cells according to the disclosed method. The analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2. 1 (Beckman Coulter). The populations of interest were manually gate.

Example 6

Intracellular Workflow II Protocol

[00114] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound and intracellular markers of T-cell activation on whole blood samples.

[00115] In this example, (using the method as depicted in Figure ID) for staining intracellular T-cell markers, 250 pL of whole blood was incubated wit brefeldin (dried or liquid) and an antigen (dried or liquid) for 3-5 hours. Next, extracellular staining was performed. Then each sample was incubated with 400 pL OptiC Lysis solution for 10 min, followed by 1.25 mL of Intraprep Permeabilization Reagent 1 (Rl) (Beckman Coulter) followed by 30 additional minutes of incubation. Next, each sample was washed then received 1.5 mL of Intraprep Permeabilization Reagent 2 (R2) followed by 10 minutes of incubation. Additional reconstituted staining markers were added (if dried, they were first reconstituted in 1.5 mL) on the cell pellet, followed by 45 minutes of incubation. Next, 3 mL of IntraPrep Permeabilization Reagent 3 (R3) at IX was added to the stained cells and the samples were centrifuged at 500g for 5 mm and the supernatant was removed. Then, 250 mL of R3 at IX was added to reconstitute cell pellet. The samples were analyzed by flow cytometry.

[00116] As depicted in Figures 7A-7D, the whole blood samples were stained for CD3, CD4, CD8, IFNy, CD19, CD45, CD154, IL-4, IL-10, TNFa, and/or IL-17 and activated with SARS-CoV-2-Spike Glycoprotein, CMV Global, CMV pp65, Allergen Mix, or PMA/ionomycin. Figures 7A-7D show the resulting flow cytometry analysis of T, B, and NK-cells according to the disclosed method. Figures 7E-7H show the resulting average significant differences in marker expression across multiple whole blood samples. The analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2. 1 (Beckman Coulter). The populations of interest were manually gated.

Example 7

Varying Whole Blood Volumes

[00117] The methods of the present disclosure allow the direct detection of rare antigen-specific lymphocytes by identifying membrane-bound markers of T-cell activation on whole blood samples of varying volumes. In this example (using the method as depicted in Figure IB) for staining extracellular T-cell markers, 250 pL, 500 pL, or 1000 pL of whole blood from three different patients was incubated for 20 hours at 37°C with antigens (SPIKE JPT or CEFX, no antigen as negative control) and antibodies specific for CD107a and CD154. At the end of the incubation, the samples were stained for 20 minutes at RT (room temperature) with the remaining antibodies (CD8, CD3, CD137, CCR7, CD69, CD56, and CD45RA). Next, Optilyse C (1.5 mL, 3 mL, or 6 mL-depending on original whole blood volume) was added and incubated for 10 minutes at RT. Next, samples were centrifuged at 250g, supernatant was removed, and the pellet was reconstituted in PBS (1.5 mL, 3 mL, or 6 mL-depending on original whole blood volume) for analysis by flow cytometry.

[00118] As depicted in Figure 8A-8C, the whole blood samples were stained for CD3, CD4, CD45RA, CD8, CD154, CD69, CD107a, and/or CD137 and not activated, activated with SPIKE JPT or activated with CEFX. Figure 8A shows the flow cytometry gating of the three donor samples (500 pl whole blood sample is shown in Figure 8A, but no significant different was noticed between the 250 pL, 500 pL, or 1000 pL of whole blood). Figures 8B-8C show the resulting flow cytometry analysis of T-cells according to the disclosed method of CD4+ T-cells from the three whole blood samples (Figure 8B) and CD8+ T-cells of the three whole blood sample (Figure 8C) (500 pl whole blood sample is shown in Figure 8A-8C, but no significant different was noticed between the 250 pL, 500 pL, or 1000 pL of whole blood). The analysis of the flow cytometry data was performed using Kaluza Analysis Software version 2.1 (Beckman Coulter). The populations of interest were manually gated.

[00119] The following numbered clauses define further example aspects and features of the present disclosure:

1. A method for determining antigen-specific T-cell responses in a subj ect, the method comprising: a) contacting a whole blood sample from the subj ect with at least one antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before step (a), concurrent with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T-cells responding to the antigen contact.

2. The method of clause 1, wherein the at least one antigen is an activator of T- cells. 3. The method of any one of clauses 1-2, wherein the mixture is analyzed by flow cytometry.

4. The method of any one of clauses 1-3, wherein the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof.

5. The method of any one of clauses 1-4, further comprising adding at least one reagent to the mixture of step (b).

6. The method of any one of clauses 1-5, further comprising a step of incubating the mixture after step (a), before step (b), after step (b), or any combination thereof.

7. The method of any one of clauses 1-6, further comprising obtaining a cell concentrate from the mixture after the step of incubating the mixture.

8. The method of any one of clauses 1-7, wherein the step of obtaining a cell concentrate from the mixture comprises: a) adding a lysing reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate.

9. The method of any one of clauses 1-8, wherein the step of obtaining a cell concentrate from the mixture further comprises: a) adding a fixation reagent to the mixture.

10. The method of any one of clauses 1-9, wherein the at least one staining reagent and/or the at least one antigen is a dry reagent. 11. The method of any one of clauses 1-10, wherein the dry reagent is provided in at least one pre-filled reagent vessel.

12. The method of any one of clauses 1-11, wherein the dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of extracellular T-cell markers.

13. The method of any one of clauses 1-12, wherein the staining reagent comprises monoclonal antibody reagents for identification of extracellular T-cell markers include CD2, CD3, CD4, CD8, GDI la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, or any combination thereof.

14. The method of any one of clauses 1-13, wherein the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of intracellular T-cell markers.

15. The method of any one of clauses 1-14, wherein the staining reagent comprises monoclonal antibody reagents for identification of intracellular T-cell markers and include IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-Y, TGF (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, or any combination thereof.

16. The method of any one of clauses 1-15, wherein the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours. 17. The method of any one of clauses 1-16, wherein the whole blood sample amount is from about 10 pL to about 10 mL.

18. The method of any one of clauses 1-17, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of activation markers of CD4+ T-cells, and/or CD8+ T-cells, and/or the presence of intracellular cytokines and/or transcription factors.

19. The method of any one of clauses 1-18, wherein the activation markers of CD4+ T-cells comprise CD69, CD154, CD137 and CD107a and wherein the activation markers of CD8+ T-cells comprise CD69, CD154, CD137 and CD107a.

20. The method of any one of clauses 1-19, wherein the at least one antigen comprises at least one virus-specific antigen.

21. The method of any one of clauses 1-20, wherein the at least one virus-specific antigen is a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, Cytomegalovirus (CMV), Respiratory' Syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), Monkey Pox (MP), or Human Papilloma virus (HPV).

22. The method of any one of clauses 1-21, wherein the full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprises a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof.

23. The method of any one of clauses 1-22, wherein the at least one antigen comprises at least one parasite-specific antigen, at least one allergen-specific antigen, or at least one bacteria-specific antigen. 24. The method of any one of clauses 1-23, wherein the method excludes a step of isolation, concentration, and/or extraction of peripheral blood mononuclear cells (PBMCs).

25. Use of the method of any one of clauses 1-24 to analyze antigen-specific T-cell response of a subject before and/or after vaccination.

26. Use of the method of any one of clauses 1-24 for monitoring an immune status of a subject prior to or after an incident of viral infection.

27. A method for determining antigen-specific T-cell responses in a subject, the method comprising: a) contacting a whole blood sample from the subj ect with at least one antigen to form a mixture; b) contacting the mixture with a staining reagent for CD 107a and a staining reagent for CD154; c) incubating the mixture; d) contacting the mixture with at least one additional staining reagent; e) adding a lysing reagent to the mixture; f) washing and concentrating the mixture to obtain the cell concentrate; and g) analyzing the mixture to identify T-cells responding to the antigen contact.

28. A method of determining an anti-virus immune response to SARS-CoV-2 in a subject, the method comprising: a) contacting a whole blood sample from the subj ect with at least one SARS-CoV-2 antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before step (a), concurrent with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T-cells responding to the SARS-CoV-2 antigen contact.

29. The method of any one of clauses 27-28, wherein the mixture is analyzed by flow cytometry.

30. The method of any one of clauses 27-29, further comprising adding at least one reagent to the mixture of step (b).

31. The method of any one of clauses 27-30, further comprising a step of incubating the mixture after step (a), before step (b), after step (b), or any combination thereof.

32. The method of any one of clauses 27-31, further comprising obtaining a cell concentrate from the mixture after the step of incubating the mixture.

33. The method of any one of clauses 27-32, wherein the step of obtaining a cell concentrate from the mixture comprises: a) adding a lysing reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate.

34. The method of any one of clauses 27-33, wherein the step of obtaining a cell concentrate from the mixture further comprises: a) adding a fixation reagent to the mixture.

35. The method of any one of clauses 27-34, wherein the at least one staining reagent and/or the at least one antigen is a dry reagent. 36. The method of any one of clauses 27-35, wherein the dry reagent is provided in at least one pre-filled reagent vessel.

37. The method of any one of clauses 27-36, wherein the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of extracellular T-cell markers.

38. The method of any one of clauses 27-37, wherein the extracellular T-cell markers include CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, and/or any combination thereof.

39. The method of any one of clauses 27-38, wherein the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of intracellular T-cell markers.

40. The method of any one of clauses 27-39, wherein the staining reagent comprises monoclonal antibody reagents for identification of intracellular T-cell markers and include IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-Y, TGF (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, or any combination thereof.

41. The method of any one of clauses 27-40, wherein the step of incubating the mixture comprises incubation at about 37°C for about 30 minutes to about 24 hours.

42. The method of any one of clauses 27-41, wherein the whole blood sample amount is from about 10 pL to about 10 mL. 43. The method of any one of clauses 27-42, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of activation markers of CD4+ T-cells, and/or CD8+ T-cells, and/or the presence of intracellular cytokines and/or transcription factors.

44. The method of any one of clauses 27-43, wherein the activation markers of CD4+ T-cells comprise CD69, CD154, CD137 and CD107a and wherein the activation markers of CD8+ T-cells comprise CD69, CD154, CD137 and CD107a.

45. The method of any one of clauses 27-44, wherein the at least one antigen is a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, derived from SARS-CoV-2.

46. The method of any one of clauses 27-45, wherein the full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprises a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof.

47. The method of any one of clauses 27-46, wherein the method excludes a step of isolation, concentration, and/or extraction of peripheral blood mononuclear cells (PBMCs).

48. Use of the method of any one of clauses 27-47 to analyze antigen-specific T- cell response of a subject before and/or after vaccination.

49. Use of the method of any one of clauses 27-47 for monitoring an immune status of a subject after an incident of viral infection.

50. The use of method of any one of clauses 27 -47 for monitoring an immune status of a subject prior to an incident of viral infection. 51. A kit for analyzing an antigen-specific T-cell response in a sample comprising: at least one antigen; and at least one staining reagent.

52. The kit of clause 51 further comprising: at least one pre-filled reagent vessel.

53. The kit of any one of clauses 51-52, wherein the at least one antigen or the at least one staining reagent is a dry reagent.

54. The kit of any one of clauses 51-53, wherein the at least one dry reagent is provided in at least one pre-filled reagent vessel.

55. The kit of any one of clauses 51-54, wherein the at least one antigen is an activator of T-cells.

56. The kit of any one of clauses 51-55, wherein the at least one antigen comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof.

57. The kit of any one of clauses 51-56 further comprising: a) a lysing reagent; b) a permeabilization; and c) a fixation reagent.

58. The kit of any one of clauses 51-57, wherein the dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of extracellular T-cell markers. 59. The kit of any one of clauses 51-58, wherein the staining reagent comprises monoclonal antibody reagents for identification of extracellular T-cell markers include CD2, CD3, CD4, CD8, CDlla, CD25 (IL2RA), CD27, CD28, CD31 (PECAM1), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CCR10, CD197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, or any combination thereof.

60. The kit of any one of clauses 51-59, wherein the at least one dry reagent contained within a pre-filled dry reagent vessel comprises monoclonal antibody reagents that allow identification of intracellular T-cell markers.

61. The kit of any one of clauses 51-60, wherein the staining reagent comprises monoclonal antibody reagents for identification of intracellular T-cell markers and include IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGFP (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, or any combination thereof.

62. The kit of any one of clauses 51-61, wherein the at least one antigen comprises at least one virus-specific antigen.

63. The kit of any one of clauses 51-62, wherein the at least one virus-specific antigen is a full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS-CoV, MERS, Cytomegalovirus (CMV), Respiratory Syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), Monkey Pox (MP), or Human Papilloma virus (HPV).

64. The kit of any one of clauses 51 -63, wherein the full-length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2 comprises a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof.

65. The kit any one of clauses 51-64, wherein the at least one antigen comprises at least one parasite-specific antigen, at least one allergen-specific antigen, or at least one bacteria-specific antigen.

66. Use of the kit of any one of clauses 51-65 to analyze antigen-specific T-cell response of a subject before and/or after vaccination.

67. Use of the kit of any one of clauses 51-65 for monitoring an immune status of a subject prior to or after an incident of viral infection.

68. A method for determining antigen-specific T-cell responses in a subject, the method comprising: a) contacting a whole blood sample from the subj ect with at least one antigen to form a mixture, wherein the whole blood sample amount is from about 10 pL to about 10 mL, wherein the at least one antigen is an activator of T-cells and comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof; b) contacting the whole blood sample with at least one staining reagent, wherein the staining reagent comprises monoclonal antibody reagents for identification of extracellular T-cell markers include CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PEC AMI), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD 194 (CCR4), CD 195 (CCR5), CD 196 (CCR6), CCR10, CD 197 (CCR7), CD200, CD279 (PD-1), CD294

(PTGDR2 or CRTH2), HLA-DR, 0X40, or any combination thereof, wherein the contacting comprises incubation for about 30 minutes to about 24 hours, wherein the contacting the whole blood sample with at least one staining reagent occurs before step (a), concurrent with step (a), after step (a), or any combination thereof; and c) analyzing the mixture by flow cytometry to identify T-cells responding to the antigen contact.

69. The method of clause 68, further comprising adding at least one reagent to the mixture of step (b).

70. The method of any one of clauses 68-69, further comprising a step of incubating the mixture after step (a), before step (b), after step (b), or any combination thereof.

71. The method of any one of clauses 68-70, further comprising obtaining a cell concentrate from the mixture after the step of incubating the mixture.

72. The method of any one of clauses 68-71, wherein the step of obtaining a cell concentrate from the mixture comprises: a) adding a lysing reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate.

73. The method of any one of clauses 68-72, wherein the step of obtaining a cell concentrate from the mixture further comprises adding a fixation reagent to the mixture.

74. The method of any one of clauses 68-73, wherein the at least one staining reagent and/or the at least one antigen is a dry reagent. 75. The method of any one of clauses 68-74, wherein the dry reagent is provided in at least one pre- fdled reagent vessel.

76. The method of any one of clauses 68-75, wherein the staining reagent comprises monoclonal antibody reagents for identification of intracellular T-cell markers and include IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGF (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, or any combination thereof.

77. The method of any one of clauses 68-76, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of activation markers of CD4+ T-cells, and/or CD8+ T cells, wherein the activation markers of CD4+ T-cells comprise CD69, CD 154, CD 137 and CD 107a and wherein the activation markers of CD8+ T- cells comprise CD69, CD154, CD137 and CD107a.

78. The method of any one of clauses 68-77, wherein the at least one antigen comprises at least one virus-specific antigen, wherein the at least one virus-specific antigen is a full- length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS- CoV, MERS, Cytomegalovirus (CMV), Respiratory Syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), Monkey Pox (MP), or Human Papilloma virus (HPV), a spike protein (S), subunit S 1 of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof.

79. The method of any one of clauses 68-78, wherein the method excludes a step of isolation, concentration, and/or extraction of peripheral blood mononuclear cells (PBMCs).

80. Use of the method of any one of clauses 68-79 for monitoring an immune status of a subject prior to or after an incident of viral infection. 81. A method for determining antigen-specific T-cell responses in a subject, the method comprising: a) contacting a whole blood sample from the subj ect with at least one antigen to form a mixture; b) contacting the mixture with a staining reagent for CD 107a and a staining reagent for CD154; c) incubating the mixture; d) contacting the mixture with at least one additional staining reagent; e) adding a lysing reagent to the mixture;

1) washing and concentrating the mixture to obtain the cell concentrate; and g) analyzing the mixture to identify T-cells responding to the antigen contact.

82. A method of determining an anti-virus immune response to SARS-CoV-2 in a subject, the method comprising: a) contacting a whole blood sample from the subject with at least one SARS-CoV-2 antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before step (a), concurrent with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T-cells responding to the SARS-CoV- 2 antigen contact.

Claims

What is claimed is:

1. Method for determining antigen-specific T-cell responses in a subject, the method comprising: a) contacting a whole blood sample from the subj ect with at least one antigen to form a mixture, wherein the whole blood sample amount is from about 10 pL to about 10 mL, wherein the at least one antigen is an activator of T-cells and comprises a full-length protein, a processed protein, a peptide, a pool of peptides, a complex mixture of full-length and/or partial proteins, a protein derivative, a single epitope, a partial proteome, a raw extract, a mixture of polypeptides and other biomolecules, or any combination thereof; b) contacting the whole blood sample with at least one staining reagent, wherein the staining reagent compnses monoclonal antibody reagents for identification of extracellular T-cell markers include CD2, CD3, CD4, CD8, CDl la, CD25 (IL2RA), CD27, CD28, CD31 (PEC AMI), CD38, CD45, CD45RA, CD45RO, CD54, CD56 (NCAM1), CD57, CD58, CD62L (SELL), CD69, CD95, CD103, CD107a, CD122, CD127, CD137, CD152 (CLTA4), CD154, CD161 (KLRB1) CD183 (CXCR3), CD 194 (CCR4), CD 195 (CCR5), CD 196 (CCR6), CCR10, CD 197 (CCR7), CD200, CD279 (PD-1), CD294 (PTGDR2 or CRTH2), HLA-DR, 0X40, or any combination thereof, wherein the contacting comprises incubation for about 30 minutes to about 24 hours, wherein the contacting the whole blood sample with at least one staining reagent occurs before step (a), concurrent with step (a), after step (a), or any combination thereof; and c) analyzing the mixture by flow cytometry to identify T-cells responding to the antigen contact.

2. The method of claim 1, further comprising adding at least one reagent to the mixture of step (b).

3. The method of claim 1, further comprising a step of incubating the mixture after step (a), before step (b), after step (b), or any combination thereof.

4. The method of claim 3, further comprising obtaining a cell concentrate from the mixture after the step of incubating the mixture.

5. The method of claim 4, wherein the step of obtaining a cell concentrate from the mixture comprises: a) adding a lysing reagent to the mixture; b) adding a permeabilization reagent to the mixture; c) staining the mixture with a staining reagent; and d) washing and concentrating the mixture to obtain the cell concentrate.

6. The method of claim 5, wherein the step of obtaining a cell concentrate from the mixture further comprises adding a fixation reagent to the mixture.

7. The method of claim 1, wherein the at least one staining reagent and/or the at least one antigen is a dry reagent.

8. The method of claim 7, wherein the dry reagent is provided in at least one pre- filled reagent vessel.

9. The method of claim 1 or 8, wherein the staining reagent comprises monoclonal antibody reagents for identification of intracellular T-cell markers and include IL-2, IL-4, IL-5, IL-9, IL-10, IL-13 IL-17 (IL-17A), IL-21, IL-22, IL-25, IL-26 IFN-y, TGFP (TGFB1), TNF, AHR, TBR2 (EOMES), FOXO4, POXP3, GATA3, IRF4 (MUM1), LEF1, PRDM1 (BLIMP1) RORC (RORy) STAT4, TBX21 (T-bet), TCF7, GZMA, GZMB, PERF, TNF-a, or any combination thereof.

10. The method of claim 1, wherein the step of analyzing the mixture by flow cytometry comprises analyzing the presence of activation markers of CD4+ T- cells, and/or CD8+ T cells, wherein the activation markers of CD4+ T-cells comprise CD69, CD154, CD137 and CD107a and wherein the activation markers of CD8+ T- cells comprise CD69, CD 154, CD 137 and CD 107a.

11. The method of claim 1 , wherein the at least one antigen comprises at least one virus-specific antigen, wherein the at least one virus-specific antigen is a full- length protein, protein derivative, peptide, or peptide pool derived from SARS-CoV-2, SARS- CoV, MERS, Cytomegalovirus (CMV), Respiratory Syncytial virus (RSV), Epstein-Barr virus (EBV), influenza (H1N1), Monkey Pox (MP), or Human Papilloma virus (HPV), a spike protein (S), subunit SI of spike protein (SI), subunit S2 of spike protein (S2), spike protein (S+), RBD domain of spike protein, nucleocapsid protein, membrane protein, envelope protein, or any combination thereof.

12. The method of claim 1, wherein the method excludes a step of isolation, concentration, and/or extraction of peripheral blood mononuclear cells (PBMCs).

13. Use of the method of claim 1 for monitoring an immune status of a subject prior to or after an incident of viral infection.

14. A method for determining antigen-specific T-cell responses in a subject, the method comprising: a) contacting a whole blood sample from the subj ect with at least one antigen to form a mixture; b) contacting the mixture with a staining reagent for CD 107a and a staining reagent for CD154; c) incubating the mixture; d) contacting the mixture with at least one additional staining reagent; e) adding a lysing reagent to the mixture; f) washing and concentrating the mixture to obtain the cell concentrate; and g) analyzing the mixture to identify T-cells responding to the antigen contact.

15. A method of determining an anti-virus immune response to SARS- CoV-2 in a subject, the method comprising: a) contacting a whole blood sample from the subject with at least one SARS-CoV-2 antigen to form a mixture; b) contacting the whole blood sample with at least one staining reagent, wherein the contacting the whole blood sample with at least one staining reagent occurs before step (a), concurrent with step (a), after step (a), or any combination thereof; and c) analyzing the mixture to identify T-cells responding to the SARS-CoV- 2 antigen contact.