Instant on-chip IGRA detection device and method for measuring specific T cell activation

Abstract

The present disclosure describes methods and devices for detecting SARS-CoV-2 specific T cell interferon gamma activation. By using a microfluidic chip to allow ELISpot Interferon Gamma Release Assay (IGRA) to be performed using a small amount of a whole blood sample collected from a fingertip, results can be rapidly provided in a small amount of a blood sample.

Description

[0001] Prior related application

[0002] This application claims the benefit of priority to U.S. Application No. 63 / 399,043, filed Aug. 18, 2022, the entire disclosure of which is incorporated herein by reference for all purposes.

[0003] Statement of federally sponsored research

[0004] Not applicable. Technical field

[0005] The present disclosure generally relates to devices and methods for measuring specific T cell activation, and more particularly to a point-of-care on-chip IGRA device for measuring T cell activation induced by a pathogen, vaccine, or therapy. Background art

[0006] IGRA assays measure the amount of IFN-γ produced or the number of cells secreting IFN-γ after stimulation with specific peptides from a pathogen of interest. The former method relies on ELISA to detect IFN-γ secretion to indicate the induction of antigen-specific T cells, while the latter method uses an enzyme-linked immunospot (ELISpot) assay to detect IFN-γ released and bound near activated T cells immobilized on a detection membrane. However, each assay has drawbacks that limit their feasibility for routine use in large populations to estimate vaccine efficacy. Both ELISA-based and ELISpot-based IGRA require a significant amount of fresh blood, which should be processed within 8 to 14 hours after collection for analysis in order to obtain reliable results from viable target cells. The ELISpot assay is more technically demanding than ELISA-based IGRA because they require isolation of peripheral blood mononuclear cells (PBMCs) from blood samples prior to stimulation with pathogen-specific target peptides and require scanning of the resulting PBMC culture wells to quantify the number of colored spots indicating the number of reactive cells. However, since both ELISpot and ELISA-based IGRA are technically demanding, they are typically performed in a central laboratory, which can lead to logistical aspects of sample transportation that may limit assay performance.

[0007] It is estimated that latent Mycobacterium tuberculosis (M. tb) infection affects one-third of the global population and has a 5–10% risk of progressing to TB disease, causing 1.5 million deaths annually, more than any other infectious disease except during the peak of the COVID-19 pandemic. Individuals with latent M. tb infection are at greatest risk of developing TB disease within the first two years after infection with M. tb, but the risk of disease progression may persist throughout their lives. Therefore, addressing this reservoir of latent disease is crucial for the "End TB Strategy", which aims to reduce the incidence of TB by 90% by 2035 and eliminate TB by 2050.

[0008] Current methods for diagnosing latent M. tb infection, including the tuberculin skin test (TST) and interferon-γ release assays (IGRAs), can detect immune sensitization to M. tb-derived antigens, but each method has drawbacks. Screening individuals using TST results requires the assessment of the resulting TST reaction by trained healthcare workers 48–72 hours after subcutaneous injection of M. tb-derived material. However, previous vaccination with Mycobacterium bovis BCG, non-tuberculous mycobacterial infections that may have different treatment requirements, and incorrect TST interpretation can all cause false-positive results, leading to unnecessary or ineffective treatment. IGRAs do not require repeat visits and do not produce false positives in individuals vaccinated with BCG, but require the use of whole blood samples that must be stored under controlled conditions and used within approximately 16 hours after collection. In addition, the sensitivity of both tests is reduced by factors that can weaken the immune response to their target M. tb antigens; therefore, they are less reliable when used in individuals with an impaired immune system, recent infection with M. tb, or recent receipt of live virus measles or smallpox vaccines, as all of these factors can lead to false-negative results. HIV infection is a particular concern in M. tb screening efforts because the risk of rapid progression from latent M. tb infection to TB disease is increased in individuals coinfected with HIV, contributing to one-third of HIV-related deaths. The rate of coinfection with M. tb and HIV also tends to be high in areas with a high prevalence of TB, exceeding 50% in some parts of South Africa.

[0009] CD4 T cells play an important role in the IFN-γ response induced after Mycobacterium tuberculosis infection, but the results of IGRA cannot reliably detect latent Mycobacterium tuberculosis infection in HIV-infected individuals with CD4 T cell counts ≤ 200 cells / μL. Therefore, we hypothesized that detection of increased activation markers less restricted by CD4 T cells might enhance the detection of Mycobacterium tuberculosis-specific immune responses in HIV co-infected individuals, and thus selected OX40 (TNFRSF4 / CD134) and 4-1BB (TNFRSF9 / CD137) as candidate markers for detecting Mycobacterium tuberculosis infection in Mycobacterium tuberculosis- and HIV-co-infected individuals. Compared with unactivated CD4 and CD8 T cells, OX40 is highly expressed on activated CD4 and CD8 T cells, and thus can serve as an antigen-specific T cell activation marker more resilient than CD4 T cell IFN-γ expression. Similarly, 4-1BB expression is also induced on activated CD4 and CD8 T cells, although CD8 T cells can upregulate 4-1BB faster and to a higher level than CD4 T cells. In addition, since both of these proteins are T cell surface markers (TSMs) and can thus be directly detected with labeled specific antibodies, there is no need for the cell fixation and permeabilization steps required in intracellular cytokine staining assays.

[0010] Moreover, with the continuous emergence of SARS-CoV-2 variant of concern (VOC), new methods are needed to evaluate the degree and durability of immune protection after vaccination or infection, including protection against specific VOCs. This information is crucial for the rapid assessment of vaccine effectiveness, vaccination guidelines, and public health decision-making. It can also be used to identify vulnerable populations or individuals in need of further preventive or intervention measures (including additional vaccine doses) and their responses to these interventions. Multiple studies have shown that VOCs, including B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.1.529 (Omicron), exhibit different resistance to the immune responses generated in vaccinated and previously infected individuals. For example, the currently dominant VOC strain Omicron in the United States has a spike protein that can evade neutralization by antibodies produced in vaccinated individuals and convalescent patients, with an efficiency 10 to 44 times higher than that of the Delta spike protein; and its spike protein is resistant to neutralizing antibodies in convalescent patients and vaccinated individuals, and the neutralizing antibodies may vary greatly and decline rapidly over time.

[0011] To evaluate the role of VOCs in the durability of vaccine-mediated variant protection and provide reliable data to reveal how the immune response against SARS-CoV-2 VOCs changes over time is crucial. However, the kinetics of antibody responses after infection or vaccination vary among different populations, and it has been shown that these data have limited clinical value when used to monitor the efficacy of vaccines over time. Evidence suggests that SARS-CoV-2-specific T cell immune responses remain active after the decline in neutralizing antibody titers. Studies on SARS-CoV-1 and MERS have shown that IFN-γ secretion in T cells can persist much longer than the antibody response or can occur in the absence of an antibody response, and SARS-CoV-2-specific T cell responses can be detected in the absence of detectable specific antibodies. For example, immunocompromised individuals may exhibit insufficient seroconversion rates and neutralizing antibody responses after vaccination with SARS-CoV-2 vaccines but still show significant virus-specific T cell responses, including strong T cell immunity against Omicron. Notably, Omicron can evade specific neutralizing antibodies but still activates T cell responses induced by previous vaccination or infection, and one study showed that 70-80% of the CD4 and CD8 T cell responses induced by vaccines against the spike protein of the reference strain remained against Omicron. Therefore, several studies have employed interferon-γ (IFN-γ) release assays (TGRAs) to evaluate T cell responses in vaccinated individuals and SARS-CoV-2 patients. Analysis of T cell responses to emerging SARS-CoV-2 VOCs can allow for the rapid and prospective assessment of vaccine effectiveness and provide information on the need for additional vaccine doses or the development of variant-specific vaccines.

[0012] Therefore, new IGRA methods are needed to streamline the workflow, reduce technical requirements, and decrease the execution time of these assays to improve their utility in high-throughput analysis. Microfluidic technology can simplify the assay workflow, reduce the sample volume to lower reagent costs, and minimize technical requirements and assay variability by automating key sample handling steps. Microfluidic methods have recently been used for COVID-19 diagnostic assays, and several microfluidic sensing platforms for RT-PCR, antigen, or antibody tests have received FDA emergency use authorization (EUA) approval and have been commercialized for COVID-19 diagnosis. Therefore, we hypothesized that a microfluidic IGRA could be developed to allow for the widespread application of IGRA to analyze T cell responses, including potential responses to emerging SARS-CoV-2 VOCs as well as other pathogens and immunotherapeutic targets, and thus be useful for evaluating the efficacy of vaccines over time. Summary of the Invention

[0014] Here, we describe the development and performance of a microfluidic ELISpot-based IGRA that provides a point-of-care (POC) assay for pathogen T cell responses, where the pathogen can be various pathogens capable of eliciting T cell responses, such as M.tb markers or SARS-CoV-2 target peptides. The methods and microfluidic assay platforms provided herein require less time, infrastructure, and expertise than standard IGRAs. The methods and assay platforms for M.tb detection detect at least one of a plurality of M.tb markers to improve its sensitivity in patient populations with impaired CD4 T cell responses and to streamline its detection workflow. Similar methods / assay platforms for detecting SARS-CoV-2 target peptides are also presented herein.

[0015] For M.tb, the results showed that, compared to the 24 - 48 hours required for conventional IGRA, the method of the present invention can provide reliable results on a TSM microchip device in 6 hours, which uses a fingertip whole blood micro-sample and integrates all assay steps on the assay chip, without the need for laboratory equipment or special expertise.

[0016] For SARS-CoV-2, we found that when evaluating the responses of individuals who have been vaccinated or not vaccinated against SARS-CoV-2 or have been infected or not infected with SARS-CoV-2, the microchip results are comparable to those generated by other immunoassay methods, including flow cytometry and conventional ELISpot assays. The microfluidic chip assay can provide results within 5 hours using a fingertip blood sample (∼25 μL), thus reducing the sample-to-answer time of the IGRA and reducing the sample handling and equipment requirements of the IGRA. Notably, this assay can be read using a mobile phone microscope, allowing for use in resource-limited areas.

[0017] In one aspect of the present disclosure, a method for identifying pathogen-specific T cell activation using a microfluidic chip is described. The method comprises the steps of: a) obtaining a biological sample from a subject; b) introducing an incubation mixture into the biological sample, wherein the incubation mixture comprises (i) a peptide of a target pathogen or a peptide of a target vaccine; and (ii) an antibody that specifically binds to T cells activated upon stimulation with the target pathogen peptide or the target vaccine peptide; c) detecting the presence of activated T cells in the biological sample of step b); wherein the antibody in step b) is conjugated to an enzyme or a fluorescent molecule.

[0018] In another aspect of the present disclosure, a point-of-care kit for identifying pathogen-specific T cell responses is described. The point-of-care kit includes a microfluidic chip having a plurality of microfluidic channels connecting a sample inlet to a detection chamber, wherein the detection chamber is coated with polylysine or other cell attachment enhancing reagents - gelatin. The detection chamber may also be coated with T cell-specific antibodies, such as anti-CD4 and anti-CD8 antibodies, to better capture CD4+ and CD8+ T cells in PBMC. The microfluidic channels allow sufficient time to enhance the T cell response to antigenic peptides while accelerating cytokine release and expression of T cell activation surface markers.

[0019] In another aspect of the present disclosure, a method for identifying pathogen-specific T cell activation using the microfluidic chip described herein is disclosed. The method includes: a) obtaining a biological sample from a subject; b) introducing an incubation mixture into the biological sample, wherein the incubation mixture includes (i) a peptide of a target pathogen or a peptide of a target vaccine, and (ii) an antibody specific for a cytokine or a surface marker, wherein the cytokine is secreted by T cells in the biological sample upon stimulation with the target pathogen peptide or the target vaccine peptide; c) introducing the biological sample from step b) through the sample inlet into the detection chamber in the microfluidic chip; and d) detecting the presence of the cytokine or the surface marker in the detection chamber; wherein the cytokine or surface marker-specific antibody is conjugated to an enzyme or a fluorescent molecule.

[0020] In one embodiment, the antibody that specifically binds to activated T cells is an anti-human interferon-γ antibody, or an antibody that specifically binds to a cytokine or a surface marker expressed by activated T cells.

[0021] In one embodiment, the anti-human interferon-γ antibody targets and binds to interferon γ secreted by T cells activated by a peptide from a target pathogen. The anti-human interferon-γ antibody is conjugated to an enzyme or a fluorescent molecule to produce a visual signal when binding to interferon γ. The fluorescent signal indicates the presence of IFN-γ.

[0022] In one embodiment, the method further includes step b-1): obtaining T cells in a whole blood sample through CD4- and CD8-specific antibodies.

[0023] In one embodiment, the point-of-care kit may further include an incubation container in which a peptide from a target pathogen is present. The collected biological sample can be placed in the incubation container and incubated with the peptide to activate T cells.

[0024] In one embodiment, the method includes an activation step, wherein T cells in the biological sample are activated by a target pathogen-specific peptide or vaccine or marker for a predetermined period of time.

[0025] In one embodiment, the activation step lasts for about 1 to 6 hours. In another embodiment, the activation step lasts for about 2 to 6 hours.

[0026] In one embodiment, the incubation container further comprises phorbol 12-myristate 13-acetate (PMA) and ionomycin for stimulating T cells. In another embodiment, the fluorescent molecule is fluorescein isothiocyanate (FITC) or Alexa 488.

[0027] In one embodiment, the target pathogen is SARS-CoV-2, HIV or Mycobacterium tuberculosis. Other pathogens that can trigger T cell responses can also be detected. Non-limiting pathogens include cytomegalovirus (CMV), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma gondii and Trypanosoma cruzi.

[0028] In one embodiment, the target therapeutic response is that a vaccine (SARS-CoV-2 vaccine) can trigger a T cell response. Non-limiting vaccines include cytomegalovirus (CMV), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma gondii and Trypanosoma cruzi.

[0029] Other antigen-specific T cell response assessments include CAR-T, TCR-T therapies, PD-1 / PD-L1, CTLA-4, TIM3, LAG3 or other checkpoint blockade therapies in cancer. The methods and devices of the present disclosure may include antibodies against these immune checkpoint proteins and screening for corresponding T cell responses.

[0030] In one embodiment, the target pathogen is Mycobacterium tuberculosis, and the peptides or markers that can be used to capture activated T cells include at least a portion of OX-40, 4-1BB, CD59, LAG-3, TIM3, and IL-12R, CD28, CD57, KIR, KLRG-1, CD27, PD-1, CTLA-4, IFN-γ, IL-2, IL-10, TNF-α. In one embodiment, the peptide or marker is the extracellular domain of OX-40, 4-1BB, CD59, LAG-3, TIM3, and IL-12R, CD28, CD57, KIR, KLRG-1, CD27, PD-1, CTLA-4, IFN-γ, IL-2, IL-10, TNF-α.

[0031] In one embodiment, the detection chamber in the microfluidics is treated with a CD4 or CD8 specific antibody. In one embodiment, the CD4 or CD8 specific antibody is treated by EDC-NHS chemistry.

[0032] In one embodiment, the detection chamber is coated with polylysine, and the concentration of polylysine can be from 1 μg / mL to 100 μg / mL. In another embodiment, the concentration of polylysine can be from 5 μg / mL to 50 μg / mL.

[0033] In one embodiment, the width of the microfluidic channel is between 10 μm and 200 μm, and the height is between 10 μm and 200 μm. In another embodiment, the width of the microfluidic channel is between 50 μm and 150 μm, and the height is between 50 μm and 150 μm. In another embodiment, the width of the microfluidic channel is approximately 100 μm, and the height is approximately 100 μm.

[0034] In one embodiment, the size of the detection chamber is 1 to 10 mm 2 . In one embodiment, the size of the detection chamber is 10 mm x 3 mm x 0.1 mm.

[0035] In one embodiment, the biological sample is a whole blood sample from a finger prick. This is different from conventional ELISpot, in which peripheral blood mononuclear cells (PBMCs) must be separated first before testing.

[0036] In one embodiment, the volume of the whole blood sample is less than 1 mL. In one embodiment, the volume of the whole blood sample is less than 100 μL. In another embodiment, the volume of the whole blood sample is less than 50 μL.

[0037] In one embodiment, the peptides of the target pathogen include a SARS-CoV-2 spike peptide pool and BEI NR-52402. However, other peptide pools can also be used as long as they represent peptides from pathogens that can trigger T cell specific responses commonly encountered. Non-limiting examples include peptides from cytomegalovirus (CMV), influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma, Trypanosoma cruzi, or tumor-specific antigen peptides from NY-ESO-1, HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), MUC1, and EGFR.

[0038] In one embodiment, the first interferon-γ specific antibody is M700-A from Endogen. However, other interferon-γ specific antibodies can also be used.

[0039] In one embodiment, the mixture of the biological sample and the incubation mixture is introduced into the reaction chamber of the point-of-care testing device at a flow rate of 5 μl / min to 20 μl / min.

[0040] In one embodiment, the cytokine is IL-2, IL-4, IL-17, or TNFα. However, other cytokines can also be used.

[0041] In one embodiment, the surface marker is at least a part of OX-40, 4-1BB, CD59, LAG-3, TIM3, and IL-12R, CD28, CD57, KIR, KLRG-1, CD27, PD-1, CTLA-4, IFN-γ, IL-2, IL-10, or TNF-α.

[0042] In one embodiment, the part of OX-40 is the extracellular domain of OX-40, having the following amino acid sequence:

[0043]

[0044] In one embodiment, the part of 4-1BB is the extracellular domain of 4-1BB, having the following amino acid sequence:

[0045]

[0046] In one embodiment, the part of CD59 is the extracellular domain of CD59, having the following amino acid sequence:

[0047]

[0048] In one embodiment, the portion of LAG-3 is the extracellular domain of LAG-3 and has the following amino acid sequence:

[0049]

[0050] In one embodiment, the portion of TIM3 is the extracellular domain of TIM3 and has the following amino acid sequence:

[0051]

[0052] In one embodiment, the portion of CD28 is the extracellular domain of CD28 and has the following amino acid sequence:

[0053]

[0054] In one embodiment, the portion of KIR is the extracellular domain of KIR and has the following amino acid sequence:

[0055]

[0056] In one embodiment, the portion of KLRG-1 is the extracellular domain of KLRG-1 and has the following amino acid sequence:

[0057]

[0058] In one embodiment, the portion of CD27 is the extracellular domain of CD27 and has the following amino acid sequence:

[0059]

[0060] In one embodiment, the portion of PD-1 is the extracellular domain of PD-1 and has the following amino acid sequence:

[0061]

[0062] In one embodiment, the portion of CTLA-4 is the extracellular domain of CTLA-4 and has the following amino acid sequence:

[0063]

[0064] In one embodiment, the portion of IL2Ra is the extracellular domain of IL2Ra and has the following amino acid sequence:

[0065]

[0066] In one embodiment, the anti-4-1BB antibody herein is the Cd137(4-1BB) monoclonal antibody (4B4(4B4-1)), FITC, eBioscience 11-1379-42.

[0067] In one embodiment, the anti-OX-40 antibody used herein is the Cd134(OX40) monoclonal antibody (ACT35(ACT-35)), FITC, eBioscience 11-1347-42.

[0068] As used herein, "cytokine" refers to any one of a variety of substances secreted by certain cells of the immune system and having an effect on other cells, such as interferons, interleukins, and growth factors.

[0069] "Surface marker" as used herein refers to a special protein or peptide expressed on the cell surface or a carbohydrate attached to the cell membrane, which is usually conveniently used as a marker for a specific cell type.

[0070] As used herein, "interferon-gamma" or "IFN-γ" refers to a dimeric soluble cytokine belonging to type II interferons. IFN-γ is crucial for innate and adaptive immunity against viral, certain bacterial, and protozoan infections. IFN-γ is an important activator of macrophages and an inducer of major histocompatibility complex class II molecule expression. IFN-γ is mainly produced by natural killer cells (NK) and natural killer T cells (NKT) (as part of the innate immune response), and once antigen-specific immunity is formed (as part of the adaptive immune response), it is produced by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells.

[0071] As used herein, "OX-40" refers to TNF receptor superfamily member 4, also known as TNFRSF4, ACT35, CD134, IMD16, or TXGP1L. The protein encoded by this gene is a member of the TNF receptor superfamily. This receptor has been shown to activate NF-κB by interacting with the adaptor proteins TRAF2 and TRAF5.

[0072] As used herein, "4-1BB" refers to TNF receptor superfamily member 9, also known as TNFRSF9, ILA, CD137, CD2137, or IMD109. The protein encoded by this gene is a member of the TNF receptor superfamily. This receptor promotes the clonal expansion, survival, and development of T cells. It can also induce the proliferation of peripheral monocytes, enhance T cell apoptosis induced by activation triggered by TCR / CD3, and regulate CD28 co-stimulation to promote Th1 cell responses.

[0073] As used herein, "CD59" is also known as 1F5, EJ16, EJ30, EL32, G344, MIN1, MIN2, MIN3, MIRL, HRF20, MACIF, MEM43, MIC11, MSK21, 16.3A5, HRF-20, MAC-IP or p18-20. This is a cell surface glycoprotein that regulates complement-mediated cell lysis and is involved in lymphocyte signal transduction. The protein is a potent inhibitor of the complement membrane attack complex, and it binds to complement C8 and / or C9 during the assembly of this complex, thereby inhibiting the incorporation of multiple copies of C9 into the complex, which is essential for the formation of the osmolytic pore.

[0074] As used herein, "LAG-3" refers to lymphocyte activation protein 3. The LAG-3 protein belongs to the immunoglobulin (Ig) superfamily and is a type I transmembrane protein of 503 amino acids that includes four extracellular Ig-like domains (designated D1 to D4).

[0075] As used herein, "TIM3" refers to hepatitis A virus cellular receptor 2, also known as CD366, KIM-3, SPTCL, TIMD3, Tim-3, TIMD-3 or HAVcr-2. The protein encoded by this gene belongs to the immunoglobulin superfamily and the TIM protein family.

[0076] As used herein, "IL-12R" consists of interleukin 12 receptor beta 1 (IL-12Rβ1) and interleukin 12 receptor beta 2 (IL-12Rβ2) chains and mediates signal transduction, which involves the recruitment of Janus family tyrosine kinase 2 and signal transducer and activator of transcription (STAT) 4.

[0077] As used herein, "CD28" refers to the protein encoded by this gene, which is essential for T cell proliferation and survival, cytokine production, and T helper type 2 development. It is also known as Tp44.

[0078] As used herein, "CD57" refers to beta-1,3-glucuronosyltransferase 1, also known as NK1, HNK1, LEU7, GLCATP or GLCUATP. The protein encoded by this gene is a member of the glucuronosyltransferase gene family.

[0079] As used herein, "KIR" refers to killer cell immunoglobulin-like receptor, which is a member of a group of regulatory molecules found in subsets of lymphocytes.

[0080] As used herein, "KLRG-1" refers to killer cell lectin-like receptor G1, also known as 2F1, MAFA, MAFA-L, CLEC15A, MAFA-2F1, or MAFA-LIKE. The protein encoded by this gene belongs to the killer cell lectin-like receptor (KLR) family, which is a group of transmembrane proteins that are preferentially expressed in NK cells.

[0081] As used herein, "CD27" refers to a member of the TNF receptor superfamily. This receptor is essential for the generation and long-term maintenance of T cell immunity.

[0082] As used herein, "PD-1" refers to programmed cell death protein 1, also known as CD279, SLEB2, hPD-1, hPD-I, or hSLE1. Programmed cell death protein 1 (PDCD1) is an immunosuppressive receptor expressed on activated T cells; it is involved in regulating T cell function, including that of effector CD8+ T cells.

[0083] As used herein, "CTLA-4" refers to cytotoxic T lymphocyte-associated protein 4, also known as CD, GSE, GRD4, ALPS5, CD152, IDDM12, or CELIAC3. This gene is a member of the immunoglobulin superfamily and encodes a protein that transmits inhibitory signals to T cells. The protein contains a V domain, a transmembrane domain, and a cytoplasmic tail.

[0084] As used herein, "IL-2" refers to interleukin 2, also known as TCGF or lymphokine. This gene is a member of the interleukin 2 (IL2) cytokine subfamily, which includes IL4, IL7, IL9, IL15, IL21, erythropoietin, and thrombopoietin. The protein encoded by this gene is a secreted cytokine that is produced by activated CD4+ and CD8+ T lymphocytes and is important for the proliferation of T and B lymphocytes.

[0085] As used herein, "IL-10" refers to interleukin 10, also known as CSIF, TGIF, GVHDS, or IL10A. The protein encoded by this gene is a cytokine that is mainly produced by monocytes and can also be produced by lymphocytes to a lesser extent.

[0086] As used herein, "TNF-α" refers to tumor necrosis factor α, which is a pro-inflammatory cytokine that plays an important role in the pathogenesis of various diseases.

[0087] As used herein, a "microfluidic device" refers to a detection device whose core is the microfluidic behavior of fluids, achieving precise control and manipulation within a small geometrically constrained scale (usually sub-millimeter level), where surface forces dominate over volume forces at said small scale. A microfluidic chip is a molded or engraved microchannel pattern. This microchannel network integrated into the microfluidic chip is connected to the macroscopic environment through several through-holes of different sizes machined on the chip. Fluids are injected into and discharged from the microfluidic chip through these channels. The fluids are guided, mixed, separated, or manipulated to achieve multiplexing, automation, and high-throughput systems. The design of the microchannel network must be precisely and elaborately formulated to achieve the desired characteristics. Microfluidic technology has multiple advantages: faster reaction time, higher analysis sensitivity, better temperature control, better portability, easier automation and parallelization, and the ability to integrate multiple laboratory procedures in one device.

[0088] As used herein, a "conjugated" antibody refers to a protein labeled with a compound or dye for tracking its interaction with a specific antigen. Fluorescent dyes such as Alex and DyLight fluor can be used in immunofluorescence assays. These fluorescent dyes can absorb and emit light of different wavelengths for different labeling purposes. Antibodies conjugated with fluorescent dyes can be used in immunoassay tests such as flow cytometry, ELISA, Western blotting, and fluorescence microscopy.

[0089] As used herein, "stimulating" or "activating" a T cell refers to the binding of a specific ligand that triggers biochemical signals in the T cell, including the production of interferon γ.

[0090] Unless the context otherwise requires, when the terms "comprising" is used in a claim or specification, the word "a" or "an" means one or more.

[0091] The term "about" means the stated value plus or minus the magnitude of the measurement error, or plus or minus 10% if no measurement method is specified.

[0092] The use of the term "or" in a claim is used to mean "and / or", unless explicitly indicated to refer only to alternative options or the alternative options are mutually exclusive.

[0093] The terms "comprising", "having", "containing", and "including" (and their variants) are open conjunctions that allow the addition of other elements when used in a claim.

[0094] The phrase "consisting of" is closed, excluding all additional elements.

[0095] The phrase "consisting essentially of" excludes additional substantial elements, but allows the inclusion of non-substantial elements that do not materially change the nature of the invention.

[0096] The following abbreviations are used in this article:

[0097]

[0098] Brief Description of the Drawings

[0100] Figure 1A . Schematic diagram of the standard and microfluidic chip ELISPOT assays.

[0101] Figure 1B . IFN-γ-independent assessment of T cell activation.

[0102] Figure 1C . Thawed PBMC aliquots, with or without PMA / ionomycin stimulation, were cultured in poly-L-lysine-coated glass-bottom wells for 24 hours and then incubated with biotinylated secondary antibody, streptavidin-HRP, and chromogenic (red) HRP substrate, or incubated with Hoescht 33342 dye and fluorescently labeled IFN-γ-specific antibody. The white scale bar represents 75 μm.

[0103] Figure 1D -E. PBMCs (~2x 10 5 ) were seeded into wells of microtiter plates coated with or without poly-L-lysine and stained with Hoechst 33342 to quantify the cell density of captured cells, or induced with PMA / ionomycin for 4 hours, stained with Hoechst 33342 and specific antibodies against IFN-γ, OX40, and 4-1BB, and then the total cell number and percentage of activated T cells were quantified using a fluorescence plate reader. Positive control (PC) wells were not washed to remove non-adherent or weakly adherent cells.

[0104] Figure 1F . One-way two-sided parametric ANOVA and Tukey's post hoc test were performed to analyze the differences between poly-L-lysine-coated and uncoated well values and (F) PMA-stimulated and unstimulated well values. Data are represented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significant difference analyzed by two-sided Mann-Whitney U test.

[0105] Figure 2A-C. Activating T cells with a pool of SARS-CoV-2 spike peptides. Cryopreserved PBMCs from donors who received (A) 3 doses of SARS-CoV-2 or (B-C) 0-3 doses of vaccine were incubated with (A) PMA / ionomycin and / or (A-C) a pool of SARS-CoV-2 peptides for no more than 24 hours for stimulation, and then the IFN-γ level was evaluated by (A) ELISA, (B) flow cytometry, or (C) ELISpot.

[0106] Figure 2D -F. Freshly isolated PBMCs from unvaccinated and vaccinated (3 doses) donors were incubated for 24 hours with or without (D-E) a pool of SARS-CoV-2 peptides or (F) peptides derived from SARS-CoV-2, Mycobacterium tuberculosis (Mtb) proteins CFP-10 and ESAT-6, or HIV-1 p24, stained with Hoechst 33342, and incubated with an IFN-γ-specific antibody. The total cell number and the percentage of activated T cells were analyzed using a fluorescence plate reader. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; or ns, no significant difference between the indicated groups by (b) one-way parametric ANOVA and Tukey's post hoc test or (e-f) Mann-Whitney U test.

[0107] Figure 3A -F. Evaluating the performance of the ELISpot assay on a chip. PBMC samples (~2 x 10 6 cells) were isolated from individuals with no history of HIV infection who received three doses of vaccine, and the IFN-γ responses to SARS-CoV-2 spike or HTV-1 p24 (nonspecific control) peptides were analyzed in the PBMC samples using (A) our ELISpot assay on a chip, (B) flow cytometry, and (C) the ELISpot assay. (D) Correlation of flow cytometry and ELISpot assay on a chip data. (E-F) ELISpot assay results on a chip, where the assay results (e) were from a finger-prick whole blood sample of an individual pretreated with or without RBC buffer, or (f) were from finger-prick whole blood samples of eight HIV-negative individuals more than six months after receiving the second or third dose of vaccine without RBC lysis. A t-test was performed to compare the stimulatory effects of the HIV-p24 or SARS-CoV-2 peptide pool. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ns, no significant difference by two-sided Mann-Whitney U test.

[0108] Figure 4A-D. Activated T cell counting on the glass surface. PBMCs were captured in wells coated with different concentrations of polylysine and uncoated wells, seeded with 2x 10 5 PBMCs, induced with PMA / ionomycin for 4 hours, stained with Hoechst 33342, and incubated with AlexaFluor488-(A), PE-labeled OX-40 (B), or APC-labeled 4-1BB (C-D) specific antibodies respectively. The activated T cell count in the wells was analyzed using a fluorescence plate reader. Positive control (PC) wells represent the signals detected in wells that were not washed to remove non-adherent or weakly adherent cells.

[0109] Figure 5A -C. Fabrication of PDMS microfluidic chips. (A) A silicon wafer coated with SU8 epoxy resin was covered with a photomask containing the device design, exposed to ultraviolet light, and washed with SU8 developer to remove the unactivated SU8 around the device design. Then a 10:1 PDMS-to-curing agent mixture was poured onto this master mold wafer and then heated at 60 °C for 5 hours. (B) 50 μg / ml polylysine was coated on the glass surface for 30 minutes and washed with deionized water. (C) The cured PDMS device was cut from the wafer and bonded to a polylysine-coated glass slide that had been previously exposed to oxygen plasma to generate a complete device.

[0110] Figure 6A -B. Flow cytometry gating of blood cell samples. Examples of IFN-γ responses detected by flow cytometry analysis of approximately 2x 10 6 PBMCs isolated from HIV-negative SARS-CoV-2 vaccine recipients (3 doses) after 24 hours of exposure to (A) SARS-CoV-2 spike peptide or (B) HIV-1 p24 (non-specific control) peptide. Scatter plots show total PBMC scatter and lymphocyte gating (left panel) and the distribution of IFN-γ negative and IFN-γ positive (gated population) within lymphocyte gating (right panel).

[0111] Figure 7A -E. Correlation between on-chip IGRA results and conventional assays. (A-D) Correlation between on-chip ELISpot and flow cytometry results among (A) SARS-Cov-2, (B) second, and (C) third vaccine dose groups and (D) vaccinated individuals who had breakthrough infections. (E) Correlation between on-chip ELISpot assay and standard ELISpot assay results. Data are represented as Spearmann / Pearson r values.

[0112] Figure 8A -K. Microchip reaction for T cell capture.

[0113] Figure 9A -G. Cumulative responses of surface markers 4-1BB and OX-40.

[0114] Figure 10A -B. Comparison of IFN-γ responses with 4-1BB and OX40 to TB infection.

[0115] Figure 11A -H. Blood-based detection method enables one-step diagnosis of TB infection. DETAILED DESCRIPTION OF THE INVENTION

[0117] Enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemical assay that uses a solid-phase type of enzyme immunoassay to detect the presence of a ligand (such as a protein) in a liquid sample using an antibody against the protein to be measured. A sample with an unknown amount of antigen is immobilized on a solid-phase support. After the antigen is immobilized, a detection antibody specific for the antigen is added to form a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can be made detectable through a secondary antibody linked by enzyme bioconjugation. Between each step, the ELISA plate is usually washed with a mild detergent solution to remove any non-specifically bound proteins or antibodies. After the final washing step, the plate is developed by adding an enzyme substrate, producing a visible signal that indicates the amount of antigen in the sample.

[0118] Enzyme-linked immunosorbent spot assay (ELISpot) is a detection method centered on quantitatively measuring the cytokine secretion rate of individual cells. This method uses antibodies to detect protein analytes (i.e., cytokines) and is very similar to ELISA. The mechanism of ELISpot begins with coating wells with analyte-specific monoclonal antibodies. The second step is incubating cells in the wells, during which time the cells are allowed to react with any present stimulant and secrete cytokines. Since the cells are surrounded by cytokine-specific monoclonal antibodies coated on the well walls, the cytokines secreted by the incubated cells will start to attach to the antibody through specific epitopes. After washing the wells to remove unbound cells and unwanted substances, since the cytokines are still attached to the first set of antibodies used, a biotinylated cytokine-specific detection antibody is added to the wells to bind to the remaining cytokines in the wells. Then, a streptavidin-enzyme conjugate is added to the wells to bind to the detection antibody. The cytokine-specific detection antibody added to the wells in the previous step can bind to this newly added streptavidin-enzyme conjugate because biotinylation can create a strong affinity between the biotin on the cytokine-specific detection antibody and the streptavidin on the conjugate. Finally, a substrate is added to the wells, and an insoluble precipitate is formed by the catalysis of the enzyme conjugate, forming spots in the wells. The spots can be read on an automated ELISpot reader or manually counted under a microscope.

[0119] Interferon-γ (IFN-γ) is an important cytokine mainly produced by immune system cells, including innate lymphocytes such as natural killer cells and innate lymphoid cells, as well as adaptive immune cells such as Th1 cells and CD8+ cytotoxic T lymphocytes. Signals induced by the IFN-γ receptor activate the Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) pathway, inducing the expression of several genes with essential immune effector functions. In addition, IFN-γ also plays a key role in regulating the functions of specialized tissue cells, affects progenitor and stem cells, and is involved in tissue and organ functions under homeostatic, immune, and pathological conditions. Interferon-γ release assays (IGRAs) are medical tests used to diagnose certain infectious diseases. Most of these tests have been developed for the field of tuberculosis diagnosis. For example, in patients with cutaneous drug adverse reactions, peripheral blood lymphocytes were stimulated with the drug causing the reaction, and positive test results were produced for half of the test drugs.

[0120] Currently, there are two IFN-γ release assays available for tuberculosis diagnosis: QuantiFERON-TB Gold and T-SPOT.TB. QuantiFERON-TB Gold (licensed in the United States, Europe, and Japan) quantifies the amount of IFN-γ produced in response to Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens, which are distinguishable from antigens present in BCG and most other non-tuberculous mycobacteria. T-SPOT.TB is a form of ELISpot, a variant of ELISA (licensed in Europe, the United States, Japan, and China). This method determines the total number of individual effector T cells expressing IFN-γ.

[0121] T cell activation requires extracellular stimulatory signals, which are mainly mediated by the T cell receptor (TCR) complex. The TCR recognizes antigens on major histocompatibility complex molecules in cooperation with CD4 or CD8 co-receptors. After recognition, the TCR induces a signal cascade that propagates signals through various molecules and second messengers. TCR signaling involves multiple signaling pathways, including the Ras-extracellular signal-related kinase (ERK)-activator protein (AP)-1 pathway, inositol trisphosphate (IP3)-Ca2+-nuclear factor of activated T cells (NFAT) pathway, protein kinase C (PKC)θ-IκB kinase (IKK)-nuclear factor (NF)-κB pathway. Among them, the NFAT pathway plays a crucial role in inducing IFN-γ from activated T cells ( Figure 1B ).

[0122] After TCR activation, T-bet induces the production of IFN-γ, and the activation of transcription factors Hix and Runx3 contributes to the induction of STAT5, which also triggers the production of IFN-γ. IFN-γ released from TB-infected T cells is usually evaluated using IGRA. However, IGRA cannot detect the full T cell response to any given antigen because only subsets of T cells, including CD4 T helper cells, are mainly responsible for the production of IFN-γ. We sought to develop a platform that could evaluate the activation response of the entire T cell repertoire, enabling the determination of T cell activation in an IFN-γ-independent manner.

[0123] To find an IFN-γ-independent T cell assessment, we started with T cell co-stimulatory signals (4-1BB and OX-40), also known as T cell surface markers (TSM). Researchers have found that stimulating Ras GTP exchange molecules triggers a kinase activity cascade involving Ras, MEK, and ERK kinases ( Figure 1B ), leading to the activation of multiple transcription factors, including activator protein-1 (AP-1) and transcription factor-nuclear factor κB (NF-κB). These activated transcription factors regulate the transcription of many tumor necrosis factor (TNF) receptor superfamily genes, including 4-1BB and OX40. We deployed these TSMs to develop a platform that could evaluate the full range of T cell activation in an IFN-γ-independent pathway.

[0124] The application of such a platform for diagnosing tuberculosis infection (TBI) has been demonstrated, where a one-step technique is employed to place the collected fingertip blood volume on a microfluidic chip, activating and staining T cells positive for surface markers for fluorescence detection ( Figure 1B ). We compared our method with intracellular IFN-γ staining at similar activation times and found that our method obtained similar or even better responses in evaluating T cell activation. Compared with single cytokine IFN-γ detection, multiplex T cell surface marker expression analysis allows several additional hypothesis testing pathways, such as direct one-step detection of activated T cells, comprehensive assessment of T cell activation in the entire T cell repertoire, and frequency distribution of surface marker expression at the shortest T cell activation time.

[0125] The present disclosure provides a new method for detecting T cell-specific interferon-γ responses that uses only a small amount of blood sample and has a rapid turnaround time. Specifically, the method of the present disclosure requires only 25 μL of whole blood sample and does not require the prior isolation of peripheral blood mononuclear cells (PBMC). The method of the present disclosure can provide results within 5 hours or less, while traditional ELISpot takes several days to complete. The method of the present disclosure utilizes a microfluidic device to reduce the sample volume required for detection.

[0126] The present invention takes the activation of SARS-CoV-2 specific T cells and the activation of tuberculosis specific T cells as examples. However, this is merely exemplary, and the present invention can be widely applied to other pathogens that cause interferon-γ secretion for T cell activation. The following examples are only for illustrative purposes and are not intended to unduly limit the scope of the appended claims.

[0127] Although the SARS-CoV-2 peptides are used as examples, the inventors envision that the devices and methods described herein are applicable to screening other pathogens and / or antigens suitable for immunotherapy. Non-limiting examples include cytomegalovirus (CMV), influenza, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma, and Trypanosoma cruzi.

[0128] In addition, although the detection employed is ELISpot-based IGRA, other T cell responses can also be evaluated. Non-limiting examples include CAR-T, TCR-T therapies, PD-1 / PD-L1, CTLA-4, TIM3, LAG3, or other checkpoint blockade therapies in cancer. CAR-T (chimeric antigen receptor T cell therapy) is a treatment method in which a patient's T cells are engineered in the laboratory to express a chimeric antigen receptor to target cancer cells. TCR-T (T cell receptor engineered T cell therapy) is similar to CAR-T, except that the cells are engineered to express a T cell receptor. PD-1 (programmed cell death protein-1) is an immune checkpoint on T cells that, when bound to PD-L1, prevents T cells from attacking other cells in the body (including cancer cells), so by targeting PD-1 or PD-L1, the attack of T cells on cancer cells can be enhanced. CTLA-4 (cytotoxic T lymphocyte associated protein-4), TIM-3 (T cell immunoglobulin and mucin domain-3), and LAG3 (lymphocyte activation gene-3) are other immune checkpoints that have shown promise in cancer treatment.

[0129] Example 1 - Development and Optimization of a Microfluidic ELISPOT IGRA System

[0130] Compared with ELISA-based IGRA, ELISpot assays have higher requirements for procedures and equipment, but are more easily adapted to microfluidic assay workflows because, in some assay designs, they require fewer liquid handling steps. The ELISpot microfluidic workflow can be divided into several basic steps: blood collection, stimulation of T cells with pathogen-specific peptides, and capture, staining, and analysis of activated T cells, most of which can be completed on a microfluidic chip, thus greatly simplifying the ELISpot workflow (Figure 1(a)).

[0131] Since microfluidic chips are typically constructed on glass or plastic substrates, and ELISpot assays are usually performed on membranes that allow local capture of IFN-γ released by activated cells, we first evaluated the ability to detect discrete aggregates of colorimetric substrates produced by activated T cells immobilized on the glass surface. This analysis found that chromophore aggregates could be detected to identify sites of IFN-γ-expressing cells, but the weak and diffuse nature of these signals did not allow accurate quantification of the number of activated cells (data not shown). However, analysis of intercellular IFN-γ expression in fixed and permeabilized PBMC samples by hybridization with IFN-γ-specific fluorescent antibodies revealed distinct foci of activated cells, which were detected only in PBMC samples pre-activated with phorbol myristate acetate (PMA) and ionomycin ( Figure 1C ). PBMC binding was improved by pre-coating the assay slides with polylysine ( Figure 1D ), which is widely used to enhance cell capture, including lymphocyte capture, by forming electrostatic interactions with anionic molecules on the cytoplasmic membrane. Polylysine titration analysis found that 5 μg / mL polylysine was sufficient to maximize average PBMC adhesion and capture (approximately 4.5×10 4 PBMC / mm 2 ), which was twice the number of cells captured on untreated slides (approximately 2.2×10 4 PBMC / mm 2 ), capturing approximately 58% of the input PBMCs (approximately 7.7×10 4 PBMC / mm 2 ). Figure 1E )

[0132] Next, we evaluated the ability to detect cell activation using three biomarkers of T cell activation (intracellular IFN-γ, surface OX-40, and 4-1BB expression) after stimulating the captured PBMCs with or without PMA / ionomycin for 24 hours. This analysis found that for each biomarker, the percentage of unstimulated PBMCs that were positive was low; the percentage of 4-1BB-positive cells did not increase after PMA / ionomycin stimulation (Figure 4), while the percentage of stimulated IFN-γ-positive cells was higher than the OX-40-positive cell values and had less variability (Figure IF), and thus we selected IFN-γ as the activation biomarker in all subsequent experiments.

[0133] Example 2 - Detection of antigen-specific T cell activation response on the glass surface

[0134] The extended incubation time required in standard ELISpot assays is a major problem for high-throughput analysis. We next isolated PBMCs from individuals vaccinated with three doses of SARS-CoV-2 RNA vaccine and evaluated the shortest time required to detect IFN-γ secreted by these PBMCs. Standard ELISA detected maximal IFN-γ secretion within 4 hours after PMA / ionomycin stimulation, but significant IFN-γ secretion was not detected until 24 hours after stimulation with a SARS-CoV-2-derived peptide pool ( Figure 2A ). Flow cytometry analysis was then performed to measure the proportion of SARS-CoV-2-reactive T cells present in PBMC fractions from unvaccinated and vaccinated individuals ( Figure 2B ). A low rate of reactive cells (0.01%) was detected in unvaccinated PBMC samples after 24-hour incubation, and this rate gradually increased in individuals vaccinated with two and three doses (3.76% and 5.41%, respectively). These differences were even more apparent when these samples were analyzed using standard ELISpot IGRA, where the PBMC response rate in unvaccinated individuals was only approximately 5 IFN-γ-secreting clusters, compared with 36 and 77 clusters in individuals vaccinated with two or three doses ( Figure 2C ).

[0135] Given that our microfluidic ELISpot assay uses fluorescent antibodies to detect intracellular IFN-γ expression, we next evaluated its ability to detect the rate of cell activation in PBMCs isolated from unvaccinated and fully vaccinated (3-dose vaccine) individuals and incubated with a SARS-CoV-2-derived peptide pool or PMA / ionomycin for 24 hours. This analysis determined that the rates of cell activation in PBMCs from fully vaccinated (Pfizer) and unvaccinated / unexposed individuals were approximately 20% and 5%, respectively, while PMA / ionomycin tended to activate approximately 20% of PBMCs in both groups ( Figure 2D -E). Notably, the frequency of IFN-γ-positive cells in the unvaccinated group was significantly higher than the results of flow cytometry analysis using a similar intracellular IFN-γ staining procedure (20% vs. 0.01%). This difference suggests that there are differences in the samples analyzed in these studies, or differences in the sensitivity, activation potency, or analytical sensitivity of these two procedures. However, the frequency of IFN-γ-positive PBMCs detected in this analysis was confirmed to be pathogen-specific, as the frequency of PMBC activation detected when these cells were incubated with peptides from other human pathogens (e.g., TB and HIV) from donors who had not been exposed or vaccinated was not different from the activation frequency measured with unstimulated PBMCs ( Figure 2E ).

[0136] Example 3 - Performance of the Microfluidic Chip ELISPOT IGRA

[0137] These cell capture, stimulation, and analysis steps were then combined into an integrated microfluidic assay procedure for evaluating the on-chip assay performance. Approximately 2×10 6 PBMCs (data not shown) were added to the wells of the microfluidic chip (Figure 5), and these PBMCs were captured on the polylysine layer and then cultured in medium with peptides for 24 hours, fixed, permeabilized, and incubated with Hoescht 33342 and IFN-γ specific fluorescent antibodies for 20 minutes. Subsequently, the fluorescence microscope images of the labeled cells on the chip were analyzed to evaluate PBMC activation. The background of cell activation detected for the unstimulated PBMC samples in this chip analysis was similar and significantly lower than the previous detection; the percentage of cells stimulated after incubation with the SARS-CoV-2 peptide pool was also similar, with no difference between the vaccinated and / or infected groups ( Figure 3A ). ELISpot and flow cytometry analyses of these samples ( Figure 3B and 6) yielded similar results but showed a moderate gradual increase in the groups according to the number of antigen exposure events ( Figure 3B -C). Notably, the average percentage of IFN-γ positive cells detected in all groups using on-chip analysis (5.3 ± 4.2%) was higher than the flow cytometry detection result (1.8 ± 1.3%), and the latter also analyzed intracellular IFN-γ expression. However, the two sets of data showed good correlation, while the correlation with the standard ELISpot data was much smaller ( Figure 3D and 7). Flow cytometry and chip readings were most consistent for the 3-dose vaccine group and least consistent for the infected group (Figure 7).

[0138] Example 4 - Microfluidic IGRA Analysis of Fingerstick Whole Blood Samples

[0139] ELISpot assays isolate and culture PBMCs from >5 mL of venous blood, which makes it unsuitable for POC testing or use in resource-limited settings. Therefore, we evaluated whether our ELISpot assay could be performed on fingerstick blood volumes (∼25 μL) with a 4-hour peptide incubation step with or without an erythrocyte (RBC) removal step. Compared with whole blood samples, RBC lysis increased the number of captured PBMCs but also increased the IFN-γ response under control peptides, resulting in a corresponding decrease in the induction ratio relative to control peptides in these samples (2.2-fold vs. 1.25-fold)( Figure 3E) Subsequently, the fingertip whole blood samples were analyzed. The samples were collected from individuals with no history of HIV infection and were collected more than six months after the individuals received the third dose of the vaccine. As a result, a similar degree of specific induction (2.4 ± 0.8-fold induction) was detected in all samples, and all samples except one reached significance ( Figure 3F ). The average percentage of IFN-γ positive cells detected in this analysis (3.8%) was lower than the percentage (9.6%) observed in the in vitro ELISpot assay using isolated PBMC samples from other vaccinated individuals, but this was balanced by the reduced sample variance.

[0140] Immunoassays that separately detect the presence or titer of specific antibodies against pathogen-derived factors and the percentage and activity of T cells that respond to these factors provide important but different information that can be used to evaluate the effectiveness of an individual's potential immune response. Specific antibody detection methods are simple and easy to perform and can be easily used in most cases, so they are often suitable for use as POC tests, but may not provide a reliable picture of immunity because the circulating antibody response can decline long before the induced immunity is lost. IGRA may help to address this issue, but is not suitable for high-throughput use or in resource-limited settings and is therefore not applicable for large-scale assessment of individual immune responses. Here, we demonstrate that an improved ELISpot IGRA can be used to address the drawbacks of traditional ELISA because this improved method can be performed using fingertip blood instead of venous blood volume; whole blood can be analyzed without processing the sample to isolate PBMC; and the results can be read using a fluorescence microscope or plate reader within approximately 5 hours after sample collection.

[0141] The ELISpot assay format was selected for this analysis because this assay format measures the proportion of T cells that respond to the selected pathogen-derived factor, providing a direct measure of the cell population that can respond to this pathogen. The more commonly used ELISA-based IGRA measures the relative degree of cytokine response, thus integrating the number of available cells and the degree of their induced cytokine response. Unlike the ELISpot and flow cytometry assay data, which show good correlation despite substantial differences, the ELISpot- and ELISA-based IGRA do not show strong correlation.

[0142] The standard ELISpot assay detects the number of cells secreting the factor, so this factor needs to be bound at its release site for subsequent detection to estimate the number of signal-positive cells in a known number of input cells, which is problematic for high-throughput applications in low-resource settings.

[0143] Conventional ELISpot assays analyze the deposition of dye foci on polyvinylidene difluoride (PVDF) membranes, but this readout method employs a sandwich ELISA where IFN-γ released by activated cells is captured on the PVDF membrane at its release location and hybridized with an enzyme-conjugated IFN-γ specific antibody to allow for in situ conversion and binding of a colorimetric substrate. This requires the use of multiple wash steps, careful control of incubation and reaction times, and a readout device that can capture high magnification images irradiated with a high-intensity light source, thereby complicating the assay workflow, increasing equipment requirements, and reducing its utility in resource-limited settings. Replacing this method with a balanced intracellular staining workflow used in flow cytometry can simplify and reduce the stringency of its readout procedure, which visualizes the relative cell activation rate by co-detecting nuclear staining without the need to analyze a predefined number of PBMCs. For this analysis, there is no need to remove RBCs as the process does not significantly reduce PBMC binding and seems to increase non-specific cell activation, thereby reducing the relative degree of specific induction. However, anucleated RBCs are not included in the cell count and thus do not affect the calculated percentage of cell activation.

[0144] Standard ELISpots assays also include assessing the number of IFN-γ positive cells in live PBMCs at known standard concentrations, which requires cell isolation, determination of cell viability, and long-term culturing in consistent amounts. All these requirements increase the complexity and render these assays impractical in many analytical settings. However, our modified ELTSpot assay uses fingertip whole blood micro-samples, thereby eliminating the need for a trained phlebotomist for venipuncture and subsequent isolation of PMBCs, while allowing for direct detection of the number of IFN-γ positive and total PBMCs present in the analyzed sample from the captured assay images. Given the limited likelihood of variability in sample collection and processing procedures, it can also be expected that cell viability should not affect the percentage of IFN-γ positive cells in this method.

[0145] Although this rapid and simplified ELISpot assay has removed most of the barriers limiting the widespread use of IGRA, several aspects can be further optimized to improve assay performance. For example, the assay does not distinguish between groups of individuals with different exposure histories to the target antigen through infection and / or past infection (which can be detected by standard assays). This may be due to the relatively low number of cells captured on the microplate wells, loss of activated T cells during the washing steps, and / or sampling bias during image capture and analysis. Improving the precision and reproducibility of such assays may be important for enhancing the ability to sensitively track the persistence of acquired T cell responses to pathogen-derived antigens and the relative amount of protective immunity retained over time. Several methods (alone or in combination) can be used to obtain enhanced precision. Cell capture can be enhanced by coating the surface of the detection wells with a polylysine coating, but this electrostatic interaction method is not T cell-specific and may limit the ability to reproducibly capture and retain T cells from whole blood during detection. Conjugating CD4 and / or CD8 specific antibodies to the surface of the detection wells can increase the number of T cells captured from the induced and stained responses of the cells under the chip and their surface retention rate, thereby reducing detection variability. This method may increase cell clustering events, potentially masking the total number of cells and / or positive cells present in the detection. However, the effect of clustering can be offset by fabricating detection chips with antibody spots in discrete arrays. Finally, the ideal form of this ELISpot assay would be read by an inexpensive portable device to allow for point-of-care analysis in resource-limited settings. We have previously developed an inexpensive fluorescence smartphone microscope and application that can be used to read other chip-based assays and can be modified to read, analyze, and report the results of our current ELISpot assay. The technology can also be modified slightly to allow for the recovery of activated cells for single-cell intracellular or secreted protein analysis.

[0146] In summary, the ELISpot of the present invention can be used as a platform for rapidly and inexpensively analyzing T cell responses to specific antigens, using fingertip whole blood micro-samples without the need for extensive equipment or technical expertise. The platform allows for high-throughput analysis of T cell responses to pathogen-derived antigens as a measure of the potential immunity acquired after prior exposure through infection or vaccination. For example, the SARS-CoV-2 peptide pool can be modified by adding specific mutant peptides to assess potential resistance to new variants of these pathogens. Alternatively, variants of this method can also be used to measure memory B cell responses. This ability should allow for large-scale assessment of acquired immune responses, which is beneficial for evaluating the effectiveness of vaccines against existing and emerging infectious diseases and can also contribute to a better understanding of certain chronic infections.

[0147] Example 5 - T cell enrichment and activation time in diagnostic results

[0148] To develop a clinically useful single-step whole blood assay for tuberculosis infection, several prerequisites were considered. We needed to define the entire T cell population by a common labeling method that could cover an expanded TB patient population. We also needed to reduce background signals and specifically detect recently activated T cell responses. We evaluated our test method to detect discrete foci of a colorimetric substrate produced by activated T cells immobilized on a glass surface. However, the weak and diffuse nature of these signals did not allow accurate quantification of the number of activated cells. Therefore, we optimized the labeling method by using the fluorescent dye Hoescht 33342 to fluorescently label the nuclear DNA of all T cells. This method was expected to be unbiased for different cell types and allowed us to calculate the proportion of surface marker-positive T cells and identify staining artifacts in non-Hoechst-labeled objects.

[0149] To maximize T cell counting on the reaction chamber, we investigated the effect of flow rate on T cell attachment within the chip ( Figure 8A ). The successful and firm adhesion of target cells in the biofunctional reaction chamber depends on the formation of bonds that can immobilize the cells to resist hydrodynamic loads and shear stresses that induce bond dissociation. Continuous exposure to stress can affect the shape, proliferation, migration, or protein expression of T cells, thus affecting their ultimate function. Therefore, it is important to control the flow rate during cell introduction and buffer flow. High flow rates will result in fewer captured cells, promoting cell shedding and rolling on the surface, while low flow rates will result in the capture of many normal cells while capturing the desired cells. We optimized the flow rate to 10 μl / min to balance shear and binding forces and maximize specific binding to target cells. We labeled all T cells captured in the reaction chamber with Hoescht 33342 dye for identification. We evaluated different flow rates (5 μl / min, 10 μl / min, 20 μl / min, 60 μl / min, 95 μl / min, and 120 μl / min) and found that the cell count in the reaction chamber decreased significantly when the flow rate was higher than 20 μl / min, while flow rates lower than 5 μl / min resulted in non-specific cell attachment.

[0150] Next, we optimized the surface chemistry to maximize the T cell response obtained from the microchip. The polylysine-coated microfluidic chip captured not only T cells but also other peripheral blood cells, masking the T cell-specific signal in this assay. Other methods, such as using glutaraldehyde to immobilize T cell-specific antibodies on the chip, require additional blocking steps to reduce non-specific binding of peripheral cells to aldehyde sites. Glutaraldehyde itself causes autofluorescence signals, increasing the background noise of the detection assay. Therefore, we optimized the T cell capture method on the chip by using EDC-NHS chemistry with CD4 and CD8 specific antibodies. When CD4 and CD8 T cell-specific antibodies were immobilized on the chip surface, a higher percentage of biomarker-positive cells was found compared to non-specific attachment of all PBMCs using a polylysine coating( Figure 8B ).

[0151] To ensure the specificity of our sensor platform, we performed positive and negative control experiments by activating T cells with TB-specific antigens on 10 T-SPOT.TB positive PBMC samples( Figure 8C ). Phytohemagglutinin (PHA) was used as a positive control to stimulate TSPOT.TB positive PBMC samples, while the reaction without T cell activation was considered a negative control. TSPOT.TB positive PBMCs were stimulated with Ebola-specific peptides to evaluate non-specific reactions. Non-specific stimulation showed a similar response to the negative control, validating the specificity of our sensor platform.

[0152] Finally, we compared the T cell response obtained from the optimized microfluidic chip with that directly obtained from PBMCs by liquid-phase detection. Significantly higher responses were obtained after T cell activation using the microfluidic chip compared to liquid-phase-based detection( Figure 8D ). These findings indicate that the high specificity of receptor-ligand binding provides an extremely sensitive means for T cell manipulation, selection, and T cell-based diagnostics.

[0153] Next, we aimed to determine the shortest activation time required to induce surface marker expression (specifically 4-1BB and OX-40) on T cells( Figure 8E ). The anti-4-1BB antibody used here is the Cd137(4-1BB) monoclonal antibody (4B4(4B4-1)), FITC, eBioscience 11-1379-42. The anti-OX-40 antibody used here is the Cd134(OX40) monoclonal antibody (ACT35(ACT-35)), FITC, eBioscience 11-1347-42.

[0154] We conducted this study on PBMC samples from 30 TSPOT.TB positive patients, which were stimulated with different activation times (2, 4, 6, and 8 hours) and TB-specific peptide antigens (CFP-10 / ESAT-6). To ensure specificity, we also included positive control (PHA stimulation) and negative control (unstimulated) experiments ( Figure 8E - 8K ). Our results showed that the T cell activation marker (4-1BB and OX-40) responses induced by a 2-hour activation time might not be sufficient to achieve a significant change in the 4-1BB and OX-40 responses induced by CFP10 / ESAT6 stimulation compared to the negative control baseline value ( Figure 8F - 8G ). However, as the stimulation time increased (4 hours, 6 hours, 8 hours) ( Figure 8H - 8K ), more T cells were activated and expressed surface markers on their surface. Starting from the 2-hour activation time, the expression of surface markers increased dynamically over time. At the 4-hour activation time, compared with the negative control, T cells provided a sharply changing response, which distinguished them from the basal signal (negative control response). Therefore, we selected a T cell activation time of 4 to 6 hours for further clinical testing.

[0155] Example 6 - Cumulative responses of surface immune markers 4-1BB and / or OX-40

[0156] First, the efficacy of the newly developed platform in diagnosing tuberculosis infection was tested in an HIV-negative PBMC clinical cohort. For this purpose, T cells were enriched on the microchip surface, and the responses of surface markers were evaluated after 6 hours of stimulation. A total of 20 TSPOT.TB positive and 20 TSPOT.TB negative PBMC samples obtained from Houston Methodist were tested, and TB-specific peptides including CFP-10 and ESAT-6 were used to activate T cells. We tested the intracellular IFN-γ as well as the surface expression of 4-1BB and OX-40 using this platform.

[0157] Next, using on-chip technology, the cumulative responses of the 4-1BB and OX-40 expression combinations were evaluated by adding a mixture of fluorescently labeled antibodies against each biomarker tested. As Figure 9A shown, we observed an overlap between the TSPOT+ group and the TSPOT- group in the individual 4-1BB signal and OX-40 signal, while we found that the signals from the TSPOT+ group and the TSPOT- group were significantly different and non-overlapping in the intracellular IFN-γ signal and the cumulative 4-1BB+OX40 signal. Based on this observation, we tested the cumulative responses of 4-1BB and OX-40 on subsequent samples, rather than their individual responses.

[0158] In addition, the direction of the linear relationship between biomarker responses (intracellular IFN-γ, 4-1BB, and OX-40) and TSPOT results was also evaluated. The covariance matrix ( Figure 9B ) indicated that the cumulative responses of intracellular IFN-γ and (4-1BB + OX40) were more closely correlated with TSPOT counts compared to the responses of the individual surface markers 4-1BB or OX40 (Pearson R coefficients of 0.96 and 0.95, respectively), further supporting the fact that the cumulative response of (4-1BB + OX-40) could be a potential strategy for diagnosing TB infection.

[0159] The cumulative responses of intracellular IFN-γ and TSM (4-1BB and OX-40) showed strong correlations with the TSPOT.TB results in the clinical cohort (r2 values of 0.92 and 0.90, respectively) ( Figure 9C -D). We further analyzed the data using a receiver operating characteristic curve (ROC) regression model to retrospectively measure the overall predictive performance of our assay ( Figure 9E -F). The area under the ROC curve (AUC) is a measure of the accuracy or discriminative ability of the assay, and ROC curve analysis shows the trade-off between sensitivity and specificity for the tests performed in the assay.

[0160] This assay showed high specificity (approximately 91%, 95% CI) for the cumulative surface marker (4-1BB + OX-40) response, and the sensitivity of the cumulative surface marker response (100%, 95% CI) was significantly higher than that of intracellular IFN-γ (85.71%, 95% CI) ( Figure 9G ). In addition, the data indicated that a single-step assessment of the cumulative surface marker expression on activated T cells was sufficient to diagnose tuberculosis infection. This eliminated the need to analyze the responses of individual TSM (4-1BB or OX40) and the fixation or permeabilization steps required for intracellular cytokine detection. In summary, this newly developed chip platform has the potential to revolutionize the diagnosis of tuberculosis infection, with high sensitivity and specificity for single-step point-of-care testing.

[0161] Example 7: Evaluation of an Immunocompromised TB Patient Cohort Using an On-Chip Assay

[0162] Next, the microchip assay of the present invention was evaluated for its response in a cohort of immunocompromised patients. A total of 37 samples were collected at Texas Children's Hospital during the period from 2021 to 2023. Among all the samples, 16 samples were from patients and the remaining 11 samples were from healthy individuals. We blindly tested this set of samples using our microchip assay and evaluated the microchip results based on the patients' clinical diagnosis, sputum culture, Gene Xpert, and IGRA results. Each collected sample was divided into four categories for testing, including a positive control (stimulated with PHA), a negative control (no activation), and the microchip results of the cumulative (4-1BB+OX40) response after stimulation with the (CFP10 / ESAT6) peptide and the intracellular IFN-γ response after stimulation with the (CFP10 / ESAT6) peptide.

[0163] The results showed that the intracellular IFN-γ responses in the LTBI positive group and the LTBI negative group could not be clearly distinguished. However, a significant difference in the (4-1BB+OX40) response was observed between the LTBI positive group and the LTBI negative group ( Figure 10A ). This further supported our study and demonstrated the advantage of using the cumulative (4-1BB+OX40) response as a biomarker for LTBI diagnosis. Among the 11 patient samples tested clinically for LTBI, a total of 4 samples showed a negative IGRA status but were positive in clinical diagnosis, Gene Xpert, and sputum culture, indicating that the role of IGRA testing was limited for these 4 patients ( Figure 10B ). The microchip test results for these patients were positive for TBI, demonstrating that the cumulative (4-1BB+OX-40) response was beneficial for this group of patients with poor response to IGRA testing. These 4 patients were further diagnosed with pulmonary pleural coccidioidomycosis, pulmonary cavitary lesions, strongyloidiasis, and primary immunodeficiency. The overall results indicated that the diagnosis based on the cumulative (4-1BB+OX-40) had the potential for patients who benefited limitedly from IGRA.

[0164] Example 8: Blood-based assay for one-step diagnosis of tuberculosis infection

[0165] Traditional PBMC-based assay tests require long-term culture of PBMC isolated from a large volume of venous blood, so it is very difficult to use in resource-limited settings. To solve this problem, the microchip assay of the present invention directly tests from a fingertip blood sample volume (about 25 μL) without an intermediate red blood cell lysis step ( Figure 11A)。This detection method uses specific antibodies against CD4 and CD8 T cells to capture these cells on the sensor surface, while removing other components in the blood through a washing step. We clinically tested 20 patients using our assay in a blinded manner. The blood samples were from Ochsner Health, and we were initially unaware of their clinical information and the QuantiFERON (QFT) gold plus test results. The intracellular IFN-γ and cumulative responses of 4-1BB and OX-40, compared with the (QFT) gold plus results of this clinical cohort ( Figure 11B ) showed strong correlations (r2 values were 0.91 and 0.88, respectively).

[0166] We further analyzed the data using a receiver operating characteristic (ROC) regression model to retrospectively measure the overall predictive performance of this assay ( Figure 11C and 11D ). The area under the ROC curve (AUC) value of intracellular IFN-γ was found to be 0.84, and the area under the ROC curve value of the cumulative (4-1BB and OX40) was 0.94, indicating that the cumulative (4-1BB + OX40) response had better diagnostic discrimination ability in TBI diagnosis than intracellular IFN-γ. In addition, we observed that the cumulative (4-1BB + OX40) response rate in QFT gold plus positive samples was significantly higher than the intracellular IFN-γ response rate ( Figure 11E ).

[0167] The ROC regression model measured the overall predictive performance of this microchip detection method for direct blood sample detection. We found that the sensitivities of this blood-based assay for intracellular IFN-γ and cumulative surface marker responses (4-1BB + OX40) were similar, 78% and 77% respectively, and the specificities were both 100% ( Figure 11F ). Importantly, compared with the PBMC-based detection method, the blood-based analysis provided higher specificity (100%, CI 95%) for intracellular IFN-γ and cumulative surface marker responses ( Figure 11G ). To determine the response rates of whole blood and PBMCs in the same sample group, QFT gold plus TBI positive samples in this group were selected, and the responses obtained from blood samples and PBMCs isolated from the corresponding blood samples were compared. We observed that the intracellular IFN-γ and cumulative (4-1BB + OX40) response rates in whole blood samples were significantly increased compared with isolated PBMCs. This microchip assay showed that the intracellular IFN-γ response rate in whole blood samples increased by approximately 2% compared with PBMC samples ( Figure 11G)。For the (4-1BB+OX40) cumulative response, microchip assays showed that the blood-based assay increased the response by nearly 4-5% compared to the PBMCs-based assay. Figure 11H )。In summary, we were able to successfully perform our microchip assay using direct blood samples without an RBC lysis step, enabling one-step, rapid, ultrasensitive, and point-of-care TBI diagnosis.

[0168] It has been shown that since only T cell subsets including CD4 T helper cells are mainly responsible for IFN-γ production, IGRA cannot detect the entire T cell response against any given antigen. Latent TB-infected individuals with low peripheral lymphocyte (CD4 T cell) counts, immunocompromise, or extrapulmonary TB infection derive limited benefit from IGRA. Additionally, the patient population in the active phase of TB infection cannot benefit from IGRA because a sharp increase in regulatory T (Treg) cells in the active TB infection state inhibits the release of IFN-γ by helper T (Th) cells. The reduced ratio of Mycobacterium tuberculosis-specific CD4 T cells that produce Th1, Th2, and Th17 cytokines, including IFN-γ, is the main reason for the limited benefit that HIV-infected individuals derive from IGRA.

[0169] Furthermore, there are still significant clinical gaps in TB infection screening among the HIV-infected patient population, which increases the overall TB incidence and related mortality. Additionally, common problems observed for TST and IGRA include sample maintenance and handling, the requirement for 2-10 mL of blood (limiting its use in very young children), long diagnostic times (sample-to-result intervals exceeding 24 hours), and errors during blood sample collection or transportation, which further affect diagnostic accuracy. Reduced access to TB diagnosis and treatment is one of the main causes of TB-related deaths. To address these problems with existing TBI diagnostic technologies, we initiated research and developed a point-of-care technology independent of IFN-γ.

[0170] Methods

[0171] Patient population: Whole venous blood and fingerstick blood samples were obtained from the adult population with SARS-COV-2 infection and / or vaccination who participated in our study at Children's Hospital of New Orleans.

[0172] Under the Tulane University Biomedical Institutional Review Board, subjects or families with suspected or confirmed SARS-CoV-2 infection were recruited from the Greater New Orleans community (Federalwide Assurance number FWA00002055, study number 2020-585). Enrolled subjects completed a study questionnaire regarding infection and demographic information and provided blood samples.

[0173] For fingerstick blood analysis research, healthy SARS-COV-2 vaccinated adults aged 21 to 41 years were recruited according to a protocol approved by the Institutional Review Board of Tulane University. Written informed consent was obtained from each participant prior to study participation. A SARS-COV-2 screening questionnaire and information regarding vaccination status were also obtained. Blood samples were collected from the fingertips of each participant using a touch lancet (BD 355594), and 400 - 800 μL of blood was collected into lithium heparin microtainers (BD 365965) and then immediately processed for ELISpot analysis on the chip. The participants did not report any adverse reactions or sequelae of infection.

[0174] PBMC isolation: PBMC were isolated from cryopreserved leukapheresis samples (Stemcell Technologies) or whole blood samples. Venous blood samples were collected in EDTA tubes and 15 volumes of cold (4 °C) isotonic ammonium chloride solution were added. The samples were inverted and mixed for 10 minutes at room temperature using a rotary mixer set at approximately 500 rpm to lyse red blood cells, and then centrifuged at 250 g for 10 minutes. The cell pellet was then resuspended in 1 mL of PBS, and this cell suspension layer was placed on top of 10 mL of Ficoll-Paque PLUS medium (Cytiva 17144002) in a 15 mL centrifuge tube and centrifuged at 500 g for 20 minutes in a Swinging buck rotor to isolate PBMC according to the manufacturer's instructions. The isolated PBMC were suspended in 5 mL of AIM V cell culture medium (Fisher Scientific 31-035-025), the cells were stained with 0.4% trypan blue solution, an aliquot was analyzed to determine the viable cell concentration, and the cell suspension was adjusted to a final concentration of 3x10 6 / mL in AIM V cell culture medium (Fisher Scientific 31-035-025), mixed with 40% fetal bovine serum and 20% dimethyl sulfoxide, and then stored in the vapor phase of a liquid nitrogen dewar.

[0175] Blood was collected from the subjects, and plasma and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation in Leukosep tubes (Greiner Bio One) and Ficol-Paque PREMIUM 1.078 g / ml (Cytiva). The PBMC were washed, counted, and suspended at a density of Ix10 7 cells / ml in FBS-10% DMSO. Aliquots of the cells were frozen at -80 °C in a Nalgene Mr.Frosty container (Nalgene Labware, Rochester, NY) and then finally stored in liquid nitrogen.

[0176] PBMC Stimulation: Aliquots of cryopreserved PBMCs were rapidly thawed in a 37°C water bath, mixed with an equal volume of RPMI-1640 medium heated to 37°C, and then centrifuged at 400 g for 5 minutes. The cell pellet was washed with 2 mL of RPMI-1640, resuspended in 150 μL of RPMI-1640, and analyzed by trypan blue exclusion to assess cell viability, and then supplemented with RPMI-1640 to a final working concentration of approximately 3 x 10 6 viable cells / mL. Samples with a cell viability < 70% were excluded from the analysis. PBMCs were seeded in 6-well cell culture plates at a concentration of 1 x 10 6 to 2 x 10 6 viable cells / well, with the specific concentration determined by different assay types, and then stimulated with 10 ng / mL phorbol 12-myristate 13-acetate (PMA, Sigma P1585) and 1 μg / mL ionomycin (STEM CELL 73722) or 1 μg / mL of the indicated peptide or peptide pool (BEI NR-52402) for the specified time at 37°C.

[0177] Flow Cytometry: Aliquots of PBMCs suspended in AIM V cell medium (2 x 10 6 / mL) were cultured overnight in 24-well culture plates, then stimulated with PMA and ionomycin (10 ng / mL and 1 μg / mL respectively) or SARS-CoV-2 or HIV-p24 peptide pools (1 μg / mL) for 24 hours, and 1 ng / mL of IFN-γ transport blocker was added 2 hours after the start of induction. After stimulation, the PBMCs were centrifuged at 500 g for 5 minutes, washed with PBS, and then resuspended in 100 μL of IC fixation buffer and permeabilization buffer (eBioscience 00-8222-49 and 00-8333) for 10 minutes, and then incubated in PBS / 10% BSA solution supplemented with 1 μg / ml AlexaFluor488-labeled IFN-γ specific antibody (eBioscience 50-168-09) for 20 minutes. Flow cytometry analysis was performed on gated cells using an Attune flow cytometer (Thermo Scientific), the IFN-γ positive cell signal was captured in the FITC / GFP channel, and the captured data was analyzed and quantified using Flow Jo software (v10.04).

[0178] IGRA ELISA: PBMCs (2 x 10 4) Incubate for the specified time at 37°C in X mL of RPMI-1640 medium supplemented with 1 μg / mL SARS-COV-2 peptide pool (BEI NR-52402), PMA, and ionomycin (10 ng / ml and 1 μg / ml), or without adding anything, with the wells containing only RPMI serving as negative controls. Aspirate the culture supernatant from each well and store it at -80°C for future ELISA analysis.

[0179] After incubation, aspirate the medium from the wells into a new 98-well plate. At this time, add the SARS-COV-2 peptide pool at a final concentration of 1 μg / mL to the stimulated group. At 4, 6, 8, 10, 12, and 24 hours, remove the supernatant and store it at -80°C for subsequent ELISA analysis.

[0180] The IGRA ELISA plates are prepared as follows: 96-well MaxiSorp plates (Nunc 44-2404-21) are incubated overnight at 4°C with 100 μL of a PBS solution of 1 μg / ml human IFN-γ-specific antibody (Endogen, M700-A). Then wash these plates 6 times with PBS / 0.05% Tween 20 (PBST), block them with 200 μl of 1% BSA / PBS for 1 hour at room temperature, then wash, dry, and store at 4°C until use. Thaw the aliquots of cryopreserved PBMC culture supernatant and transfer them to the assay plates in triplicate (50 μL / well) and incubate for 1 hour at room temperature. Add 50 μL of IFN-γ-biotinylated antibody (Endogen, M-701B) diluted 1:1000 in 2% FBS / IXPBS to each well and incubate for 1 hour at room temperature. Wash and dry the plates, then add 50 μL / well of Poly-HRP streptavidin (Pierce, N200, diluted 1:5000 in 1% BSA / 1X PBS) and incubate for 30 minutes at room temperature in the dark. After that, wash and dry the plates. Add 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB, Thermo Scientific 34029) solution and observe the color development. After sufficient color development (~10 minutes), add 50 μL / well of stop solution (2.5N H2SO4) and read the plates at OD450.

[0181] ELISPOT: Coat a FilterScreen plate (Millipore MAIPS4510) with anti-human IFN-γ (Endogen, M700-A, 1 mg / ml) at a concentration of 1 μg / ml and store it overnight at 4°C. The next day, wash the plate 6 times with wash buffer (I×PBS + Tween 20 diluted 1:2000) and pat dry. Block the wells with 200 μl of 1% BSA / 1X PBS for 1 hour at room temperature. Then inoculate 2X10 3 PBMCs into the plate and stimulate them with PMA-ionomycin (10 ng / ml and 1 μg / ml), SARS-CoV-2 spike peptide pool (1 μg / mL), or HIV-p24 peptide (1 μg / mL). Add 100 μl of IFN-γ-biotinylated antibody (Endogen, M-701B) diluted 1:1000 in 1% BSA / IX PBS to each well and incubate for 1 hour at room temperature. Wash and dry the plate, then add 100 μl / well of Poly-HRP streptavidin (Pierce, N200, diluted 1:5000 in 1% BSA / 1X PBS) and incubate for 30 minutes at room temperature in the dark. Then add 100 μl / well of 3-amino-9-ethylcarbazole (AEC, BD557630) and incubate for 15 minutes at room temperature. Wash the entire plate with DI water, separate the bottom to dry completely overnight. Scan the spots using a CTL-Immunospot S6 Universal Analyzer (ImmunoSpot) and count them using the Dual Color ELISPOT Enzymatic software (ImmunoSpot).

[0182] Chip fabrication

[0183] Silicon wafers with a microfluidic design are fabricated according to the previously described method. A polydimethylsiloxane (PDMS) mold with the said design is fabricated from this silicon wafer (Figure 4). Mix the PDMS elastomer with the curing agent at a ratio of 10:1 and then pour it onto the silicon wafer. The curing agent crosslinks the elastomer and forms a rigid structure, which will cure into a complete chip. To accelerate the PDMS curing rate, place the PDMS mold in an oven at 60°C for 5 hours. After the elastomer is completely cured, remove the mold from the silicon wafer for chip assembly. Perform plasma treatment on the PDMS chip and a 1 mm glass coverslip to allow the formation of silanol functional groups, which can form strong covalent bonds with each other to create a fluid seal to form the microfluidic channels.

[0184] ELISpot assay on chip:

[0185] PMA-ionomycin (10 ng / ml and 1 μg / ml), SARS-CoV-2 spike peptide pool (1 μg / mL), or HTV-p24 peptide (1 μg / mL) was added to 25 μL of whole blood and then incubated at 37 °C for 4 hours. The blood samples were fixed with IC fixation buffer (eBioscience TM 00-8222-49) and permeabilized with permeabilization buffer (eBioscience TM 00-8333) at 25 °C for 20 minutes, and then stained with 1 μg / ml anti-IFN-γ-Alexa488 (eBioscience 50-168-09) and 0.1 μg / mL Hoechst 33342 at 25 °C for 20 minutes. Detection on the chip was performed as described above.

[0186] Image capture and analysis: Images of PBMCs bound to the microfluidic chamber were acquired using an EVOS TM M5000 imaging system (manufactured by Invitrogen, Thermo Fisher Scientific, Madrid, Spain), scale bar: 300 μm. Images (10X) of stained PBMCs reflect the total cell population and IFN-γ-positive cells. A green fluorescence signal was obtained when Alexa 488 bound to intracellular IFN-γ. The blue fluorescence signal from Hoechst 33342 represents the total cell count. All experiments were performed in triplicate. Four different random regions in the microfluidic chamber were selected each time to acquire images. All data acquired on the EVOS TM M5000 imaging system were analyzed using ImageJ software.

[0187] Cell counting: The total cell count and the proportion of IFN-γ-positive cells were quantified using the National Institutes of Health (NIH) Image J image analysis software. The images were converted to 8-bit grayscale. The lower threshold was set to 70 and the upper threshold was set to 255. Cell counts with a size range of 1 to 100 (pixels) and a roundness of 0.00 - 1.00 were analyzed.

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Claims

1. A method for identifying pathogen - specific T - cell activation, comprising the following steps: a) Obtaining a biological sample from a subject; b) Introducing an incubation mixture into the biological sample, wherein the incubation mixture comprises (i) peptides of a target pathogen or peptides of a target vaccine, and (ii) an antibody that specifically binds to T cells activated upon stimulation with the peptides of the target pathogen or the target vaccine; c) Detecting the presence of activated T cells in the biological sample from step b); wherein the antibody in step b) is conjugated to an enzyme or a fluorescent molecule.

2. The method according to claim 1, wherein the biological sample is a whole - blood sample.

3. The method according to claim 2, wherein the biological sample is not subjected to peripheral blood mononuclear cell (PBMC) isolation or red - blood - cell removal.

4. The method according to claim 2, wherein the volume of the whole - blood sample is less than 1 mL.

5. The method according to claim 2, further comprising the following step b - 1) after step b): b - 1) Obtaining T cells in the whole - blood sample by CD4 - and CD8 - specific antibodies.

6. The method according to claim 1, wherein the target pathogen is SARS - CoV - 2, HIV, Mycobacterium tuberculosis, cytomegalovirus (CMV), influenza, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein - Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma, or Trypanosoma cruzi.

7. The method according to claim 6, wherein the peptides of the target pathogen comprise peptides from the SARS - CoV - 2 spike peptide pool, Mycobacterium tuberculosis, BEI NR - 52402, cytomegalovirus (CMV), influenza, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein - Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma, Trypanosoma cruzi, or tumor - specific antigen peptides from NY - ESO - 1, HER2, PSA, TRP - 2, EpCAM, GPC3, mesothelin (MSLN), MUC1, EGFR, OX - 40, CD59, LAG - 3, TIM3, IL - 12R, CD28, CD57, KIR, KLRG - 1, CD27, PD - 1, or CTLA - 4.

8. The method according to claim 1, wherein in step b), at least one of the following substances is further introduced into the biological sample: phorbol 12 - myristate 13 - acetate (PMA) and ionomycin.

9. The method according to claim 1, wherein the antibody is an OX - 40 or 4 - 1BB - specific antibody.

10. The method according to claim 1, wherein the antibody binds to IFN - γ and is M700 - A from Endogen.

11. The method according to claim 1, wherein step b) is carried out for 1 to 6 hours.

12. A point - of - care test kit for identifying pathogen - specific T - cell responses, comprising: a) A microfluidic device having a plurality of microfluidic channels connecting a sample inlet to a detection chamber; b) wherein the detection chamber is coated with polylysine.

13. The point-of-care test kit according to claim 12, further comprising an incubation container having a peptide from a target pathogen or a peptide from a target vaccine, wherein a biological sample is introduced into the incubation container.

14. The point-of-care test kit according to claim 13, wherein the detection chamber further comprises at least one of the following: an anti-human IFN-γ antibody, an anti-4-1BB antibody, and an anti-OX-40 antibody, wherein the antibody is conjugated to an enzyme or a fluorescent molecule.

15. The point-of-care test device according to claim 13, wherein the target pathogen is SARS-CoV-2, HIV, Mycobacterium tuberculosis, cytomegalovirus (CMV), influenza, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma, or Trypanosoma cruzi.

16. The point-of-care test device according to claim 13, wherein the target vaccine is a vaccine against SARS-CoV-2, Mycobacterium tuberculosis, cytomegalovirus (CMV), influenza, respiratory syncytial virus (RSV), herpes simplex virus (HSV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma, or Trypanosoma cruzi.

17. The point-of-care test kit according to claim 13, wherein the incubation container further comprises phorbol 12-myristate 13-acetate (PMA) and ionomycin.

18. A method for identifying pathogen-specific T cell activation, comprising the steps of: a) obtaining a biological sample from a subject; b) introducing an incubation mixture into the biological sample, wherein the incubation mixture comprises (i) a peptide from a target pathogen or a peptide from a target vaccine, and (ii) an antibody specific for a cytokine or a surface marker, wherein the cytokine is secreted by T cells in the biological sample after being stimulated by the peptide from the target pathogen or the peptide from the target vaccine; and c) detecting the presence of the cytokine or the surface marker in the reaction chamber of the point-of-care test kit of claim 12; wherein the cytokine or surface marker specific antibody is conjugated to an enzyme or a fluorescent molecule.

19. The method according to claim 18, wherein the cytokine is IL-2, IL-4, IL-17, or TNFα.

20. The method according to claim 18, wherein the surface marker is at least a part of OX-40, 4-1BB, CD59, LAG-3, TIM3, IL-12R, CD28, CD57, KIR, KLRG-1, CD27, PD-1, CTLA-4, IFN-γ, IL-2, IL-10, or TNF-α.

21. The method according to claim 18, wherein the mixture in step b) is introduced into the reaction chamber at a flow rate of 5 μl / min to 20 μl / min.

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