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  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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  • G01N33/56911—Bacteria
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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  • G01N2333/01—DNA viruses
  • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
  • G01N2333/035—Herpes simplex virus I or II
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  • G01N2333/01—DNA viruses
  • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
  • G01N2333/04—Varicella-zoster virus
  • G01N2333/045—Cytomegalovirus
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  • G01N2333/01—DNA viruses
  • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
  • G01N2333/05—Epstein-Barr virus
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
  • G01N2333/135—Respiratory syncytial virus
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
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  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa

Definitions

• the disclosure generally relates to device and method for measuring specific T-cell activation, and more specifically to point-of-care on-chip IGRA device for measuring the T-cell activation by a pathogen, vaccine or therapy.
• IGRA assays measure either the amount of IFN-y produced or the number of cells that secrete IFN-y after stimulation with specific peptides derived from a pathogen of interest.
• the former approach relies on ELISA to detect IFN-y secretion indicative of antigen-specific T cell induction, while the latter employs an enzyme-linked immunosorbent spot (ELISpot) assay to detect IFN-y released and bound in proximity to activated T cells immobilized on a detection membrane.
• ELISpot enzyme-linked immunosorbent spot
• ELISpot assays are more technically demanding than ELISA-based IGRAs since they require that peripheral blood mononuclear cells (PBMCs) be isolated from blood samples prior to stimulation with pathogen-specific target peptides and that the resulting PBMC culture reaction wells be scanned to quantify the number of colorimetric spots that indicate the number of responsive cells.
• PBMCs peripheral blood mononuclear cells
• both ELISpot and ELISA-based IGRAs can be technically demanding and are thus typically performed at central laboratories so that sample shipping logistics can be a limiting factor in assay performance.
• Latent Mycobacterium tuberculosis (M.tb) infections are estimated to affected one third of the world population, and have a 5 - 10% risk for progression to TB disease, which is responsible for 1.5 million deaths each year, more than any other infectious disease, except during the height of the COVID- 19 pandemic.
• Individuals with latent M.tb-infections are at the greatest risk for TB disease within the first two years after M.tb infection but can remain at risk for disease progression throughout their lifetime. Addressing this latent disease reservoir is thus critical for the End TB Strategy to reduce TB incidence 90% by 2035 and eliminate TB by 2050.
• TSTs tuberculin skin tests
• IGRAs interferon gamma release assays
• IGRAs do not require repeat visits and do not produce false positives for BCG-vaccinated individuals, but utilize whole blood samples that must be maintained under controlled conditions and used within ⁇ 16 hours of collection.
• the sensitivity of both tests is also reduced by factors that attenuate immune responses to their targeted M.tb antigens and are thus less reliable when used on individuals with compromised immune systems, recent M.tb infections, or recent live-virus measles or smallpox vaccinations, all of which can induce false negative results.
• HIV infection is a particular concern for M.tb screening efforts since HIV co-infected individuals are at increased risk for rapid progression from latent M.tb infection to TB, which is responsible for one third of HIV-related deaths.
• M.tb and HIV co-infection rates are also often high in regions with high endemic TB rates, and exceed 50% in parts of southern Africa.
• CD4 T-cells play an essential role in the IFN-y response induced following M.tb infection but IGRA results cannot reliably detect latent M. tb infections in HIV-infected individuals who have CD4 T-cells counts ⁇ 200 cells/pL.
• detection of activation markers increases that are less restricted to CD4 T-cells could enhance detection of /b-specific immune responses in HIV co-infected individuals, selecting 0X40 (TNFRSF4/CD134) and 4- IBB (TNFRSF9/CD137) as candidate markers to detect M.tb infections in individuals with M.tb and HIV co-infections.
• 0X40 is highly expressed activated versus nonactivated CD4 and CD8 T-cells and may thus serve as a more resilient marker of antigen-specific T-cell activation than CD4 T- cell IFN-y expression.
• 4- IBB expression is also induced on both activated CD4 and CD8 T-cells, although CD8 T cells can upregulate 4-1BB more rapidly and to a higher level than CD4 T cells.
• both these proteins are T-cell surface markers (TSMs) and thus can be directly detected with labeled specific antibodies, eliminating the need for the cell fixation and permeabilization steps required in intracellular cytokine staining assays.
• VOCs SARS-CoV-2 variants of concern
• the spike protein of Omicron currently the dominant VOC strain in the US, can evade neutralization by antibodies produced by vaccinated individuals and convalescent patients with 10- to 44-fold higher efficiency than the Delta spike protein, and its spike protein is resistant to neutralizing antibodies of convalescent patients and vaccinated individuals, which can be highly variable and rapidly decrease with time.
• immunocompromised individuals can exhibit inadequate seroconversion rates and neutralizing antibody responses following SARS-CoV-2 vaccination, but still reveal significant virus-specific T-cell responses, including strong T cell immunity to Omicron.
• Omicron can evade specific neutralizing antibodies, but still activate T-cell responses induced by prior vaccination or infection, with one study indicating that 70-80% of the vaccine-induced CD4 and CD8 T-cell response to the spike protein of the reference strain was retained for Omicron.
• IFN- y interferon-gamma
• TGRAs interferon-gamma
• Analysis of T-cell responses to emerging SARS-CoV-2 VOCs may allow rapid prospective evaluation of vaccine efficacy and inform the need for additional vaccine doses or the development of variant-specific vaccines.
• Microfluidic techniques can simplify assay workflows, reduce sample volumes to decrease reagent costs, and minimize technical requirements and assay variation by automating key sample handling steps.
• Microfluidic approaches have recently been employed for COVTD-19 diagnostic assays and several microfluidic sensing platforms for RT- PCR, antigen or antibody tests have received Emergency Use Authorization (EUA) approval from the FDA and been commercialized for COVID-19 diagnosis.
• EUA Emergency Use Authorization
• a microfluidic IGRA could be developed to permit broad application of IGRAs to analyze T-cell responses that could be used to evaluate vaccine efficacy over time, including potential responses against emerging SARS-CoV-2 VOCs, as well as other pathogens and targets for immunotherapies.
• microfluidic ELISpot- based IGRA that provides point-of-care (POC) analysis of T-cell responses to various pathogens that are capable of eliciting T-cell response, for example, M. tb markers or SARS-CoV-2 target peptides.
• POC point-of-care
• a method and microfluidic assay platform that requires less time, infrastructure and expertise than standard TGRAs is proposed herein.
• the method and assay platform for M. tb detection detects at least one of multiple M. tb markers to improve its sensitivity in patient populations with impaired CD4 T-cell response and simplify its detection workflow. Similar method/assay platform is also proposed for detecting SARS-CoV-2 target peptides.
• microchip results were comparable to those produced by other immunoassay methods, including flow-cytometry and conventional ELISpot assay results, when evaluating the response of individuals who had or had not been vaccinated against or infected with SARS-CoV-2.
• This microfluidic chip assay provides results within 5 hours using fingerstick blood samples ( ⁇ 25 pL) thereby reducing both the sample-to-answer time and the sample handling and equipment requirements for IGRA. Notably, this assay is readable using a cellphone microscope permitting utilization in resource limited areas.
• a method of identifying pathogen-specific T-cell activation using a microfluidic chip 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) peptides of a pathogen of interest or peptides of a vaccine of interest; and (ii) an antibody binding specific to activated T-cells upon stimulation by the peptides of the pathogen of interest or by the peptides of the vaccine of interest; c) detecting presence of activated T-cells in the biological sample from step b); wherein the antibody in step b) is conjugated with an enzyme or a fluorescent molecule.
• a point-of-care kit for identifying pathogenspecific T-cell response.
• the point-of-care kit comprises a microfluidic chip having a plurality of microfluidic channels connecting a sample inlet to a detection chamber, wherein the detection chamber is coated with poly-lysine or other cell attachment enhancing reagents, gelatin.
• the detection chamber can also be coated with T-cell specific antibodies such as anti-CD4 and anti-CD8 antibodies in order to better capture CD4+ and CD8+ T-cells within PBMCs.
• the microfluidic channels allow sufficient time to enhance the T-cell response against antigen peptides, while speeding up cytokine release of T-cell activation surface marker expression.
• a method of identifying pathogen-specific T- cell activation using a microfluidic chip described herein comprises: 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 pathogen of interest or peptides of a vaccine of interest, and (ii) an antibody specific to a cytokine or a surface marker, wherein the cytokine is secreted by T-cells in the biological sample upon stimulation by the peptides of the pathogen of interest or by the peptides of the vaccine of interest; c) introducing the biological sample from step b) into the detection chamber in the microfluidic chip through the sample inlet; and d) detecting presence of the cytokine or the surface marker in the detection chamber; wherein the cytokine- or surface marker-specific antibody is conjugated with an enzyme or a fluorescent molecule.
• the antibody binding specific to activated T-cells are antihuman interferon-gamma antibodies, or antibodies binding specific to cytokines or surface markers expressed by activated T-cells.
• the anti-human interferon-gamma antibodies target and bind to interferon gamma secreted by T-cells after being activated by peptides coming from a pathogen of interest.
• the anti-human interferon-gamma antibodies are conjugated with an enzyme or fluorescent molecules in order to generate visual signals upon binding to interferon gamma.
• the fluorescent signals to indicate the presence of fFN-y.
• the method further comprising step b-1): obtaining T-cells in the whole blood sample by CD4 and CD8 specific antibodies.
• the point-of-care kit can further comprise an incubation container within which peptides from a pathogen of interest are present.
• the collected biological sample can be introduced into the incubation container and incubate with the peptides in order for the T-cells to be activated.
• the method comprises an activation step, wherein the T-cells in the biological sample is activated by the pathogen-specific peptides or vaccines of interest or markers for a predetermined period of time.
• the activation step lasts about 1 to 6 hours. In another embodiment, the activation step lasts about 2 to 6 hours.
• the incubation container further comprises phorbol 12- myri state 13 -acetate (PMA) and ionomycin for T-cell stimulation.
• the fluorescent molecule is Fluorescein isothiocyanate (FITC) or Alexa Fluor® 488.
• the pathogen of interest is SARS-CoV-2, HIV or M. tuberculosis.
• Other pathogens may elicit T-cell responses can also be detected.
• Non-limiting pathogens include Cytomegalovirus (CMV), Influenza, Respiratory syncytial virus (RSV), herpes simplex virus (HSV), Hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma gondii, and Trypanosoma cruzi.
• treatment response of interest is vaccines (SARS-CoV-2 vaccine) may elicit T cell response.
• Non-limiting vaccines include Cytomegalovirus (CMV), Influenza, Respiratory syncytial virus (RSV), herpes simplex virus (HSV), Hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma gondii, and Trypanosoma cruzi.
• Other antigen-specific T-cell response evaluation assay comprises CAR-T, TCR-T therapy, PD-1/PD-L1, CTLA-4, TIM3, LAG3 or other checkpoint blockage treatment in cancer.
• the method and device of this disclosure can include antibodies against these immune checkpoint proteins and screen for corresponding T-cell responses.
• the pathogen of interest is M. tb and the peptide or markers that can be used to capture the activated T-cells comprises 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-y, IL-2, IL-10, TNF-cr.
• the peptide or makers are extracellular domain of OX- 40, 4-1BB, CD59, LAG-3, TIM3, and TL-12R, CD28, CD57, KIR, KLRG-1 , CD27, PD-1, CTLA-4, IFN-y, IL-2, IL- 10, TNF-cr.
• the detection chamber in the microfluidic is treated with CD4- or CD8-specific antibodies.
• the CD4- or CD8-specific antibodies are treated via EDC-NHS chemistry.
• the detection chamber is coated with poly-lysine, and the concentration of the poly-lysine can range from 1 pg/mL to 100 pg/mL. In another embodiment, the concentration of the poly-lysine can range from 5 pg/mL to 50 pg/mL.
• the microfluidic channel has a width between 10pm and 200 pm, and a height between 10pm and 200pm. In another embodiment, the microfluidic channel has a width between 50pm and 150 pm, and a height between 50pm and 150pm. In another embodiment, the microfluidic channel has a width of about 100pm, and a height of about 100pm.
• the detection chamber has a size of 1 to 10 mm 2 . In one embodiment, the detection chamber has a size of 10 mm x 3 mm x 0.1 mm.
• the biological sample is whole blood sample from fingerstick. This is different from the conventional ELISpot where peripheral blood mononuclear cells (PBMCs) must first be isolated before being subject to the test.
• PBMCs peripheral blood mononuclear cells
• the amount of the whole blood sample is less than 1 mL. In one embodiment, the amount of the whole blood sample is less than 100 pL. In another embodiment, the amount of the whole blood sample is less than 50 pL.
• the peptides of the pathogen of interest include SARS-CoV-2 spike peptide pool and BEI NR-52402.
• SARS-CoV-2 spike peptide pool and BEI NR-52402.
• other peptide pools can also be used, as long as they represent the commonly encountered peptides from the pathogen that may elicit T-cell specific response.
• Non-limiting examples include peptides from Cytomegalovirus (CMV), Influenza, Respiratory syncytial virus (RSV), herpes simplex virus (HSV), Hepatitis B virus (HBV), Epstein-Barr virus (EBV), Listeria, Salmonella, Plasmodium, Toxoplasma gondii, Trypanosoma cruzi, or tumor specific antigen peptide from NY-ESO-1, HER2, PSA, TRP-2, EpCAM, GPC3, mesothelin (MSLN), MUC1 and EGFR.
• CMV Cytomegalovirus
• RSV Respiratory syncytial virus
• HSV herpes simplex virus
• HBV Hepatitis B virus
• EBV Epstein-Barr virus
• Listeria Salmonella
• Plasmodium Plasmodium
• Toxoplasma gondii Trypanosoma cruzi
• MSLN mesothelin
• the first interferon-gamma-specific antibody is M700-A from Endogen.
• other interferon-gamma-specific antibody can also be used.
• the mixture of the biological sample and the incubation mixture is introduced into the reaction chamber of the point-of-care device at a flow rate between 5pl/min to 20pl/min.
• the cytokine is IL-2, IL-4, IL- 17 or TNFa. However, other cytokines may also be used.
• LAG-3, TIM3, and IL-12R CD28, CD57, KIR, KLRG-1, CD27, PD-1, CTLA-4, IFN-y, IL-2, IL- 10, or TNF-cr.
• the portion of OX-40 is the extracellular domain of OX-40, having the following amino acid sequence: LH CVGDTYPSND RCCHECRPGN
• GPSTRPVEVP GGRA SEQ ID NO. 1.
• the portion of 4-1BB is the extracellular domain of 4-1BB, having the following amino acid sequence: LQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQ (SEQ ID NO. 2).
• the portion of CD59 is the extracellular domain of CD59, having the following amino acid sequence: MGIQGGSVLFGLLLVLAVFCHSGHSL QCYNCPNPTADCKTAVNCSSDFDACLITKAGLQVYNKCWKFEHCNFNDVTTRLRENEL TYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFLAAAWSLHP (SEQ ID NO 3)
• the portion of LAG-3 is the extracellular domain of LAG-3, having the following amino acid sequence: LQPGAEVPVVWA
• the portion of TIM3 is the extracellular domain of TIM3 having the following amino acid sequence: MTPWLGLIVLLGSWSLGDWGAEACTCSP SHPQDAFCNSDIVIRAKVVGKKLVKEGPFGTLVYTIKQMKMYRGFTKMPHVQYIHTEA SESLCGLKLEVNKYQYLLTGRVYDGKMYTGLCNFVERWDQLTLSQRKGLNYRYHLGC NCKIKSCYYLPCFVTSKNECLWTDMLSNFGYPGYQSKHYACIRQKGGYCSWYRGWAP PDKSIINATDP (SEQ ID NO. 5).
• the portion of CD28 is the extracellular domain of CD28, having the following amino acid sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSR EFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQ TDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO 6).
• the portion of KIR is the extracellular domain of KIR, having the following amino acid sequence: IPFLEQNNFSPNTRTQKARHCGHCPEEWITYSNSCYYI GKERRTWEESLLACTSKNSSLLSIDNEEEMKFLASILPSSWIGVFRNSSHHPWVTINGLAF KHKIKDSDNAELNCAVLQVNRLKSAQCGSSMIYHCKHKL (SEQ ID NO. 7).
• the portion of KLRG-1 is the extracellular domain of KLRG- 1, having the following amino acid sequence: LCQGSNYSTCASCPSCPDRWMKYGNHCY YF S VEEKD WNS SLEFCL ARD SHLL VTTDNQEMSLLQ VFL SE AFCWTGLRNNSGWRWED GSPLNFSRISSNSFVQTCGAINKNGLQASSCEVPLHWVCKKCPFADQALF (SEQ ID NO. 8).
• the portion of CD27 is the extracellular domain of CD27, having the following amino acid sequence: ATPAPKSCPERHYWAQGKLCCQMCEP GTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAEC ACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQT LADFRQLPARTLSTHWPPQRSLCSSDFIR (SEQ ID NO. 9).
• the portion of PD-1 is the extracellular domain of PD-1, having the following amino acid sequence: FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRND SGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO. 10).
• the portion of CTLA-4 is the extracellular domain of CTLA- 4, having the following amino acid sequence: KAMHVAQPAVVLASSRGIASFVC EYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQ GLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD (SEQ ID NO. 11).
• the portion of IL2Ra is the extracellular domain of IL2Ra, having the following amino acid sequence: ELCLYDPPEVPNATFKALSYKNGTILNCECK RGFRRLKELVYMRCLGNSWSSNCQCTSNSHDKSRKQVTAQLEHQKEQQTTTDMQKPT QSMHQENLTGHCREPPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQRGPAISICKMKC GKTGWTQPQLTCVDEREHHRFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETF VLTMEYK (SEQ ID NO. 12).
• the anti-4-lBB antibody herein is Cdl37 (4-1BB) Monoclonal Antibody (4B4 (4B4-1)), FITC, eBioscience 11-1379-42.
• the anti-OX-40 antibody used herein is Cdl34 (0X40) Monoclonal Antibody (ACT35 (ACT-35)), FITC, eBioscience 11-1347-42.
• cytokine refers to any of a number of substances, such as interferon, interleukin, and growth factors, which are secreted by certain cells of the immune system and have an effect on other cells.
• surface marker refers to special proteins or peptides expressed on the surface of cells or carbohydrates attached to the cell membrane that often conveniently serve as markers of specific cell types.
• IFN-y refers to a dimerized soluble cytokine that belongs to the type II interferons.
• IFN-y is critical for innate and adaptive immunity against viral, some bacterial and protozoan infections. IFN-y is an important activator of macrophages and inducer of maj or histocompatibility complex class II molecule expression. IFN- y is produced predominantly by natural killer cells (NK) and natural killer T cells (NKT) as part of the innate immune response, and by CD4 Thl and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops as part of the adaptive immune response.
• NK natural killer cells
• NKT natural killer T cells
• CTL cytotoxic T lymphocyte
• 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-kappaB through its interaction with adaptor proteins TRAF2 and TRAF5.
• 4- IBB 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 contributes to the clonal expansion, survival, and development of T cells. It can also induce proliferation in peripheral monocytes, enhance T cell apoptosis induced by TCR/CD3 triggered activation, and regulate CD28 co-stimulation to promote Thl cell responses.
• CD59 is also known as 1F5, EJ16, EJ30, EL32, G344, MINI, MIN2, MIN3, MIRL, HRF20, MACIF, MEM43, MICH, MSK21, 16.3A5, HRF-20, MAC -IP, or pl 8-20.
• This is a cell surface glycoprotein that regulates complement-mediated cell lysis, and it is involved in lymphocyte signal transduction.
• This protein is a potent inhibitor of the complement membrane attack complex, whereby it binds 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 necessary for osmolytic pore formation.
• LAG-3 refers to lymphocyte-activation protein 3.
• the LAG-3 protein which belongs to immunoglobulin (Tg) superfamily, comprises a 503-amino acid type I transmembrane protein with four extracellular Ig-like domains, designated DI to D4.
• TIM3 refers to hepatitis A virus cellular receptor 2, also known as CD366, KIM-3, SPTCL, TIMD3, Tim-3, TIMD-3, or HAVcr-2.
• CD366 hepatitis A virus cellular receptor 2
• KIM-3 hepatitis A virus cellular receptor 2
• SPTCL hepatitis A virus cellular receptor 2
• TIMD3 Tim-3
• TIMD-3 Tim-3
• HAVcr-2 HAVcr-2
• IL-12R is composed of interleukin 12 receptor beta 1 (IL-12Rpi) and Interleukin 12 receptor beta 2 (IL-12RP2) chains, and mediates signal transduction, which involves the recruitment of Janus family tyrosine kinase 2 and signal transducer and activator of transcription (STAT)4.
• IL-12Rpi interleukin 12 receptor beta 1
• IL-12RP2 Interleukin 12 receptor beta 2
• CD28 refers to a protein encoded by this gene that is essential for T-cell proliferation and survival, cytokine production, and T-helper type-2 development. It is also known as Tp44.
• CD57 refers to beta-1, 3-glucuronyltransferase 1, also known as NK1, HNK1, LEU7, GLCATP or GLCUATP. The protein encoded by this gene is a member of the glucuronyltransferase gene family.
• KIR refers to killer cell immunoglobulin-like receptors, which are members of a group of regulatory molecules found on subsets of lymphoid cells.
• KLRG-1 refers to killer cell lectin like receptor Gl, 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 preferentially expressed in NK cells.
• CD27 refers to a member of the TNF-receptor superfamily. This receptor is required for generation and long-term maintenance of T cell immunity.
• PD-1 refers to programmed cell death 1, also known as CD279, SLEB2, hPD-1, hPD-I, or hSLEl.
• Programmed cell death protein 1 (PDCD1) is an immune- inhibitory receptor expressed in activated T cells; it is involved in the regulation of T-cell functions, including those of effector CD8+ T cells.
• CTL-4 refers to cytotoxic T-lymphocytes 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 which transmits an inhibitory signal to T cells.
• the protein contains a V domain, a transmembrane domain, and a cytoplasmic tail.
• 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, IL 15, IL21, erythropoietin, and thrombopoietin.
• the protein encoded by this gene is a secreted cytokine produced by activated CD4+ and CD8+ T lymphocytes, that is important for the proliferation of T and B lymphocytes.
• IL- 10 refers to interleukin 10, also known as CSIF, TGIF, GVHDS, or IL10A.
• the protein encoded by this gene is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes.
• TNF-a refers to tumor necrosis factor alpha, which is a proinflammatory cytokine with an important role in the pathogenesis of several diseases.
• microfluidic device refers to a testing device that focuses on microfluidic behavior of fluids for precise control and manipulation in geometrically constrained small scale (typically sub-millimeter) at which surface forces dominate volumetric forces.
• a microfluidic chip is a pattern of microchannels, molded or engraved. This network of microchannels incorporated into the microfluidic chip is linked to the macro-environment by several holes of different dimensions hollowed out through the chip.
• microfluidics have diverse assets: faster reaction time, enhanced analytical sensitivity, enhanced temperature control, portability, easier automation and parallelization, integration of lab routines in one device.
• conjugated antibody refers to tagging on a protein, compound or dye in order to track its interaction with specific antigens.
• Fluorescent dyes such as Alex and DyLight fluor can be used in immuofluorescent assays. They can absorb and emit light at different wavelength for different labeling purposes.
• Antibodies conjugated with fluorescent dyes are used in immunoassays such as flow cytometry, ELISA, Western blot and fluorescence microscopy.
• the "simulation” or “activation” of T-cells refers to the binding of specific ligands to trigger biochemical signals in T-cells, including the production of interferon gamma.
• FIG. 1A Schematics for standard and microfluidic chip ELISPOT assays.
• FIG. IB IFN- y independent evaluation of T-cells activation.
• FIG. 1C Thawed PBMC aliquots stimulated with or without PMA/ionomycin were cultured for 24 h in glass bottom wells coated with polysine, then incubated with a biotinylated secondary antibody, streptavidin-HRP and a chromogenic (red) HRP substrate or (Hoescht 33342 dye and a fluorescently tagged IFN-y-specific antibody. White size bars indicate 75 pm.
• FIG. 1D-E The PBMC aliquots stimulated with or without PMA/ionomycin were cultured for 24 h in glass bottom wells coated with polysine, then incubated with a biotinylated secondary antibody, streptavidin-HRP and a chromogenic (red) HRP substrate or (Hoescht 33342 dye and a fluorescently tagged IFN-y-specific antibody. White size bars indicate 75 pm.
• FIG. 1D-E The PBMC aliquots stimulated with or without PMA/ionomycin
• PBMCs (-2x 105) were seed on microplate wells coated with and without polylysine and stained with Hoechst 33342 to quantify the cell density of captured cells, or induced with PMA/ionomycin for 4 h, stained Hoechst 33342 and specific antibodies to IFN-y, 0X40, and 4- IBB, after which total cell numbers and activated T cell percentages were quantified using a fluorescent plate reader. Positive control (PC) wells were not washed to remove non- or weakly adherent cells.
• PC Positive control
• FIG. IF One-Way two-sided parametric ANOVAs with Tukeys post-test were performed to analyze differences between the polylysine-coated and uncoated well values and (F) PMA-stimulated and unstimulated well values. Data indicate Mean ⁇ SD; *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001; ns, no significant difference when analyzed by two-sided Mann- Whitney U-test.
• FIG. 2A-C T cell activation with SARS-CoV-2 spike peptide pool.
• Cryopreserved PBMCs from blood donors who received (A) 3 SARS-CoV-2 or (B-C) 0-3 vaccine doses were simulated by incubation with (A) PMA/ionomycin and/or (A-C) a SARS-CoV-2 peptide pool for up to 24 h, after which IFN-y levels were evaluated by (A) ELISA, (B) Flow cytometry, or (C) ELISpot.
• FIG. 2D-F Freshly isolated PBMCs from unvaccinated and vaccinated (3 doses) donors were incubated with or without (D-E) a SARS-CoV-2 peptide pool, or (F) peptides derived from SARS-CoV-2, the M. tuberculosis (Mtb) proteins CFP-10 and ESAT-6 or HIV-1 p24 for 24 h, stained with Hoechst 33342, and incubated with IFN-y-specific antibody.
• D-E SARS-CoV-2 peptide pool
• F peptides derived from SARS-CoV-2
• Mtb M. tuberculosis
• FIG. 3A-F Evaluation of on-chip ELISpot assay performance. Analysis the IFN-y response to SARS-CoV-2 spike or HTV-1 p24 (non-specific control) peptides in PBMC samples ( ⁇ 2x 106 cells) isolated from individuals without a history of HIV infection who had received three vaccine doses when analyzed by (A) our on-chip ELISpot, (B) flow cytometry, and (C) ELISpot assays. (D) Correlation of flow cytometry and on-chip ELISpot data.
• E-F On-chip ELISpot assays results from fingerstick whole blood samples (e) after pre-treatment with or without RBC buffer for one individual or (f) without RBC lysis for eight HIV-negative individuals more than six months after their second or third vaccine dose, t-test was performed to compare HIV-p24 or SARS-CoV-2 peptide pool stimulation. Data indicate Mean ⁇ SD;*, p ⁇ 0.05; **, p ⁇ 0.01; ns, no significant difference by two-sided Mann-Whitney U-test.
• FIG. 4A-D Activated T cell counting on glass surface.
• PBMC capture on well coated with and without different polylysine concentrations seeded with 2x 105 PBMCs, induced with PMA/ionomycin for 4 h, and stained with Hoechst 33342 and incubated with AlexaFluor488- (A), PE-tagged OX-40 (B) or APC tagged 4-1BB (C-D) specific antibodies, respectively.
• Wells were analyzed for activated T cell counts using a fluorescent plate reader. Positive control (PC) wells indicate signal detected in wells that were not washed to remove non- or weakly adherent cells.
• PC Positive control
• FIG. 5A-C PDMS microfluidic chip fabrication.
• A Silicon wafers coated with SU8 Epoxy are then covered with a photomask containing the device design, exposed to ultraviolet light, and washed with SU8 developer to remove inactivated SU8 surrounding the device design. Then a 10:1 PDMS-to-curing agent mixture is poured onto the master wafer and then heated at 60°C for 5 h.
• B 50 pg/ml polylysine was coated on glass surface for 30 min and washed with DI water
• C Solidified PDMS device is cut from the wafer and bound to the polylysine coated glass slide previously exposed to oxygen plasma to generate the complete device.
• FIG. 6A-B Flow cytometry gating of blood cells samples.
• Scatterplots indicate the total PBMC scattering and lymphocyte gate (left panels) and the distribution of the IFN-y-negative and IFN-y-positive (gated population) in the lymphocytes gate (right panels).
• FIG. 7A-E Correlation of On-Chip IGRA results with traditional assays.
• A-D Correlations of on-chip ELTSpot and flowcytometry results among the (A) SARS-Cov-2, (B) second and (C) third vaccine dose groups and (D) the vaccinated individuals with breakthrough infections.
• E Correlation of on-chip ELISpot assay and standard ELISpot assay results. Data indicate Spearmann/Pearson r-values.
• FIG. 8A-K Microchip response to T-cell capture.
• FIG. 9A-G Cumulative response of surface marker 4- IBB and OX-40
• FIG. 10A-B Comparison of IFN-y with 4-1BB and 0X40 response to TB infection.
• FIG. 11 A-H Blood-based assay for enabling single-step diagnosis of TB infection.
• Enzyme-linked immunosorbent assay is a commonly used analytical biochemistry 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 antibodies directed against the protein to be measured.
• a ligand such as a protein
• the sample with an unknown amount of antigen is immobilized on a solid support, and after the antigen is immobilized, a detection antibody specific to the antigen is added to form a complex with the antigen.
• the detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation.
• the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound.
• the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
• Enzyme-linked immune absorbent spot is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion of a single cell. It utilizes antibodies to detect a protein analyte (the cytokine), much like ELISA.
• the mechanism of ELISpot starts with coating the wells with analyte-specific monoclonal antibodies. The second step is to incubate cells within the wells, during which the cells are allowed to react to any present stimuli and secrete the cytokine.
• cytokine specific monoclonal antibodies that coat the walls of the wells
• cytokines that has been secreted by the incubated cells will start to attach to the antibodies at a specific epitope.
• biotinylated cytokine-specific detection antibodies are then added to the well to bind to any cytokine that is left in the well, as the cytokine is still attached to the first set of antibodies used. Streptavidin-enzyme conjugate is then added to the wells to bind with the detection antibodies.
• the present invention is exemplified with respect to SARS-CoV-2 specific T-cell activation, as well as tuberculosis-specific T-cell activation.
• SARS-CoV-2 specific T-cell activation as well as tuberculosis-specific T-cell activation.
• this is exemplary only, and the invention can be broadly applied to other pathogens that elicit T-cell activated interferon-y secretion.
• the following examples are intended to be illustrative only, and not unduly limit the scope of the appended claims.
• CMV Cytomegalovirus
• RSV Respiratory syncytial virus
• HSV herpes simplex virus
• HBV Hepatitis B virus
• EBV Epstein-Barr virus
• Listeria Salmonella, Plasmodium, Toxoplasma gondii, and Trypanosoma cruzi.
• PD-1 programmed cell death protein- 1
• CTLA-4 cytotoxic T-lymphocyte-associated protein-4
• TIM-3 T-cell immunoglobulin and mucin domain-3
• LAG3 lymphocyte activation gene-3
• ELISpot assays have greater procedural and equipment requirements than ELISA- based IGRAs, but are more readily adapted to a microfluidic assay workflow, since they can require fewer liquid handling steps in certain assay designs.
• the ELISpot microfluid workflow can be broken down into a few basic steps: blood collection, stimulation of T cells with pathogenspecific peptides, and the capture, staining, and analysis of activated T cells, most which can be accomplished on a microfluidic chip to greatly simplify the ELISpot workflow ( Figure 1(a)).
• polylysine titration analysis found 5 pg/mL polylysine was sufficient to maximize mean PBMC adherence and capture (-4.5* 10 4 PBMC / mm 2 ), doubling the cells captured on untreated slides ( ⁇ 2.2* 10 4 PBMC / mm 2 ), to capture -58% of the input PBMCs (-7.7* 10 4 PBMC / mm 2 ) ( Figure IE).
• the frequency of IFN-y-positive cells in the unvaccinated group was substantially higher (20% compared to 0.01%) than in the flow cytometry analysis, which used a similar intracellular IFN-y staining procedure. This difference suggests either differences in the samples analyzed in these studies, or differences in the sensitivity, activation efficacy, or analytical sensitivity of these two procedures.
• the frequency of IFN-y-positive PBMCs detected in this analysis was found to be pathogen specific, since PMBC activation frequencies detected when these cells were incubated with peptides from other human pathogens to which their donors had not been exposed or vaccinated (e.g. TB and HIV) were not different from those measured with unstimulated PBMCs ( Figure 2E).
• Microfluidic chip ( Figure 5) wells were loaded with ⁇ 2> ⁇ 10 6 PBMCs (data not shown), which were captured on a polylysine layer and then cultured in peptide-spiked culture media for 24 h, fixed, permeabilized and incubated with Hoescht 33342 and an IFN-y-specific fluorescent antibody for 20 min, after which on-chip fluorescent microscope images of labeled cells were analyzed to evaluate PBMC activation.
• ELISpot assays isolate and culture PBMCs from > 5 mL of venous blood, which renders them unsuitable for POC tests or use in resource limited settings. We therefore evaluated whether our ELISpot assay, could be performed with fingerstick blood draw volumes ( ⁇ 25 pL) with or without a red blood cell (RBC) removal step using a 4 h peptide incubation step. RBC lysis increased the number of captured PBMCs versus whole blood samples, but also increased the IFN- y response to a control peptide, resulting in a corresponding decrease specific to control peptide induction ratio (2.2-fold versus 1.25-fold) in these samples ( Figure 3E).
• Immunoassays that respectively detect the presence or titer of specific antibodies to pathogen-derived factors and the percentage and activity of T cells that respond to these factors provide important, but divergent, information that is useful in evaluating the efficacy of an individual’s potential immune response.
• Specific antibody assays are straightforward and can be readily employed in most settings, and are thus often suitable for use as POC tests, but may not provide a reliable picture of immunity as circulating antibody responses can wane long before the loss of inducible immunity.
• IGRAs are potentially useful to address this question, but are not suitable for high-throughput use or use in resource limited settings and thus are not practical for the evaluation of individual immune response at large scale.
• ELISpot assay approach was chosen for this analysis since this assay format measures the fraction of T-cell that are responsive to a selected pathogen-derived factor, and thus provides a direct measure of the cell population available to respond to this pathogen.
• ELISA- based IGRAs which are more commonly used, measure the relative degree of the cytokine response, and thus integrate the number of available cells and the extent of their inducible cytokine response. ELISpot and ELISA-based IGRA do not exhibit strong correlation, unlike ELISpot and flow cytometry assay data, which demonstrates good correlation, albeit with substantial variation.
• Standard ELISpot assays detect the number of cells secreting a factor and thus require this factor be bound at the site of its release for subsequent detection in order to estimate the number of signal-positive cells in a known number of input cells, are problematic for use at high-throughput in low resources settings.
• Standard ELISpots assays also evaluate the number of IFN-y-positive cells within a known and standard concentration of viable PBMCs requiring isolation of the cells, determination of cell viability, and extended culturing at consistent amounts. All of these requirements add complexity that renders these assays impractical for use in many analysis settings.
• our revised ELTSpot assay employs fingerstick whole blood microsamples, eliminating the need for a trained phlebotomist to perform a venous blood draw and then need to isolate PMBCs, while the number of IFN-y-positive and total PBMCs present in analysis sample can be directly detected from captured assay images. Given the limited opportunity for variation in the sample collection and processing procedure, it can also be assumed that cell viability should not influence the IFN-y-positive cell percentages in this approach.
• the assay does not distinguish between groups of individuals who have different exposure histories to the targeted antigen through infection and/or been infection, which can be detected with standard assays. This may be due to the relatively small number of cells captured on the microwell, the loss of activated T cells during the washing step, and/or sampling bias during image capture and analysis. Improving the precision and reproducibility of such assay measurements may be important to improve the ability to sensitively track the durability of acquired T cell responses to specific pathogen-derived antigens and the relative amount of protective immunity retained with the passage of time. Enhanced precision could be obtained using several approaches, either alone or in combination.
• this ELISpot serves as a platform to rapidly and inexpensively analyze T cell responses to specific antigens using fingerstick whole blood microsamples, without requiring significant equipment or technical expertise.
• This platform should allow high-throughput analysis of T cell responses to specific pathogen derived antigens as a measure of potential immunity following previous exposure via infection or vaccination.
• the potential resistance to new variants of these pathogens can be evaluated by modify SARS-CoV-2 peptide pool by adding specific mutated peptides.
• a variant of this approach could also be adapted to measure memory B cell responses. This capacity should permit large-scale evaluation of acquired immune responses to benefit the evaluation of vaccine effectiveness for existing and emerging infectious diseases and may also facilitate improved understanding of some chronic infections.
• T-SPOT.TB positive PBMC samples were used as a positive control to stimulate TSPOT.TB positive PBMC samples, and the response without T-cell activation was considered a negative control.
• PHA Phytohemagglutinin
• the TSPOT.TB positive PBMCs were stimulated with an Ebola-specific peptide to evaluate nonspecific responses. The nonspecific stimulation showed a similar response to the negative control, validating the specificity of our sensor platform.
• the anti-4-lBB antibody used herein is Cdl37 (4-1BB) Monoclonal Antibody (4B4 (4B4-1)), FITC, eBioscience 11-1379-42.
• the anti-OX-40 antibody used herein is Cdl34 (0X40) Monoclonal Antibody (ACT35 (ACT-35)), FITC, eBioscience 11-1347-42.
• T-cells were enriched on the microchip surface and evaluated the response of surface markers after 6 hours of stimulation.
• a total of 20 TSPOT.TB positive and 20 TSPOT.TB negative PBMC samples obtained from Houston Cincinnati were tested, using TB specific peptides including CFP-10 and ESAT-6 to activate T- cells.
• Subjects or households with suspected or confirmed SARS-CoV-2 infection were recruited from the Greater New La community under Tulane Biomedical Institutional Review Board (federal-wide assurance number FWA00002055, under study number 2020-585). Enrolled subjects completed a study questionnaire regarding infection and demographic information and provided a blood sample.
• PBMC isolation PBMCs were isolated from frozen leukophoresis samples (Stemcell Technologies) or whole blood samples. Venous blood samples were collected in EDTA tubes and supplemented with a 15 x volume of cold (4°C) isotonic ammonium chloride solution, mixed by inversion at room temperature for 10 minutes using a rotary mixer set to -500 rpm to allow RBC lysis, and then centrifuged at 250g for 10 minutes.
• Isolated PBMCs were resuspended in 5 mL AIM V cell culture media (Fisher Scientific 31-035-025), aliquots were analyzed to determine viable cell concentrations by staining cells with a 0.4% Trypan Blue solution, and cells suspensions adjusted to a final concentration of 3 x 10 6 /mL in AIM V cell culture media (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.
• PBMCs peripheral blood mononuclear cells
• PBMCs peripheral blood mononuclear cells
• FBS-10% DMSO FBS-10% DMSO
• PBMC Stimulation Cryopreserved PBMC aliquots were rapidly thawed in a 37°C water bath, mixed with an equal volume of RPMI-1640 media warmed to 37°C, and then centrifuged at 400g for 5 minutes. Cell pellets were washed with 2 mL of RPMI-1640, resuspended in 150 pL RPMI-1640 and analyzed by Trypan Blue exclusion to evaluate cell viability, and then supplemented with RPMI-1640 to a final working concentration of ⁇ 3 x 10 6 viable cells/mL. Samples that had cell viabilities ⁇ 70% were excluded from analysis.
• PBMCs were plated in 6 well cell culture plates at a concentration of 1 x 10 6 to 2 x io 6 viable cells/well as specified by different assay types, and then stimulated with 10 ng/mL phorbol 12-myri state 13 -acetate (PMA, Sigma P1585) and 1 pg/mL ionomycin (STEM CELL 73722) or 1 pg/mL of the indicated peptide or peptide pools (BEI NR-52-/02) at 37°C for the specified times.
• PMA phorbol 12-myri state 13 -acetate
• STEM CELL 73722 1 pg/mL ionomycin
• BEI NR-52-/02 1 pg/mL of the indicated peptide or peptide pools
• PBMCs aliquots suspended in AIM V cell culture media (2 x 10 6 /mL) were cultured overnight in 24 well culture plates overnight before and then stimulated for 24 h with PMA and iomyocin (10 ng/mL and 1 pg/mL, respectively) or a SARS-CoV-2 or HIV- p24 peptide pool (1 pg/mL), with 1 ng/mL IFN-y transport blocker added 2 h after the start of induction.
• PMA and iomyocin 10 ng/mL and 1 pg/mL, respectively
• SARS-CoV-2 or HIV- p24 peptide pool (1 pg/mL
• PBMCs were pelleted by centrifugation at 500g for 5 min, PBS washed, and then resuspended in 100 pL of IC Fixation Buffer and Permeabilization Buffer (eBioscience 00-8222-49 and 00-8333) for 10 min, then incubated in a PBS/10% BSA solution supplemented with I g/ml of an AlexaFluor488-labeled IFN-y-specific antibody (eBioscience 50- 168-09) for 20 min.
• IC Fixation Buffer and Permeabilization Buffer eBioscience 00-8222-49 and 00-8333
• Flow cytometry analyses were performed using an Attune Flow Cytometer (Thermo Scientific) gating cells, capturing IFN-y-positive cell signal in the FITC/GFP channel, and analyzing and quantifying captured data with Flow Jo software (vl0.04).
• IGRA ELISAs PBMCs (2 x io 4 ) were cultured for the indicated times at 37°C in X mL RPMI-1640 media supplemented with a 1 pg/mL SARS-COV-2 peptide pool (BEI NR- 52402) PMA and ionomycin (lOng/ml and Ipg/ml), or no added material, with an RPMI only well included as a negative control. Culture supernatants were pipetted from each well and stored at -80°C for future ELISA analysis.
• the media was pipetted from wells into a new 98 well plate. 1 pg/mL final concentration of SARS-COV-2 peptide pool was added to the stimulation group at this time. At 4, 6, 8, 10, 12 and 24 hours, the supernatant was removed and stored at -80°C for future ELISA.
• IGRA ELISA plates were generated by incubating 96 well MaxiSorp plates (Nunc 44-2404-21) withlOO pL of Ipg/ml PBS solution of human IFN-y-specific antibody (Endogen, M700-A) overnight at 4°C. These plates were then washed 6 times with PBS/0.05% Tween 20 (PBST), blocked with 200pl of 1% BSA/PBS for 1 h at room temperature, and then PBST washed, dried, and stored at 4°C until use. Cryopreseved PBMC culture supernatant aliquots were thawed and transferred to assay plates in triplicate (50pL/well) and incubated at room temperature for 1 h.
• PBST PBS/0.05% Tween 20
• IFN-y-biotin-labeled antibody (Endogen, M-701B) diluted at 1 :1000 in 2% FBS/ IX PBS was added to each well and incubated at room temperature for 1 hour. Plates were washed and dried before pipetting 50pl/well of Poly-HRP streptavidin (Pierce, N200) diluted at 1 :5000 in 1% BSA/1X PBS and incubated at room temperature for 30 minutes in the dark. Afterward, the plate was washed and dried for a final time. lOOpl/well of 3,3',5,5'-Tetramethylbenzidine (TMB, Thermo Scientific 34029) solution was added, and color development was observed. After adequate color development ( ⁇ 10 minutes) 50 pl/well of stop solution (2.5 N H 2 SO .) was added and plates were read at OD450.
• TMB 3,3',5,5'-Tetramethylbenzidine
• ELISPOT Filter Screen Plates (Millipore MAIPS4510) were coated with antihuman IFN-y (Endogen, M700-A, Img/ml) at Ipg/ml and stored overnight at 4°C. The following day the plate was washed 6 times with washing buffer (IX PBS + 1 :2000 diluted Tween 20) and tapped dry. Wells were blocked with 200pl of 1% BSA/1X PBS for 1 hour at room temperature.
• 2X10 3 PBMC were then seeded into plates and stimulated with PMA-ionomycin (lOng/ml and Ipg/ml), SARS-CoV-2 Spike peptide pool (Ipg /mL) or HIV-p24 peptide (1 pg/mL).
• PMA-ionomycin laspasmodic acid
• SARS-CoV-2 Spike peptide pool Ipg /mL
• HIV-p24 peptide 1 pg/mL
• the silicon wafer with the microfluidic design was fabricated based on previously described methods.
• Polydimethylsiloxane (PDMS) molds of the design were fabricated from the silicon wafer ( Figure 4).
• the PDMS elastomer was mixed with a curing agent at a 10: 1 ratio and pour over the silicon wafer.
• the curing agent allows for the elastomer to crosslink and form a rigid structure that will solidify into a complete chip.
• the PDMS mold is placed in an oven at 60 °C for 5 hours. After the elastomer has completely solidified, the molds are removed from the silicon wafer to be used for chip assembly.
• Plasma treatment of a PDMS chip and a 1mm glass slide allows for the formation of silanol functional groups that can form strong covalent bonds with each other to create a fluid-tight seal that forms that microfluidic channel.
• PMA-ionomycin (lOng/ml and Ipg/ml), SARS-CoV-2 Spike peptide pool (Ipg /mL) or HTV-p24 peptide (1 pg/mL) were added into 25 pL whole blood then incubated at 37 °C for 4 hours.
• the blood samples were fixed with IC Fixation Buffer (eBioscienceTM 00-8222-49) and Permeabilization Buffer (eBioscienceTM 00-8333) at 25 °C for 20 min then stained with 1 pg/ml anti-IFN-y-Alexa488 (eBioscience 50-168-09) and 0.1 pg/ mL Hoechst 33342 at 25 °C for 20 min.
• Image capture and analysis Images of PBMCs attached microfluidic chamber were obtained using an EVOSTM M5000 Imaging System, Invitrogen by Thermo Fisher Scientific, Madrid, Spain, Scale bar: 300pm. Images (10X) of the stained PBMCs are representative of the total cell population and the IFN-y positive cells. The green fluorescence signal was obtained, when Alexa 488 binds to intracellular IFN-y. The blue fluorescence signal from Hoechst 33342 represents the total cells counts. All the experiments were conducted in triplicate. Each time four different random areas from the microfluidic chamber were chosen to obtain the images. All data acquired on EVOSTM M5000 Imaging System were analyzed using the ImageJ software.
• Cell counting The total cell counts and IFN-y positive cells ratio were quantified using the National Institutes of Health (NIH) Image I image-analysis software. The images were converted to 8-bit greyscale. The lower threshold value was set to 70 and the higher threshold value was set to 255. The cell counts were analyzed with the size range from 1 to 100 (Pixel) and circularity 0.00-1.00.
• NASH National Institutes of Health
• Clark RA Mukandavire C, Portnoy A, et al.

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