METHOD TO IDENTIFY INDIVIDUALS WITH T CELL IMMUNITY TO SPECIFIC INFECTIOUS AGENTS
Methods, devices and kits for rapid identification of individuals previously infected with, or vaccinated for, a bacterial or viral pathogen, such as SARS CoV-2 virus. Pathogen specific memory T cells, such as SARS CoV-2 specific memory T cells, provide indication of prior infection, if detected. Memory T cells are assayed by exposure to specific bacterial and/or viral antigens and then tested for activation by analysis of nucleotide content.
1 . A method to identify presence of SARS CoV-2-specific T cells in a sample from a subject to ascertain prior exposure to, or vaccination with, SARS CoV-2 virus, comprising:
exposing a biological sample comprising memory T-cells from the subject to one or more peptide antigens specific for SARS CoV-2;
contacting the exposed memory T-cells with an indicator compound that associates with RNA, DNA, or both, wherein the contacting is simultaneous with or sequential to the exposing;
analyzing the memory T-cells for indicator compound.
2 . The method of claim 1 , where the biological sample is a blood sample or a fraction thereof.
3 . (canceled)
4 . The method of claim 2 , where the biological sample is a fraction of the blood sample, wherein the fraction is the buffy coat fraction or peripheral blood mononuclear cells (PBMCs) or a mixture of buffy coat fraction and PBMCs.
5 . The method of claim 1 , wherein said exposing to one or more peptide antigens specific for SARS CoV-2 comprises exposing to a solution comprising the one or more peptide antigens, the indicator compound and one or more of a buffer, an energy source for the cells, and a balanced salt solution, thereby simultaneously with said exposing, contacting the T cells with the indicator compound.
6 . The method of claim 1 , wherein the method further comprises exposing a second biological sample comprising memory T-cells to a control reagent that (i) lacks the one or more peptide antigens specific for SARS CoV-2 and (ii) comprises a control indicator compound that associates with RNA, DNA, or both, to thereby generate a control sample.
7 . The method of claim 6 , wherein the second biological sample is from a subject, and wherein the biological sample is from the same subject or wherein the second biological sample is a portion of the biological sample.
8 . The method of claim 6 , wherein the indicator compound and the control indicator compound are the same.
9 . The method of claim 6 , wherein said analyzing comprises measuring signal of indicator compound associated with memory T-cells in the biological sample and measuring signal of control indicator compound associated with memory T-cells in the control sample.
10 . The method of claim 6 , wherein said analyzing comprises measuring signal of indicator compound associated with RNA in the biological sample and measuring signal of control indicator compound associated with RNA in the second biological sample.
11 . The method of claim 1 , wherein said analyzing comprises measuring an RNA signal based on signal of indicator compound associated with RNA, measuring a DNA signal based on signal of indicator compound associated with DNA, and determining a ratio of RNA signal to DNA signal or of DNA signal to RNA signal.
12 . The method of claim 1 , wherein the indicator compound is a fluorescent dye that selectively stains RNA.
13 . The method of claim 1 , wherein said contacting comprises contacting the exposed memory T-cells with a first indicator compound that selectively stains RNA or DNA and with a second indicator compound that non-specifically stains RNA and DNA.
14 . The method of claim 1 , wherein the indicator compound has an excitation between about 330-360 nm and an emission at greater than between about 500-600 nm.
15 . The method of claim 1 , wherein the memory T-cells are CD4+ and/or CD8+ T cells.
16 . The method of claim 1 , further comprising after said contacting and before said analyzing, incubating for a period of time.
17 . The method of claim 16 , wherein said incubating is at a temperature of between about 25-40° C.
18 . The method of claim 16 , wherein said period of time is between about 10-60 minutes or between about 10-30 minutes.
19 . The method of claim 1 , wherein the one or more peptide antigens specific for SARS CoV-2 comprises between 2-20 peptide antigens specific for SARS CoV-2 or between 3-15 peptide antigens specific for SARS CoV-2.
20 . The method of claim 1 , wherein exposing further comprising exposing the biological sample to one or more peptide antigens non-specific for SARS CoV-2.
21 . The method of claim 1 , wherein the one or more peptide antigens specific for SARS CoV-2 comprise one or more of the peptides identified as SEQ ID NO: 1-SEQ ID NO: 12.
22 . A method to ascertain prior exposure of a subject to an infectious pathogen, comprising:
exposing a biological sample comprising memory T-cells from the subject to one or more peptide antigens specific for the pathogen;
contacting the exposed memory T-cells with an indicator compound that associates with RNA, DNA, or both, wherein the contacting is simultaneous with or sequential to the exposing;
analyzing the memory T-cells for indicator compound.
23 - 27 . (canceled)
28 . The method of claim 22 , wherein said exposing to one or more peptide antigens specific for the pathogen comprises exposing to a solution comprising the one or more peptide antigens, the indicator compound and one or more of a buffer, an energy source for the cells, and a balanced salt solution, thereby simultaneously with said exposing, contacting the T cells with the indicator compound.
29 . The method of claim 22 , wherein the method further comprises exposing a second biological sample comprising memory T-cells to a control reagent that (i) lacks the one or more peptide antigens specific for the pathogen and (ii) comprises a control indicator compound that associates with RNA, DNA, or both, to thereby generate a control sample.
30 - 41 . (canceled)
42 . The method of claim 22 , wherein the one or more peptide antigens specific for the pathogen comprises between 2-20 peptide antigens specific for the pathogen or between 3-15 peptide antigens specific for the pathogen.
43 . The method of claim 22 , wherein exposing further comprising exposing the biological sample to one or more peptide antigens non-specific for the pathogen.
44 . A method to identify presence of infectious agent-specific T cells in a sample from a subject to ascertain prior exposure to, or vaccination with, the infectious agent, comprising:
exposing a biological sample comprising memory T-cells from the subject to one or more peptide antigens specific for an infectious agent;
contacting the exposed memory T-cells with an indicator compound that associates with RNA, DNA, or both, wherein the contacting is simultaneous with or sequential to the exposing;
analyzing the memory T-cells for indicator compound.