WO2022260986A1 - Dosage pour évaluer l'immunité de lymphocytes t contre le sars-cov-2 et ses variants chez des individus infectés et vaccinés - Google Patents

Dosage pour évaluer l'immunité de lymphocytes t contre le sars-cov-2 et ses variants chez des individus infectés et vaccinés Download PDF

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WO2022260986A1
WO2022260986A1 PCT/US2022/032322 US2022032322W WO2022260986A1 WO 2022260986 A1 WO2022260986 A1 WO 2022260986A1 US 2022032322 W US2022032322 W US 2022032322W WO 2022260986 A1 WO2022260986 A1 WO 2022260986A1
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cov
sars
cells
sars cov
specific
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PCT/US2022/032322
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English (en)
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Stanley C. Jordan
Ruan ZHANG
Bongha SHIN
Mieko Toyoda
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Cedars-Sinai Medical Center
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8

Definitions

  • This invention relates to the detection of T-cell Immunity to SARS-COV-2.
  • SARS-CoV-2 T cell immunity in patients was indirectly measured by cytokines levels in serum or plasma after SARS-CoV-2 infection.
  • the most commonly used test to assess immunity to SARS-CoV-2 is measurement of IgG antibodies to spike protein. This assay is useful, but suffers from the rapid dissipation of IgG specific to spike protein over time. At this point we do not know the durability of T-cell responses to spike protein, but reports have shown that recall memory responses to SARS-CoV-1 were detected 17 years after infection.
  • Various embodiments of the invention provide for a method of detecting a quantity of SARS CoV-2-specific CD4+ T-cells, a quantity of CD8+ T-cells, or a quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject, comprising: contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantity of SARS CoV-2 - specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2- specific CD8+ T-cells.
  • the method can further comprise detecting the presence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.05% or greater as compared to a control.
  • Various embodiments of the invention provide for a method of detecting a quantity of SARS CoV-2-specific CD4+ T-cells, a quantity of CD8+ T-cells, or a quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject, comprising: contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantity of SARS CoV-2 - specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2- specific CD8+ T-cells, wherein the subject desires a determination regarding the presence or absence of protective immunity to SARS CoV-2.
  • Various embodiments of the invention provide for a method of detecting a likely absence of protective immunity to SARS CoV-2, comprising: contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting the likely absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2 - specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • the method can further comprise re-vaccinating the subject against SARS CoV-2.
  • Various embodiments of the invention provide for a method of determining the likelihood of contracting coronavirus disease 2019 (COVID-19), comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T- cells; and detecting a likelihood of contracting COVID-19 based on the absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2- specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject can be performed 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine or 21 days or more after the subject has had a SARS CoV-2 infection.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject can be performed 30 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine or 30 days or more after the subject has had a SARS CoV-2 infection.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject can be performed 45 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine or 45 days or more after the subject has had a SARS CoV-2 infection.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject can be performed about 21-56 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine or about 21-56 days after the subject has had a SARS CoV-2 infection.
  • Various embodiments of the invention provide for a method of vaccinating a subject against SARS CoV-2, comprising detecting a likely absence of protective immunity to SARS CoV-2 by a method described herein, or detecting a likelihood of contracting coronavirus disease 2019 (COVID-19) by a method described herein; and administering to the subject a vaccine against SARS CoV-2.
  • Various embodiments of the invention provide for a method of vaccinating a subject against SARS CoV-2, comprising obtaining the results regarding a likely absence of protective immunity to SARS CoV-2 by a method described herein, or obtaining the results regarding a likelihood of contracting coronavirus disease 2019 (COVID-19) by the method described herein; and administering to the subject a vaccine against SARS CoV-2.
  • Various embodiments of the invention provide for a method of vaccinating a subject against SARS CoV-2, comprising administering a vaccine against SARS CoV-2 to a subject who has been determined to have a likely absence of protective immunity to SARS CoV-2 by a method described herein, or determined to have a likelihood of contracting coronavirus disease 2019 (COVID-19) by a method described herein.
  • the method can further comprising adjusting one or more immunosuppressive medication that the subject is taking.
  • adjusting can comprise reducing an amount of the one or more immunosuppressive medication.
  • administering the vaccine against SARS CoV-2 can comprise administering the vaccine 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine or 21 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 can comprise administering the vaccine 30 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine or 30 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 can comprise administering the vaccine 45 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine or 45 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, administering the vaccine against SARS CoV-2 can comprise administering the vaccine about 21-56 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine or about 21-56 days after the subject has had a SARS CoV-2 infection.
  • the SARS CoV-2 peptide fragments can be fragments of SARS CoV-2 spike protein.
  • the SARS CoV-2 can be a natural isolate SARS-
  • the SARS CoV-2 can be Washington isolate of SARS-CoV- 2. coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1.
  • the SARS CoV-2 can be a SARS CoV-2 variant.
  • the SARS CoV-2 variant can be selected from the group consisting of U.K. variant, California variant, South Africa variant, Brazil variant, Delta variant, Omicron variant, subvariants/sublineages thereof and combinations thereof.
  • the period of time can be about 8 to 10 hours.
  • CD4+ T-cells measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells can comprise contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa can comprise using flow cytometry.
  • the anti-IL-2 antibodies, anti-TNF-a antibodies, anti- g-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies can each independently comprise a label.
  • the biological sample can comprise T-cells. In various embodiments, the biological sample can be whole blood.
  • the subject can be one who has received an organ transplant. In various embodiments, the subject can be immune compromised. In various embodiments, the subject can be one who has received a vaccine for SARS CoV-2. In various embodiments, the subject can be one who has been infected with SARS CoV-2. In various embodiments, the subject can be one who has not been known to be infected with SARS CoV-2 and has not received a vaccine for SARS CoV-2.
  • kits comprising: a composition comprising
  • SARS-CoV-2 peptide fragments comprising anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and instructions to use the composition and the antibodies to measure a quantity of SARS CoV-2- specific CD4+ T-cells, a quantity of SARS CoV-2-specific CD8+ T-cells, or quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells.
  • the anti-IL-2 antibodies, anti-TNF-a antibodies, anti- g-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies can each independently comprise a label.
  • the kit can further comprise brefeldin A, phytohemagglutinin (PHA), anti-CD28 antibodies, or anti-CD49d antibodies, or combinations thereof.
  • PHA phytohemagglutinin
  • anti-CD28 antibodies or anti-CD49d antibodies, or combinations thereof.
  • Figure 1 depicts how infection with the SARS-CoV-2 virus initiates CD4+/CD8+
  • T-cell activation with the production of IL-2 and TNF-a by CD4+ T-cells and TNF-a + g-IFN by CD8+ T-cells in accordance with various embodiments of the present invention.
  • This ultimately leads to development of cytotoxic T-cell responses to virally infected cells, B-cell activation by CD4+ T-cells that initiate B-cell activation and result in production of sterilizing IgG antibodies and establishment of memory in the B-cell and T-cell compartments.
  • FIG. 2 depicts an exemplary process in accordance with various embodiments of the present invention.
  • 3cc of blood collected in a sodium-heparin tube from patients with confirmed SARS-CoV-2, non-exposed individuals or patients with symptoms of SARS- CoV-2 without confirmation of infection is incubated for 9 hours with peptide pools obtained from the SARS-CoV-2 proteins.
  • the SAR-CoV-2 peptides activate T-cells in previously exposed individuals resulting in the production of IL-2/TNF-a (CD4+) or TNF-a/g- IFN(CD8+) in these cells.
  • CD4+ IL-2/TNF-a
  • TNF-a/g- IFN(CD8+) TNF-a/g- IFN(CD8+
  • Figure 3 shows that the assay of the present invention is very effective in detecting immune responses to SARS-CoV-2 spike mRNA vaccination in accordance with various embodiments of the present invention.
  • the flow cytometry plot shown in the figure was taken from two individuals who participated in the Pfizer placebo controlled vaccine trial. (See also Figure 7) They were unaware if they received the vaccine or placebo. As can be seen, both have robust T-cell responses to SARS-CoV-2 spike protein.
  • the ability to assess immune responses to vaccines is critical to determining if patients have immunity to the virus. It is also critical, in conjunction with measuring antibody to spike to determining the composition and durability of immune responses to the virus.
  • Figure 4 depicts another important utility of the SARS-CoV-2 T-cell assay of the present invention in accordance with various embodiments of the present invention.
  • the inventors can determine reactivity of patients’ T-cells with SARS-CoV-2 peptides and simultaneously assess reactivity with SARS-CoV-2 variants.
  • patients who were infected with SARS-CoV-2 or received SARS-CoV-2 mRNA vaccines exhibit robust immunity in the T-cell compartment to the Oxford (UK) and Cal.20C variants.
  • Figure 5 A depicts an exemplary CoV-2-T-Intracellular Cytokine Flow Cytometry
  • Figure 5B depicts an assay of the present invention in accordance with various embodiments of the present invention.
  • Whole blood was incubated with overlapping peptide mixtures spanning the sequence of SARS CoV-2 Spike glycoprotein together with Brefeldin A and anti-CD28/CD49d overnight in accordance with various embodiments of the present invention.
  • After 9 h of T cell activation at 37°C cells were harvested and stained for surface markers and intracellular cytokines.
  • the IL-2+TNFa+cell % in CD4+ cells and IFNy+TNFa+cell % in CD8+ cells stimulated with S peptides were enumerated and analyzed, respectively.
  • Negative and positive controls included cells not incubated with S-peptides and those stimulated with phytohemagglutinin (PHA).
  • PHA phytohemagglutinin
  • Figure 6 depicts SARS-CoV-2 T-cell Assay (TNF-a & IL-2 Production in CD4+
  • Figure 7 depicts SARS-CoV-2 T-cell Assay (TNF-a & IL-2 Production in CD4+
  • FIG. 8 shows that COVID-19 antigen-specific T-cells in normal and COVID-19 recovered patients in accordance with various embodiments of the present invention.
  • the diversity of antigen responses suggests multiple targets for CD4+ T-cells independent of spike protein which may enhance immunity.
  • Figure 9 shows CD4+T-cell immune responses to Spike protein. Transplant patients show severe deficiency here and may not have adequate immunity to CoV-2 to prevent re-infection in accordance with various embodiments of the present invention.
  • FIGS 10A-10B show the monitoring of CD4 and CD8 T cell immunity against
  • 10A T cells in bloods of unexposed individuals, patients with history of SARS-CoV-2 infection and vaccinated individuals were stimulated by the original CoV-2 Spike protein. IL-2+TNFa+ cell% in CD4 and TNFa+IFNg+ cell% in CD8 were examined. 10B. Similar to 10A, T cells in blood of patients with SARS-CoV-2 infection and vaccinated individuals were stimulated by Spike protein of original virus, UK variant, or Cal.20C variant. IL-2+TNFa+ cell% in CD4 and TNFa+IFNg+ cell% in CD8 were shown. Each dot represents one individual.
  • FIGs 11A-11B depict detection of SARS-CoV-2-specific T-cells in whole blood. Fresh whole blood from participants were stimulated by SARS-CoV-2 Spike peptides. Activated CD4+ T-cells were identified as CD45+CD3+CD4+IL-2+/TNF-a+ cells while activated CD8+ T-cell were CD45+CD3+CD8+TNF- a +/EFN-y+ cells. Also shown is Blood + PHA which is positive control and Blood only which is negative control.
  • FIGS 12A-12D depict T cell immune response in SARS-CoV-2 infected patients and vaccinated individuals.
  • 12A CD4+ and CD8+ T-cell immune responses to SARS- CoV-2 peptides from 151 patients with confirmed SARS-CoV-2 infection. T-cells were stimulated separately using 5 major CoV-2 peptides: Spike, VME, NCAP, AP3A, NS7A. Activated CD4+ and CD8+ T cells were enumerated as Figure 11A-11B. Each dot represents one individual reading.
  • 12B CD4+ and CD8+ T-cell immune responses to SARS-CoV-2 Spike peptides in healthy, infected and vaccinated individuals. 12C.
  • Figures 13A-13D depict immunogenicity of variant B.1.1.7 spike peptides.
  • 13A&13B CD4+ & CD8+ T-cell immune responses to Spike-specific peptides are shown in infected/recovered and vaccinated patients. T cells were stimulated by the original SARS-CoV-2 Spike (Wuhan) or variant B.1.1.7 Spike peptides. Activated CD4+ (13A) and CD8+ T- cell (13B) in 20 infected patients and 18 vaccinated individuals are shown. 13C&13D. The paired data for immune responses to SARS-CoV-2 Spike peptides and UK (B.1.1.7) Spike peptides for each individual are shown.
  • Figure 14A shows SARS-CoV-2 Spike-Specific CD4+/CD8+ T-cell Responses in
  • Figure 14B shows a summary of SARS-CoV-2 Spike-Specific immunity after
  • Figure 15 depicts a representative schema for revaccination in immunocompromised patients.
  • FIG. 16A-16F depicts the immune responses in SARS-CoV-2-infected patients and vaccinated individuals.
  • 16A, 16B SARS-CoV-2 Spike-specific CD4+ and CD8+ T cells in healthy, infected and vaccinated individuals. Whole blood was stimulated with Spike peptides, and T cells with dual-cytokine staining were gated. The blue line shows the 0.05% cutoff.
  • 16C The correlation of Nucleocapsid-specific IgG titers with CD4+ T cell immune responses to one or more of 5 major SARS-CoV-2 proteins in 25 patients.
  • 16D The correlation between Spike- specific CD4+ T cell immune responses and Spike-specific IgG levels in 13 patients with elevated CD4+ Spike-specific T cell immune responses (IL-2+/TNF-a+ cell% in CD4+ >0.3%).
  • 16E, 16F T cell immune responses to ancestral and variant spike peptides are shown.
  • FIG. 17A depicts detectable CD4 + /CD8 + T-cell immune responses in vaccinated patients against SARS-CoV-2 spike peptides.
  • Cells were stimulated by SARS- CoV-2 spike peptides pool (PepMix SARS-CoV-2 [spike glycoprotein, JPT) for 9 h in vitro in the presence of Brefeldin A and anti-CD28/ CD49d (BD Bioscience, San Jose, CA). Cells were stained for T-cell surface markers followed by fixation, permeabilization, and intracellular staining of cytokines (BD Bioscience, San Jose, CA).
  • Activated CD4 + T cells IL-2 + TNFa +
  • CD8 + T cells IFNy'TNFa 1
  • Figure 17B shows immune responses to SARS-CoV-2 spike peptides in 2 patients who obtained external revaccination with the Johnson & Johnson vaccine.
  • percentages of activated CD4 + T cells (IL-2 + TNFa + ) and CD8 + T cells (IFNy'TNFa 1 ) in blood are shown after first vaccination (2 doses) and after third vaccination (booster).
  • Activated CD4 + T cells (IL-2 + TNFa + ) and CD8 + T cells (IFNy'TNFa 1 ) are shown in the upper right-hand comer of each flow box.
  • FIGS 18A-18B depict deficient CoV-2 T cells in vaccinated renal transplant patients. Fresh whole blood was stimulated with SARS-CoV-2 Spike peptides overnight. CoV-2- specific CD4+ (A) and CD8+ (B) were enumerated in healthy controls and transplant recipients (Tx) >1 month post-second dose of BNT162b2 vaccine. The dotted line represents the cutoff level (0.05%) for positive CoV-2 T cells. *p ⁇ .05, ***p ⁇ .001.
  • FIGS 19A-19B show immunoglobin G (IgG) serology in vaccinated renal transplant recipients.
  • A/B Plasma was collected from healthy individuals (Controls), belatacept recipients (Bela), and Tacrolimus recipients (Tac) 1 month post-second dose of BNT162b2 vaccine.
  • the CoV-2 Spike (S) receptor binding domain (RBD)-specific IgG levels in plasma were measured by ELISA.
  • Each dot represents one individual (A) and percentages of recipients with positive IgG serology and/or CoV-2 T cells (either CD4+ or CD8+) were analyzed in (B).
  • Dotted line represents the cutoff level of 15 unit/ml for a positive IgG serology.
  • NS not significant (p > .05), ***/? ⁇ .001
  • the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein.
  • the language “about 50%” covers the range of 45% to 55%.
  • the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.
  • SARS-CoV-2 refers to a coronavirus that has a wild-type sequence, natural isolate sequence, or mutant forms of the wild- type sequence or natural isolate sequence that causes COVID-19. Mutant forms arise naturally through the virus’ replication cycles, or through genetic engineering.
  • Natural isolate as used herein with reference to SARS-CoV-2 refers to a virus such as SARS-CoV-2 that has been isolated from a host (e.g., human, bat, feline, pig, or any other host) or natural reservoir.
  • the sequence of the natural isolate can be identical or have mutations that arose naturally through the virus’ replication cycles as it replicates in and/or transmits between hosts, for example, humans.
  • SARS-CoV-2 variant refers to a mutant form of SARS-CoV-2 that has developed naturally through the virus’ replication cycles as it replicates in and/or transmits between hosts such as humans.
  • SARS-CoV-2 variants include but are not limited to U.K.
  • variant also known as 20I/501Y.V1, VOC 202012/01, B.1.1.7, or Alpha
  • South African variant also known as 20H/501Y.V2, B.1.351, or Beta
  • Brazil variant also known as P.l or Gamma
  • California variant also known as CAL.20C
  • Delta variant also known as B.1.617.2
  • Omicron variant also known as B.1.1.529
  • subvariants/sublineages BA.l, BA.1.1, BA.2, BA.3, BA.4, BA.5
  • Wildington coronavirus isolate refers to a wild-type isolate of
  • SARS-CoV-2 that has GenBank accession no. MN985325.1 as of July 5, 2020, which is herein incorporated by reference as though fully set forth in its entirety.
  • “Full dosing regimen” as used herein refers to a dosing regimen that is specific to each vaccine. For example, a dosing regimen that is recommended by the Centers for Disease Control and Prevention (CDC) or a similar agent in countries outside of the United States.
  • CDC Centers for Disease Control and Prevention
  • the timing of testing for CD4+ & CD8+ T-cell responses e.g., contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject as described herein
  • the timing of the test is counted from the day the subject receives the full dosing of the vaccine, or where indicated counted from the day the subject receives a booster dose of the vaccine.
  • the full dosing regimen was two doses, while subsequent booster dose(s) have since been recommended by the CDC.
  • the test will be performed 30 days after the subject receives the second dose if two doses are considered a full dosing regimen; or the test will be performed 30 days after the subject receives the last booster dose, if two doses plus one or more booster dose(s) is considered a full dosing regimen.
  • Ad26.CoV2.S the full dosing regimen was one dose, while a subsequent booster dose has since been recommended.
  • the test will be performed 30 days after the subject receives the first dose if one does is considered a full dosing regimen, or 30 days after the subject receives the booster dose, if one plus a booster dose is considered a full dosing regimen.
  • additional booster doses for example, BNT162b2
  • the previously used full dosing regimen is administered over two doses, 21 days apart.
  • CD4+ and CD8+ T-cell responses were deemed important.
  • Various embodiments of the present invention provide an in vitro diagnostic assay to monitor SARS-CoV-2-specific CD4 and CD8 T cells in patients or vaccinated individuals in whole blood. While antibodies response may wane in patients, T-cell immunity persists long in body after infection or vaccination. Therefore, the present invention provides a developed methodology to reliably and qualitatively measure CD4 and CD8 T-cell immunity against SARS-CoV-2. This invention can be used to determine the status and durability of T-cell immunity to SARS-CoV-2 and variants.
  • the cytokines are not secreted solely by T lymphocytes and the current method to measure the levels of cytokines in blood could not reflect the T cell immunity in vivo.
  • This invention offers a diagnosis tool to accurately detect SARS-CoV-2-reactive CD4 and CD8 T cells.
  • the sensitivity and specificity of this invention represents a dramatic improvement over other technologies such as ELISpot assays where non-quantitative assessments are made.
  • This invention addresses the challenge how to accurately measure the acquired T cell immunity in patients or vaccinated individuals against SARS-CoV-2.
  • a purpose of this invention is to provide critical T cell immunity information of patients, vaccinated individuals, or general public to clinicians and health care workers in treatments or preventing the SARS-CoV-2 infection.
  • the assay of the present invention can be used to assess T-cell immunity in immunocompromised individuals who are at higher risk for poor outcomes from SARS-CoV-2 infection. Further the assay of the present invention can be used to assess cross-reactivity of SARS- CoV-2 reactive T-cells with emerging variants.
  • the present invention includes guiding decisions about vaccine development or need for re-vaccination.
  • CoV-2 T-cell assay can distinguish unexposed from infected patients and that intense immune responses to non-spike proteins are also induced by infection. We also see the intense immune responses in two normal individuals 2 months after the Pfizer vaccine. However, poor immune responses are seen in transplant patients compared to normal individuals. This is of great concerns and has important clinical implications for this at-risk patient population. [0071] Additionally, assessing the composition, scope, and durability of protective immunity generated after SARS-CoV-2 infection or vaccination are critical for control of the pandemic and future vaccination strategies. It is likely that analyzing immune responses to SARS- CoV-2 has garnered more attention and information than any other human infection in history. Traditional assessments have included antibody responses which are often transient or rapidly declining in patients with moderate infections.
  • Various embodiments of the present invention provide for a method of detecting a quantity of SARS CoV-2-specific CD4+ T-cells, a quantity of CD8+ T-cells, or a quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject, comprising: contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantity of SARS CoV-2-specific CD4+ T- cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells.
  • the method comprises detecting a quantity of both SARS
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilizing the cells allow for detection of intracellular cytokines with, for example, labeled monoclonal antibodies. Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for a method of detecting a quantity of SARS CoV-2-specific CD4+ T-cells, a quantity of CD8+ T-cells, or a quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject, comprising: contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantity of SARS CoV-2-specific CD4+ T- cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells, wherein the subject desires a determination regarding the presence or absence of protective immunity to SARS CoV-2.
  • the method comprises measuring a quantity of both
  • SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject is performed 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 21 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 21 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 30 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 30 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 30 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 270 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 270 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 270 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 1 year or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 1 year or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 1 year or more after the subject has had a SARS CoV-2 infection.
  • it is performed about 21-84 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-84 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-84 days after the subject has had a SARS CoV-2 infection.
  • it is performed about 21-70 days after the subject received a full dosing regimen, about 21-70 days after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or about 21-70 days after the subject has had a SARS CoV-2 infection.
  • it is performed about 30 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 30 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 30 days after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed about 60-120 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 60-120 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 60-120 days after the subject has had a SARS CoV-2 infection.
  • the method further comprising detecting the presence of
  • T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.05% or greater as compared to a control.
  • the method further comprising detecting the presence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.02% or greater as compared to a control.
  • the method further comprising detecting the presence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.1% or greater as compared to a control.
  • the method further comprising detecting the presence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.15% or greater as compared to a control.
  • the method further comprising detecting the presence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.2% or greater as compared to a control.
  • the method further comprising detecting the presence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.25% or greater as compared to a control.
  • the percent positives are after deducting the background levels in blood only conditions.
  • the method comprises detecting the presence of T-cell immunity when the quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject are 0.05% or greater as compared to a control. In various embodiments, the method comprises detecting the presence of T-cell immunity when the quantity of both SARS CoV-2- specific CD4+ T-cells and CD8+ T-cells in a subject are 0.1% or greater as compared to a control. In various embodiments, the method comprises detecting the presence of T-cell immunity when the quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject are 0.2% or greater as compared to a control. In various embodiments, the method comprises detecting the presence of T-cell immunity when the quantity of both SARS CoV-2- specific CD4+ T-cells and CD8+ T-cells in a subject are 0.25% or greater as compared to a control.
  • the SARS CoV-2 peptide fragments are fragments of
  • the SARS CoV-2 peptide fragments are fragments of SARS CoV-2 variant spike protein.
  • the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence.
  • the SARS CoV-2 peptide fragments are fragments of any one or more SARS CoV-2 or SARS CoV-2 variant structural proteins, nonstructural proteins, or accessory proteins. Examples of structural proteins include E protein, M protein, the N protein, the S protein (also known as spike protein).
  • nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6.
  • assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.
  • the SARS CoV-2 is Washington isolate of SARS-CoV-
  • coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1. As such, the peptide fragments are from the Washington isolate of SARS-CoV-2.
  • the SARS CoV-2 is a SARS CoV-2 variant.
  • the peptide fragments are from the variant SARS-CoV-2.
  • SARS CoV-2 variant include but are not limited to U.K. variant, California variant, South Africa variant, Brazil variant, Delta variant, Omicron variant, subvariants/sublineages thereof and combinations thereof.
  • SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours.
  • period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
  • period of time is about 4-6 hours.
  • period of time is about 6-8 hours.
  • period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with three or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • the method comprises measuring the quantities of both
  • the method further comprises enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa. In various embodiments, the method further comprises enumerating both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • the CD8+ cells expressing IFNy and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa comprises using flow cytometry.
  • IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies each independently comprises a label.
  • Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein.
  • the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label.
  • the label is a fluorophore.
  • Exemplary fluorescent labeling reagents include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Methoxycoumarin, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R- Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430
  • the biological sample comprises T-cells.
  • the biological sample is whole blood. Additional examples of biological samples include, but are not limited to nose swab, cheek swab, saliva, sputum, pulmonary secretions, mucus, blood, serum, plasma, urine, lymph, fecal extract, intestinal fluid, amniotic fluid, and tissue sample.
  • the biological sample is less than lOmL. In various embodiments, the biological sample is less than 8mL. In various embodiments, the biological sample is less than 5mL. In various embodiments, the biological sample is less than 4mL. In various embodiments, the biological sample is less than 3mL.
  • the biological sample is less than 2mL. In various embodiments, the biological sample is about 2mL. In various embodiments, the biological sample is about 3mL. In various embodiments, the biological sample is about 4mL. In various embodiments, the biological sample is about 5mL. In various embodiments, the biological sample is about 8mL. In various embodiments, the biological sample is whole blood and is about 3mL.
  • the subject has received an organ or tissue transplant.
  • the subject has received a kidney transplant, lung transplant, heart transplant, liver transplant, pancreas transplant, stomach transplant, intestine transplant, cornea transplant, bone morrow transplant, tendon transplant, or heart valve transplant.
  • the subject has received a kidney transplant.
  • the subject is immunocompromised.
  • immunocompromised individuals include but are not limited to those who have cancer, are HIV positive, have AIDS, are taking immunosuppressive drugs, are taking anticancer drugs, are undergoing radiation therapy, or are transplant patients.
  • the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.
  • the subject has not been known to be infected with
  • SARS CoV-2 and has not received a vaccine for SARS CoV-2.
  • the subject may not be aware that he or she had been infected with SARS CoV-2; for example, the subject did not exhibit symptoms of SARS CoV-2.
  • the subject does not have detectable amounts of spike- specific IgG. In various embodiments, the subject does not have an amount of spike-specific IgG that is considered sterilizing immunity. In various embodiments, the subject does not have amount of spike-specific IgG above a control level.
  • the control level can be the mount of spike-specific IgG that is generated by a healthy individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an nonimmunocompromized individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual who is not taking any immunosuppressant drugs or is not receiveing any immunosuppressant therapies. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual is not an organ or tissue transplant recipient.
  • Various embodiments of the present invention provide for a method of detecting a likely absence of protective immunity to SARS CoV-2, comprising: contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting the likely absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • the percent positives are after deducting the background
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • the method comprises detecting the likely absence of T- cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control.
  • the method comprises detecting the likely absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control.
  • the method comprises detecting the likely absence of T-cell immunity to SARS- CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.15% compared to a control.
  • the method comprises detecting the likely absence of T-cell immunity to SARS- CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control.
  • the method comprises detecting the likely absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • the method comprises detecting the likely absence of T- cell immunity when the quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject less than 0.05% as compared to a control. In various embodiments, the method comprises detecting the likely absence of T-cell immunity when the quantity of both SARS CoV- 2-specific CD4+ T-cells and CD8+ T-cells in a subject are less than 0.1% as compared to a control. In various embodiments, the method comprises detecting the likely absence of T-cell immunity when the quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject less than 0.2% as compared to a control. In various embodiments, the method comprises detecting the likely absence of T-cell immunity when the quantity of both SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject less than 0.25% as compared to a control.
  • the method further comprising re-vaccinating the subject against SARS CoV-2.
  • the subject is given a booster dose of the SARS CoV-2 vaccine.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject is performed 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 21 days or more after the subject received a booster dose SARS CoV-2 vaccine, or 21 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 30 days or more after the subject received a full dosing regimen, 30 days or more after the subject received a booster dose SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or 30 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 1 year or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 1 year or more after the subject received a booster dose SARS CoV-2 vaccine, or 1 year or more after the subject has had a SARS CoV-2 infection.
  • it is performed about 21-84 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-84 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-84 days after the subject has had a SARS CoV-2 infection.
  • it is performed about 21-70 days after the subject received a full dosing regimen, about 21-70 days after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or about 21-70 days after the subject has had a SARS CoV-2 infection.
  • the SARS CoV-2 peptide fragments are fragments of
  • the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence.
  • the SARS CoV-2 peptide fragments are fragments of any one or more SARS CoV- 2 structural proteins, nonstructural proteins, or accessory proteins.
  • structural proteins include E protein, M protein, the N protein, the S protein (also known as spike protein).
  • nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6.
  • assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.
  • the SARS CoV-2 is a natural isolate SARS-CoV-2.
  • the peptide fragments are from the natural isolate SARS-CoV-2.
  • the SARS CoV-2 is Washington isolate of SARS-CoV-
  • coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1. As such, the peptide fragments are from the Washington isolate of SARS-CoV-2.
  • the SARS CoV-2 is a SARS CoV-2 variant.
  • the peptide fragments are from the variant SARS-CoV-2.
  • SARS CoV-2 variant include but are not limited to U.K. variant, California variant, South Africa variant, Brazil variant, Delta variant, Omicron variant, subvariants/sublineages thereof and combinations thereof.
  • SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours.
  • period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
  • period of time is about 4-6 hours.
  • period of time is about 6-8 hours.
  • period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with three or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • the method further comprises enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa. In various embodiments, the method further comprises enumerating both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • the CD8+ cells expressing IFNy and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa comprises using flow cytometry.
  • IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies each independently comprises a label.
  • Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein.
  • the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label.
  • the label is a fluorophore.
  • Exemplary fluorescent labeling reagents include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Methoxycoumarin, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R- Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430
  • the biological sample comprises T-cells.
  • the biological sample is whole blood. Additional examples of biological samples include, but are not limited to nose swab, cheek swab, saliva, sputum, pulmonary secretions, mucus, blood, serum, plasma, urine, lymph, fecal extract, intestinal fluid, amniotic fluid, and tissue sample.
  • the biological sample is less than lOmL. In various embodiments, the biological sample is less than 8mL. In various embodiments, the biological sample is less than 5mL. In various embodiments, the biological sample is less than 4mL. In various embodiments, the biological sample is less than 3mL. In various embodiments, the biological sample is less than 2mL. In various embodiments, the biological sample is about 2mL. In various embodiments, the biological sample is about 3mL. In various embodiments, the biological sample is about 4mL. In various embodiments, the biological sample is about 5mL. In various embodiments, the biological sample is about 8mL. In various embodiments, the biological sample is whole blood and is about 3mL.
  • the subject has received an organ or tissue transplant.
  • the subject has received a kidney transplant, lung transplant, heart transplant, liver transplant, pancreas transplant, stomach transplant, intestine transplant, cornea transplant, bone morrow transplant, tendon transplant, or heart valve transplant.
  • the subject has received a kidney transplant.
  • the subject is immunocompromised.
  • immunocompromised individuals include but are not limited to those who have cancer, are HIV positive, have AIDS, are taking immunosuppressive drugs, are taking anticancer drugs, are undergoing radiation therapy, are transplant patients.
  • the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.
  • the subject has not been known to be infected with
  • SARS CoV-2 and has not received a vaccine for SARS CoV-2.
  • the subject may not be aware that he or she had been infected with SARS CoV-2; for example, the subject did not exhibit symptoms of SARS CoV-2.
  • the subject does not have detectable amounts of spike- specific IgG. In various embodiments, the subject does not have an amount of spike-specific IgG that is considered sterilizing immunity. In various embodiments, the subject does not have amount of spike-specific IgG above a control level.
  • the control level can be the mount of spike-specific IgG that is generated by a healthy individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an nonimmunocompromized individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual who is not taking any immunosuppressant drugs or is not receiveing any immunosuppressant therapies. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual is not an organ or tissue transplant recipient.
  • Various embodiments of the present invention provide for a method of determining the likelihood of contracting coronavirus disease 2019 (COVID-19). It can be important to determine a subject’s risk for contracting COVID-19. For example, a subject may have received a vaccine but an amount of time has passed and thus, may be at a greater risk of contracting COVID-19. Therefore, a subject may want to know whether revaccination should be done. In another example, the subject, even though vaccinated, may not have developed a T-cell protective immune response and thus, may want to either be revaccinated or take other precautions. Immunocompromised patients and transplant patients may be in this exemplary group.
  • a subject may have been vaccinated and has a protective immune response, but with circulating SARS CoV-2 variants may want to know the likelihood of contracting COVID-19 via a SARS CoV-2 variant.
  • the test would utilize a composition comprising SARS CoV-2 variant peptide fragment(s).
  • the method comprises contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • the percent positives are after deducting the background levels in blood only conditions.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T- cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 the quantity of SARS CoV- 2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.15% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2- specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control. In various embodiments, the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.15% compared to a control. In various embodiments, the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject is performed 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 21 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 21 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 30 days or more after the subject received a full dosing regimen, 30 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or 30 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 1 year or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 1 year or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 1 year or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed about 21- 84 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-84 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-84 days after the subject has had a SARS CoV-2 infection.
  • it is performed about 60-120 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 60-120 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 60-120 days after the subject has had a SARS CoV-2 infection.
  • the SARS CoV-2 peptide fragments are fragments of
  • the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence.
  • the SARS CoV-2 peptide fragments are fragments of any one or more SARS CoV- 2 structural proteins, nonstructural proteins, or accessory proteins.
  • structural proteins include E protein, M protein, the N protein, the S protein (also known as spike protein).
  • nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6.
  • the SARS CoV-2 is a natural isolate SARS-CoV-2.
  • the peptide fragments are from the natural isolate SARS-CoV-2.
  • the SARS CoV-2 is Washington isolate of SARS-CoV-
  • coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1. As such, the peptide fragments are from the Washington isolate of SARS-CoV-2.
  • the SARS CoV-2 is a SARS CoV-2 variant.
  • the peptide fragments are from the variant SARS-CoV-2.
  • SARS CoV-2 variant include but are not limited to U.K. variant, California variant, South Africa variant, Brazil variant, Delta variant, Omicron variant and subvariants/sublineages and combinations thereof.
  • SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours.
  • period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
  • period of time is about 4-6 hours.
  • period of time is about 6-8 hours.
  • period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with three or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • the method further comprises enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa. In various embodiments, the method further comprises enumerating both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • the CD8+ cells expressing IFNy and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa comprises using flow cytometry.
  • IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies each independently comprises a label.
  • Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein.
  • the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label.
  • the label is a fluorophore.
  • Exemplary fluorescent labeling reagents include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Methoxycoumarin, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R- Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430
  • the biological sample comprises T-cells.
  • the biological sample is whole blood. Additional examples of biological samples include, but are not limited to cheek swab, saliva, sputum, pulmonary secretions, mucus, blood, serum, plasma, urine, lymph, fecal extract, intestinal fluid, amniotic fluid, and tissue sample.
  • the biological sample is less than lOmL. In various embodiments, the biological sample is less than 8mL. In various embodiments, the biological sample is less than 5mL. In various embodiments, the biological sample is less than 4mL. In various embodiments, the biological sample is less than 3mL. In various embodiments, the biological sample is less than 2mL.
  • the biological sample is about 2mL. In various embodiments, the biological sample is about 3mL. In various embodiments, the biological sample is about 4mL. In various embodiments, the biological sample is about 5mL. In various embodiments, the biological sample is about 8mL. In various embodiments, the biological sample is whole blood and is about 3mL.
  • the subject has received an organ or tissue transplant.
  • the subject has received a kidney transplant, lung transplant, heart transplant, liver transplant, pancreas transplant, stomach transplant, intestine transplant, cornea transplant, bone morrow transplant, tendon transplant, or heart valve transplant.
  • the subject has received a kidney transplant.
  • the subject is immunocompromised.
  • immunocompromised individuals include but are not limited to those who have cancer, are HIV positive, have AIDS, are taking immunosuppressive drugs, are taking anticancer drugs, are undergoing radiation therapy, are transplant patients.
  • the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.
  • the subject has not been known to be infected with
  • SARS CoV-2 and has not received a vaccine for SARS CoV-2.
  • the subject may not be aware that he or she had been infected with SARS CoV-2; for example, the subject did not exhibit symptoms of SARS CoV-2.
  • the subject does not have detectable amounts of spike- specific IgG. In various embodiments, the subject does not have an amount of spike-specific IgG that is considered sterilizing immunity. In various embodiments, the subject does not have amount of spike-specific IgG above a control level.
  • the control level can be the mount of spike-specific IgG that is generated by a healthy individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by a non- immunocompromised individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual who is not taking any immunosuppressant drugs or is not receiving any immunosuppressant therapies. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual is not an organ or tissue transplant recipient.
  • Various embodiments of the present invention provide for a method of vaccinating a subject against SARS CoV-2, comprising detecting a likelihood of contracting coronavirus disease 2019 (COVID-19) by contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2- specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T
  • the method comprises measuring the the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for a method of vaccinating a subject against SARS CoV-2, comprising detecting a likely absence of protective immunity to SARS CoV-2 by contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting the likely absence of T-cell immunity to SARS- CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2- specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control; and administering
  • the method comprises detecting a likely absence of protective immunity to SARS CoV-2 by contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting the likely absence of T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control; and administering to the subject a vaccine against SARS CoV-2.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for method of vaccinating a subject against SARS CoV-2, comprising obtaining the results regarding a likelihood of contracting coronavirus disease 2019 (COVID-19) wherein the results were obtained by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2- specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T
  • the method obtaining the results regarding a likelihood of contracting coronavirus disease 2019 (COVID-19) wherein the results were obtained by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV- 2-specific CD8+ T cells are less than 0.05% compared to a control; and administering to the subject a vaccine against SARS CoV-2.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for method of vaccinating a subject against SARS CoV-2, comprising obtaining the results regarding a likely absence of protective immunity to SARS CoV-2 wherein the results were obtained by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likely absence of T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV- 2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than
  • the method comprises obtaining the results regarding a likely absence of protective immunity to SARS CoV-2 wherein the results were obtained by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likely absence of T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control; and administering to the subject a vaccine against SARS CoV-2.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for a method of vaccinating a subject against SARS CoV-2, comprising administering a vaccine against SARS CoV-2 to a subject who has been determined to have a likelihood of contracting coronavirus disease 2019 (COVID-19), wherein the subject was determined by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T
  • the method comprises administering a vaccine against
  • SARS CoV-2 to a subject who has been determined to have a likelihood of contracting coronavirus disease 2019 (COVID-19), wherein the subject was determined by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for a method of vaccinating a subject against SARS CoV-2, comprising administering a vaccine against SARS CoV-2 to a subject who has been determined to have a likely absence of protective immunity to SARS CoV-2, wherein the subject was determined by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2- specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD
  • the method comprises administering a vaccine against
  • SARS CoV-2 to a subject who has been determined to have a likely absence of protective immunity to SARS CoV-2, wherein the subject was determined by a method comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; measuring the quantities of both SARS CoV-2- specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells; detecting a likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T- cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2- specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.02% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.1% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.15% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.2% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.25% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.02% or greater as compared to a control. In various embodiments, the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.1% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV- 2-specific CD8+ T cells are 0.15% or greater as compared to a control. In various embodiments, the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.2% or greater as compared to a control.
  • the method comprises detecting the presence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are 0.25% or greater as compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T- cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2- specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.15% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control. In various embodiments, the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV- 2-specific CD8+ T cells are less than 0.15% compared to a control.
  • the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control. In various embodiments, the method comprises detecting the likely absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T- cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 the quantity of SARS CoV- 2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.15% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2-specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantity of SARS CoV-2- specific CD4+ T-cells, the quantity of SARS CoV-2-specific CD8+ T cells, or the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.02% compared to a control. In various embodiments, the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.1% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.15% compared to a control. In various embodiments, the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.2% compared to a control.
  • the method comprises detecting a likelihood of contracting COVID-19 based on the absence of sufficient T-cell immunity to SARS-CoV-2 when the quantities of both SARS CoV-2-specific CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.25% compared to a control.
  • contacting a composition comprising SARS CoV-2 peptide fragments to the biological sample obtained from the subject is performed 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 21 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 21 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 30 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 30 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 30 days or more after the subject has had a SARS CoV-2 infection.
  • it is performed 1 year or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 1 year or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 1 year or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed about 21- 84 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-84 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-84 days after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 21 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 21 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 21 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 30 days or more after the subject received a full dosing regimen, 30 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or 30 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 45 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 45 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 45 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 60 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 60 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 60 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 90 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 90 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 90 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 120 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 120 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 120 days or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine 1 year or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 1 year or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 1 year or more after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine about 21-84 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-84 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-84 days after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine about 21-70 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-70 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-70 days after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine about 21-56 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-56 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-56 days after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine about 21-42 days after the subject received a full dosing regimen of a SARS CoV-2 vaccine, about 21-42 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 21-42 days after the subject has had a SARS CoV-2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine about 30 days after the subject received a full dosing regimen, about 30 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 30 days after the subject has had a SARS CoV- 2 infection.
  • administering the vaccine against SARS CoV-2 comprises administering the vaccine about 60-120 days after the subject received a full dosing regimen, about 60-120 days after the subject received a booster dose of a SARS CoV-2 vaccine, or about 60-120 days after the subject has had a SARS CoV-2 infection.
  • SARS CoV-2 vaccines that are administered include but are not limited to Pfizer-BioNTech BNT162b2, Modema mRNA-1273, Janssen/Johnson & Johnson Ad26.CoV2.S, AstraZeneca/Oxford ChAdOxl (AZS1222), Novavax NVX-CoV2373, CureVac/GSK CVnCoV, Gamaleya National Research Center for Epidemiology and Microbiology Gam-COVID-Vac (Sputnik V), Sinovac Biotech CoronaVac, and Sinopharm 1/2 BBIBO-CorV. Future generations of these vaccines, as well as SARS CoV-2 vaccines currently in development are also examples of SARS CoV-2 vaccines that can be administered in accordance with the methods of the present invention.
  • the SARS CoV-2 peptide fragments are fragments of
  • the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence.
  • the SARS CoV-2 peptide fragments are fragments of any one or more SARS CoV- 2 structural proteins, nonstructural proteins, or accessory proteins.
  • structural proteins include E protein, M protein, the N protein, the S protein (also known as spike protein).
  • nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6.
  • assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.
  • the SARS CoV-2 is a natural isolate SARS-CoV-2. As such, the peptide fragments are from the natural isolate SARS-CoV-2. [0183] In various embodiments, the SARS CoV-2 is Washington isolate of SARS-CoV-
  • coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1. As such, the peptide fragments are from the Washington isolate of SARS-CoV-2.
  • the SARS CoV-2 is a SARS CoV-2 variant.
  • the peptide fragments are from the variant SARS-CoV-2.
  • SARS CoV-2 variant include but are not limited to U.K. variant, California variant, South Africa variant, Brazil variant, Delta variant, Omicron variant and subvariants/sublineages and combinations thereof.
  • SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours.
  • period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
  • period of time is about 4-6 hours.
  • period of time is about 6-8 hours.
  • period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with three or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • the method further comprises enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa. In various embodiments, the method further comprises enumerating both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • the CD8+ cells expressing IFNy and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa comprises using flow cytometry.
  • IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies each independently comprises a label.
  • Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein.
  • the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label.
  • the label is a fluorophore.
  • Exemplary fluorescent labeling reagents include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Methoxycoumarin, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R- Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430
  • the biological sample comprises T-cells.
  • the biological sample is whole blood. Additional examples of biological samples include, but are not limited to nose swab, cheek swab, saliva, sputum, pulmonary secretions, mucus, blood, serum, plasma, urine, lymph, fecal extract, intestinal fluid, amniotic fluid, and tissue sample.
  • the biological sample is less than lOmL. In various embodiments, the biological sample is less than 8mL. In various embodiments, the biological sample is less than 5mL. In various embodiments, the biological sample is less than 4mL. In various embodiments, the biological sample is less than 3mL. In various embodiments, the biological sample is less than 2mL. In various embodiments, the biological sample is about 2mL. In various embodiments, the biological sample is about 3mL. In various embodiments, the biological sample is about 4mL. In various embodiments, the biological sample is about 5mL. In various embodiments, the biological sample is about 8mL. In various embodiments, the biological sample is whole blood and is about 3mL.
  • the subject has received an organ or tissue transplant.
  • the subject has received a kidney transplant, lung transplant, heart transplant, liver transplant, pancreas transplant, stomach transplant, intestine transplant, cornea transplant, bone morrow transplant, tendon transplant, or heart valve transplant.
  • the subject has received a kidney transplant.
  • the subject is immunocompromised.
  • immunocompromised individuals include but are not limited to those who have cancer, are HIV positive, have AIDS, are taking immunosuppressive drugs, are taking anticancer drugs, are undergoing radiation therapy, are transplant patients.
  • the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.
  • the subject has not been known to be infected with
  • SARS CoV-2 and has not received a vaccine for SARS CoV-2.
  • the subject may not be aware that he or she had been infected with SARS CoV-2; for example, the subject did not exhibit symptoms of SARS CoV-2.
  • the subject does not have detectable amounts of spike- specific IgG. In various embodiments, the subject does not have an amount of spike-specific IgG that is considered sterilizing immunity. In various embodiments, the subject does not have amount of spike-specific IgG above a control level.
  • the control level can be the mount of spike-specific IgG that is generated by a healthy individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by a non- immunocompromised individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual who is not taking any immunosuppressant drugs or is not receiving any immunosuppressant therapies. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual is not an organ or tissue transplant recipient.
  • Various embodiments of the present invention provide for a method of testing a vaccine’s or immune composition’s efficacy in providing a T-cell protective immune response, comprising contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantity of SARS CoV- 2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells, wherein the subject has been administered the vaccine or the immune composition.
  • the method comprises contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells, wherein the subject has been administered the vaccine or the immune composition.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample.
  • Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • Various embodiments of the present invention provide for a method of testing a vaccine’s or immune composition’s efficacy in providing a T-cell protective immune response against a SARS CoV-2 variant, comprising contacting a composition comprising SARS CoV-2 variant peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells, wherein the subject has been administered the vaccine or the immune composition.
  • the method comprises contacting a composition comprising SARS CoV-2 variant peptide fragments to a biological sample obtained from the subject for a period of time; and measuring the quantities of both SARS CoV-2-specific CD4+ T- cells and SARS CoV-2-specific CD8+ T-cells, wherein the subject has been administered the vaccine or the immune composition.
  • the method further comprises permeabilizing the cells after contacting a composition comprising SARS CoV-2 peptide fragments to a biological sample. Permeabilization can be performed, for example, by using brefeldin A. Additional examples include but are not limited to Triton X-100, Tween-20, saponins, and methanol.
  • the SARS CoV-2 variant peptide fragments are fragments of SARS CoV-2 spike protein.
  • the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence.
  • the SARS CoV-2 variant peptide fragments are fragments of any one or more SARS CoV-2 structural proteins, nonstructural proteins, or accessory proteins. Examples of structural proteins include E protein, M protein, the N protein, the S protein (also known as spike protein).
  • nonstructural proteins examples include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6.
  • assessor proteins examples include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.
  • SARS CoV-2 variants include but are not limited to U.K. variant,
  • SARS CoV-2 variant peptide fragments is in contact with the biological sample is about 8 to 10 hours.
  • period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours.
  • period of time is about 4-6 hours.
  • period of time is about 6-8 hours.
  • period of time is about 9-11 hours.
  • the period of time is about 9 hours.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • measuring the quantity of SARS CoV-2-specific CD4+ T-cells, measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV- 2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with three or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • the method further comprises enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa. In various embodiments, the method further comprises enumerating both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • the CD8+ cells expressing IFNy and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa comprises using flow cytometry.
  • IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies each independently comprises a label.
  • Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein.
  • the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label.
  • the label is a fluorophore.
  • Exemplary fluorescent labeling reagents include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Methoxycoumarin, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R- Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430
  • the biological sample comprises T-cells.
  • the biological sample is whole blood. Additional examples of biological samples include, but are not limited to nose swab, cheek swab, saliva, sputum, pulmonary secretions, mucus, blood, serum, plasma, urine, lymph, fecal extract, intestinal fluid, amniotic fluid, and tissue sample.
  • the biological sample is less than lOmL. In various embodiments, the biological sample is less than 8mL. In various embodiments, the biological sample is less than 5mL. In various embodiments, the biological sample is less than 4mL. In various embodiments, the biological sample is less than 3mL. In various embodiments, the biological sample is less than 2mL. In various embodiments, the biological sample is about 2mL. In various embodiments, the biological sample is about 3mL. In various embodiments, the biological sample is about 4mL. In various embodiments, the biological sample is about 5mL. In various embodiments, the biological sample is about 8mL. In various embodiments, the biological sample is whole blood and is about 3mL.
  • the subject has received an organ or tissue transplant.
  • the subject has received a kidney transplant, lung transplant, heart transplant, liver transplant, pancreas transplant, stomach transplant, intestine transplant, cornea transplant, bone morrow transplant, tendon transplant, or heart valve transplant.
  • the subject has received a kidney transplant.
  • the subject is immunocompromised.
  • immunocompromised individuals include but are not limited to those who have cancer, are HIV positive, have AIDS, are taking immunosuppressive drugs, are taking anticancer drugs, are undergoing radiation therapy, are transplant patients.
  • the subject has received a vaccine for SARS CoV-2 that is not specifically developed for the SARS CoV-2 variant.
  • the subject has been infected with SARS CoV-2 variant.
  • the subject does not have detectable amounts of spike- specific IgG. In various embodiments, the subject does not have an amount of spike-specific IgG that is considered sterilizing immunity. In various embodiments, the subject does not have amount of spike-specific IgG above a control level.
  • the control level can be the mount of spike-specific IgG that is generated by a healthy individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an nonimmunocompromized individual. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual who is not taking any immunosuppressant drugs or is not receiveing any immunosuppressant therapies. In various embodiments, the control level can be the mount of spike-specific IgG that is generated by an individual is not an organ or tissue transplant recipient.
  • kits comprising: a composition comprising SARS-CoV-2 peptide fragments; anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and instructions to use the composition and the antibodies to measure a quantity of SARS CoV-2-specific CD4+ T-cells, a quantity of SARS CoV-2-specific CD8+ T-cells, or quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells.
  • the kit comprises two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti- g-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies the kit comprises three or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies.
  • the kit further comprises brefeldin A, phytohemagglutinin (PHA), anti-CD28 antibodies, or anti-CD49d antibodies, or combinations thereof.
  • PHA phytohemagglutinin
  • anti-CD28 antibodies or anti-CD49d antibodies, or combinations thereof.
  • the anti-IL-2 antibodies anti-TNF-a antibodies, anti-y-
  • IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies each independently comprises a label.
  • the anti-IL-2 antibodies anti-TNF-a antibodies, anti-y-
  • IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies each independently comprises a label.
  • Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein.
  • the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label.
  • the label is a fluorophore.
  • Exemplary fluorescent labeling reagents include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Methoxycoumarin, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R- Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430
  • the SARS CoV-2 peptide fragments are fragments of
  • the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence.
  • the SARS CoV-2 peptide fragments are fragments of any one or more SARS CoV- 2 structural proteins, nonstructural proteins, or accessory proteins.
  • structural proteins include E protein, M protein, the N protein, the S protein (also known as spike protein).
  • nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6.
  • assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.
  • the SARS CoV-2 is a natural isolate SARS-CoV-2.
  • the peptide fragments are from the natural isolate SARS-CoV-2.
  • the SARS CoV-2 is Washington isolate of SARS-CoV-
  • coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1. As such, the peptide fragments are from the Washington isolate of SARS-CoV-2.
  • the SARS CoV-2 is a SARS CoV-2 variant.
  • the peptide fragments are from the variant SARS-CoV-2.
  • SARS CoV-2 variant include but are not limited to U.K. variant, California variant, South Africa variant, Brazil variant, Delta variant, Omicron variant, subvariants/sublineages thereof, and combinations thereof.
  • instructions comprise measuring the quantity of SARS
  • CoV-2-specific CD4+ T-cells measuring the quantity of SARS CoV-2-specific CD8+ T-cells, or measuring the quantities of both SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells comprises contacting the biological sample with anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, or anti-CD8 antibodies, or combinations thereof; and detecting the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • the instruction further comprises enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa.
  • instructions for detecting and/or enumerating the CD4+ cells expressing IL-2 and TNFa, the CD8+ cells expressing IFNy and TNFa, or both the CD4+ cells expressing IL-2 and TNFa and the CD8+ cells expressing IFNy and TNFa comprises instructions for using flow cytometry.
  • B cells CD19+, CD27+
  • COVID-19-specific IgG and IgM antibodies in populations with different demographics: ages, sex, underlying disease severity and vaccine responses in normal and immune compromised individuals.
  • the comparison of unexposed and recovered individuals provides an indication of Cov-2-specific T cell immunity.
  • the CoV-2 T & B cell immunity in volunteers before and after vaccination are monitored.
  • Monitoring immune responses in CD4+& CD8+ T-cells is critical as the former is required for immunologic memory and the later for cytotoxic elimination of COVID-19 infected cells.
  • Cov-2 T cell response in healthy individuals, recovered patients, transplant recipients and volunteer’s post- vaccination. We expect to enroll about total 200 individuals, including 50 CoV-2 non-exposed individuals or pre-vaccination, 50 recovered patients, 50 healthy volunteers’ post-vaccination, and 50 transplant patients with different clinical complications.
  • Whole blood will be stimulated by CoV-2 peptide pools for 9 h and cells will be surface stained for CD4 &CD8 lymphocytes, CD19+/CD27+ B-cells along with activation markers such as CD45RA and CD45RO.
  • CoV-2 reactive T cells & B-cells can be detected by their intracellular cytokine production of IFNy, TNFa, and IL-2 or MHC class II expression.
  • CD4 cells can be further analyzed for T follicular helper cells for their role in antibody production.
  • the cytolytic function of CD8 T cells can be analyzed by Granzyme B and perforin staining.
  • S SARS CoV-2 Spike glycoprotein
  • B.1.1.7 SARS CoV-2 Spike glycoprotein
  • VME virus membrane protein
  • NCAP nucleoprotein
  • APIA protein 3 A
  • N7A non-structural protein 7A
  • Brefeldin A and anti-CD28/CD49d were added and incubated for 9 hours at 37°C.
  • Negative and positive controls included cells not incubated with peptides and those stimulated with phytohemagglutinin (PHA).
  • PHA phytohemagglutinin
  • ELISA kit (Ray Biotech, GA) per the manufacture’s manual. Briefly, the 96 well plates coated with the SARS-CoV-2 SI RBD protein were incubated with plasma followed by biotinylated anti -human IgG. After washing, HRP-conjugated streptavidin was added, and spike-specific IgG was quantitated by OD450 nm reading.
  • IL-2 is a key growth factor for activated T-cells, while TNF-a and IFN-g are considered canonical inflammatory cytokines mediating effector/memory T-cell functions.
  • Analysis of cytokine production in stimulated T-cells confirmed that IL-2 and TNF-a were consistent markers for activated CD4+ T-cells, while activated CD8+ T-cells mainly produced TNF-a and IFN-g.
  • SARS-CoV-2 Spike peptide pool we were able to discern Spike-reactive T-cells by dual cytokine gating (Figure 11A-11B).
  • T-cells from SARS-CoV-2-infected or vaccinated individuals showed substantial spike-specific CD4+ and CD8+ T-cells.
  • CD4+ T-cell immunity 82%, 31 of 38
  • CD4+ T-cells demonstrated immunodominant responses to Spike peptides as previously described ( Figure 12A).
  • CD8+ T- cells showed similar responses to the 5 proteins; 66% (25 of 38) had positive CoV-2 specific CD8+ T-cells to one or more of 5 CoV-2 proteins.
  • BNT162b2 vaccine was used to compared Spike-specific T-cell immunity to 19 healthy controls, 38 infected patients, and 38 vaccinated individuals 1 month after the 2 nd vaccine dose (Figure 12B). No healthy unvaccinated individuals showed positive CD4+ T-cells against SARS-CoV-2, but infected patients and vaccinated individuals demonstrated substantial spike-specific CD4+ T-cell immunity: 76% (29 of 38) and 89% (34 of 38) respectively.
  • CD8+ T-cells from healthy controls, infected patients, and vaccinated individuals showed 21% (4 of 19), 32% (12 of 38), and 58% (22 of 38) positive immune responses against SARS-CoV-2 spike peptides, respectively. Therefore, the Pfizer BNT162b2 vaccine induced T-cell immunity to Spike-specific peptides that was equivalent or greater than that seen in infected patients after recovery.
  • T-cell immunity in SARS-CoV-2 patients in general, was associated with positive IgG serology. However, 39% of previously infected patients with positive Spike-IgG serology did not demonstrate T-cell immunity. This may represent variability in the composition of immune responses from one individual to another as was reported.
  • the B.1.1.7 variant contains the E484K mutation which renders resistance to serologic responses in infected individuals.
  • T cell immune responses to SARS-CoV-2 and variants of concern (Alpha and Delta) in infected and vaccinated individuals [0255]
  • a more comprehensive understanding of the breadth and longevity of immune responses after infection and vaccination requires analysis of cellular immunity.
  • T cell immunity in infected and vaccinated individuals identifying CD4+/CD8+ T cell cytokine responses to SARS-CoV-2 and variant peptides (Alpha, B.1.1.7 and Delta, B.1.617.2).
  • Our results demonstrate that T cells in infected or vaccinated individuals can elicit robust and cross-reactive immune responses against variants of concern (VOCs).
  • Spike-specific IgG receptor-binding domain (RBD) antibodies are evanescent and do not reflect important memory compo- nents.
  • RBD IgG receptor-binding domain
  • the emerging Delta variant is characterized by multiple mutations in the spike protein including T19R, A157-158, L452R, T478K, D614G, P681R, and D950N. It is likely that these mutations affect immune responses to important antigenic regions of the receptor-binding domain. In addition, strains with the P681R mutation have accelerated replication, increasing infectivity.
  • IL-2 and TNF-a are consistent markers for activated CD4+ T cells, while activated CD8+ T cells mainly produce TNF-a and IFN-g.
  • SARS-CoV-2 Spike peptide pool we were able to identify Spike- reactive T cells by dual-cytokine gating.
  • healthy individuals with no history of SARS-CoV-2 infection demonstrated no significant T cell response to the SARS- CoV-2 spike peptide.
  • T cells from SARS-CoV-2-infected or vaccinated individuals showed substantial spike-specific CD4+ and CD8+ T cell populations.
  • CD4+ T cell immunity 85%, 29 of 34
  • CD4+ T cells demonstrated immunodominant responses to Spike peptides (Fig. 16A).
  • CD8+ T cells showed similar responses to the 5 proteins.
  • Pfizer BNT162b2 vaccine was used to compared Spike-specific T cell immunity among 19 healthy controls, 38 infected patients, and 38 vaccinated individuals 1 month after the 2nd vaccine dose (Fig. 16A, 16B). No healthy unvaccinated individuals showed positive CD4+ T cell immunity against SARS-CoV-2, but infected patients and vaccinated individuals demonstrated substantial spike-specific CD4+ T cell immunity, with rates of 87% (33 of 38) and 89% (34 of 38), respectively.
  • CD8+ T cells from healthy controls, infected patients, or vaccinated individuals showed 21% (4 of 19), 34% (13 of 38), and 58% (22 of 38) positivity for immune responses against SARS-CoV-2 spike peptides, respectively. Therefore, the Pfizer BNT162b2 vaccine induced T cell reactivity to Spike-specific peptides that was equivalent to that seen in infected patients after recovery.
  • the Alpha (B.1.1.7) variant contains the E484K mutation, which establishes resistance to serologic responses in infected individuals.
  • VOC evades T cell immunity
  • Fig. 16E, 16F there were no significant reductions in CD4+/CD8+ T cell responses to the Alpha variant Spike peptides compared to those to the ancestral Spike peptides (mean of infected patients: 0.23% ancestral vs. 0.18% Alpha variant; mean of vaccinated individuals: 0.16% ancestral vs. 0.14% Alpha variant).
  • nearly identical CD4+ and CD8+ T cell responses to the Delta variant peptides and SARS-CoV-2 spike peptides were detected in 11 healthy BNT162b2-vaccinated individuals (Fig. 16E, 16F).
  • T cell memory induced by SARS-CoV-2 infection or vaccination produces similar immune responses against the Alpha and Delta variants. This suggests protective immunity against Alpha and Delta variant infection and possibly infection by other VOCs.
  • analysis of T cell responses to VOCs (Alpha and Delta) showed that SARS-CoV-2 infection and vaccination with BNT162b2 elicited equivalent T cell responses.
  • T cells, B cells, and plasma cells are a natural temporal evolution after infection and/or vaccination that produces populations that can rapidly be activated upon re-exposure to SARS-CoV-2 and are likely to have an important role in preventing or modifying infection by SARS- CoV-2 VOCs.
  • An important consideration in this regard is the dissipation of humoral immunity over time. IgG responses are critical for sterilizing immunity.
  • T cell immunity does require an infection to reactivate memory responses. This may result in mild or asymptomatic infections that would be considered “breakthrough” infection. Thus, the level and robust- ness of T cell memory responses would likely affect the clinical manifestations of the disease.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • T-cell immunity An important and more durable response involves cytotoxic T-cells that can eliminate virally infected cells and T helper cells, which are critical to coordinating adaptive immunity toward the virus and generating long-lasting immunologic memory.
  • T-cell immune responses in patients failing to generate spike-specific IgG may aid in a more comprehensive assessment of immunity to SARS-CoV-2, identifying patients who would no longer be considered “unvaccinated” based on negative spike-specific IgG. This likely has relevance to patients receiving B-cell directed therapies for autoimmune and hematologic diseases.
  • RBD Spike-receptor binding domain
  • SARS-CoV-2 Spike-specific T-cell assay as described herein was developed in our laboratory and was fully validated. Briefly, whole blood was incubated with 1 ug/ml SARS CoV-2 Spike glycoprotein (JPT Peptide Technologies GmbH, Berlin, Germany) in the presence of brefeldin A and anti-(TNF-a)) cells and CD8+ (TNF-a/interferon (IFN)-y) were enumerated and defined as CoV-2-specific T cells after deducting the background levels in blood only conditions. Dual cytokines (%) in CD4 cells >0.05% were considered positive. Negative and positive controls included cells not incubated with peptides and those stimulated with phytohemagglutinin (PHA).
  • PHA phytohemagglutinin
  • CMV-specific T cells were detected by cytokine flow cytometry developed in our laboratory. Briefly, whole blood was incubated with 1.75 ug/ml CMV protein pp65 peptides pool together with brefeldin A and anti-CD28/CD49d for 6 h at 37°C. The IFNy+ cell% in CD8 cells were enumerated and defined as CMV-specific cytotoxic T cells (CMV-Tc). CMV-Tc >0.20% were considered positive.
  • FITC Fluorescence Activated Cell Sorting
  • CD4 PerCP Cy5.5
  • CD 8 V450
  • CD45 V500
  • CD56 PE-CF594
  • APC fluorochrome-conjugated antibodies to IL-2
  • PE IFN-y
  • PE- Cy7 PE- Cy7
  • Kidney transplant patients evaluated in this study were maintained on tacrolimus+MMF+steroids (Tac, 52%, 32 of 61) or belatacept+MMF + steroids (Bela, 48%, 29 of 61).
  • tacrolimus+MMF+steroids Tec, 52%, 32 of 61
  • belatacept+MMF + steroids Bela, 48%, 29 of 61.
  • CMV CMV-specific T-cell immune responses in BNT162b2 vaccinated immunocompromised patients
  • T-cell immune responses could be dampened by immunosuppression post transplantation.
  • SARS-CoV-2-specific T-cell responses (CoV-2T)
  • CMV-Tc represents a memory response and all CMV-Tc+ patients were also CMV-IgG+.
  • SARS-CoV-2 Spike-RBD-specific IgG levels in 38 vaccinated transplant recipients (18 Bela + 20 TAC) were compared to 41 healthy nonimmunocompromised vaccinated individuals.
  • This patient group is likely at high risk for SARS-CoV-2 infection and are unlikely to respond to repeated vaccination, thus should be considered for passive immunotherapy with monoclonal antibodies to SARSCoV-2 Spike protein.
  • Spike IgG responses are essential for mediation of sterilizing immunity.
  • Spike IgG would bind to and eliminate virus before infection can occur.
  • the critical difference between cellular and humoral immunity is that T cells cannot prevent infection since antigen presentation is required before T-cell activation can occur.
  • SARS-CoV-2 Spike-specific CD4+/CD8+ T cells can be rapidly activated within hours of Spike-RBD exposure, as was shown in our studies, and initiate deployment of SARS-CoV-2 immunity. Since infection is required for T-cell activity, patients would likely have mild to moderate symptoms, but are unlikely to develop severe disease. In this regard, Oberhardt et al. recently showed that vaccine- induced CD8+ T cells are the primary mediators of protection after vaccination as they emerged prior to detection of neutralizing antibody and expand after booster vaccination.
  • T-cell immune responses Another important aspect of analysis of T-cell immune responses is the ability to detect immune responses to VOCs.
  • SARS-CoV-2 ancestral Spike
  • VOCs VOCs
  • SARS-CoV-2 equal Spike
  • alpha and delta Spike peptides were seen.
  • T-cell immunity confers a diverse and broadly reactive immune responses to ancestral and emerging VOCs.

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Abstract

La présente invention concerne des méthodes et des kits permettant d'évaluer l'immunité de lymphocytes T contre le SARS-CoV-2 et ses variants. L'invention concerne également des méthodes de vaccination et de revaccination.
PCT/US2022/032322 2021-06-07 2022-06-06 Dosage pour évaluer l'immunité de lymphocytes t contre le sars-cov-2 et ses variants chez des individus infectés et vaccinés WO2022260986A1 (fr)

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ANONYMOUS: "SARS-CoV-2 T Cell Analysis Kit", MILTENYI BIOTEC, 23 January 2021 (2021-01-23), XP093019301, Retrieved from the Internet <URL:https://web.archive.org/web/20210123002356/https://www.miltenyibiotec.com/US-en/lp/sars-cov-2-t-cell-analysis-kit.html#collapsepanei-1a35fe07-1332-40dd-9b61-213c41de5bc1> [retrieved on 20230131] *
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SATTLER ARNE, ANGERMAIR STEFAN, STOCKMANN HELENA, HEIM KATRIN MOIRA, KHADZHYNOV DMYTRO, TRESKATSCH SASCHA, HALLECK FABIAN, KREIS M: "SARS–CoV-2–specific T cell responses and correlations with COVID-19 patient predisposition", THE JOURNAL OF CLINICAL INVESTIGATION, B M J GROUP, GB, vol. 130, no. 12, 1 December 2020 (2020-12-01), GB , pages 6477 - 6489, XP093019345, ISSN: 0021-9738, DOI: 10.1172/JCI140965 *

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