AN ASSAY TO ASSESS T-CELL IMMUNITY TO SARS-COV-2 AND VARIANTS IN INFECTED AND VACCINATED INDIVIDUALS

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63/197,808, filed June 7, 2021, and U.S. provisional patent application No. 63/228,316, filed August 2, 2021, the entirety of both are hereby incorporated by reference.

FIELD OF INVENTION

[0002] This invention relates to the detection of T-cell Immunity to SARS-COV-2.

BACKGROUND

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] The COVID-19 pandemic caused by SARS-CoV-2 (CoV-2) infection is running rampant globally. Little is known about the nature of individual immune responses to CoV-2 infection and vaccinations. In analyzing the composition of sterilizing immunity, CD4+ & CD8+ T- cell and B-cell responses are deemed critical. A suboptimal T cell response could lead to development of non-sterilizing immunity to Cov-2 infection, possible persistence of viral shedding, followed by severe, lingering symptom in the host with the possibility of susceptibility to reinfection. This may disproportionally affect immune compromised individuals. However, persistent and unrelenting activation of T cells by COVID-19 could result in development of systemic inflammation, severe cytokine release syndrome and autoimmunity which can be life threatening. In addition, it was demonstrated that 20 to 40% of unexposed populations may contain some levels of cross-reactive T cells against CoV-2 virus. It becomes imperative to determine whether the T cell immunity against CoV-2 is induced and persist for long periods of time after vaccination and provides comparable protection to emerging variants. [0005] Heretofore, there was no available method to detect T cell immunity against

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.

[0006] As such, there is a need in the art for assessing an individual’s immune response to guide vaccinations, and treatments among other things.

SUMMARY OF THE INVENTION

[0007] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0008] 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.

[0009] In various embodiments, 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.

[0010] 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.

[0011] 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.

[0012] In various embodiments, the method can further comprise re-vaccinating the subject against SARS CoV-2.

[0013] 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.

[0014] In various embodiments, 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.

[0015] In various embodiments, 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.

[0016] In various embodiments, 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.

[0017] In various embodiments, 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In various embodiments, the method can further comprising adjusting one or more immunosuppressive medication that the subject is taking. In various embodiments, adjusting can comprise reducing an amount of the one or more immunosuppressive medication.

[0022] In various embodiments, 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. In various embodiments, 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. In various embodiments, 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.

[0023] In various embodiments, the SARS CoV-2 peptide fragments can be fragments of SARS CoV-2 spike protein.

[0024] In various embodiments, the SARS CoV-2 can be a natural isolate SARS-

CoV-2. In various embodiments, the SARS CoV-2 can be Washington isolate of SARS-CoV- 2. coronavirus having a nucleic acid sequence of GenBank accession no. MN985325.1. In various embodiments, the SARS CoV-2 can be a SARS CoV-2 variant. In various embodiments, 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.

[0025] In various embodiments, the period of time can be about 8 to 10 hours.

[0026] In various embodiments, 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 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.

[0027] In various embodiments, 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. [0028] In various embodiments, 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.

[0029] In various embodiments, the biological sample can comprise T-cells. In various embodiments, the biological sample can be whole blood.

[0030] In various embodiments, 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.

[0031] Various embodiments provide for a kit 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.

[0032] In various embodiments, 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.

[0033] In various embodiments, the kit can further comprise brefeldin A, phytohemagglutinin (PHA), anti-CD28 antibodies, or anti-CD49d antibodies, or combinations thereof.

[0034] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0035] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0036] 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.

[0037] Figure 2 depicts an exemplary process in accordance with various embodiments of the present invention. Briefly, 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. During incubation, 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. We then permeabilize the cells and stain with antibodies specific for the aforementioned cytokines and then perform flow cytometry to count SARS-CoV-2 reactive CD4+ and CD8+ T-cells (%). This test is highly specific and sensitive.

[0038] 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.

[0039] 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. As seen in this figure, 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.

[0040] Figure 5 A depicts an exemplary CoV-2-T-Intracellular Cytokine Flow Cytometry

(CoV-2-T) Assay in accordance with various embodiments of the present invention. After whole blood incubation with SARS-CoV-2 peptides and controls with brefeldin permeabilization, red cells are lysed and immune cells are detected by flow cytometry to distinguish CD8 and CD4 subsets. These cells are stained with anti-cytokine antibodies to detect IL-2+TNFa+cell % in CD4+ cells and IFNy+TNFa+cell % in CD8+ cells activated by the Spike protein and variants. The cells are enumerated and analyzed, respectively. Negative and positive controls included cells not incubated with S-peptides and those stimulated with phytohemagglutinin (PHA). Samples expressing 0.05% or greater numbers of CD4+/CD8+ T-cells expressing both cytokines are considered positive.

[0041] 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).

[0042] Figure 6 depicts SARS-CoV-2 T-cell Assay (TNF-a & IL-2 Production in CD4+

Cells) in accordance with various embodiments of the present invention. This figure shows CD4+ T-cell responses to Spike protein in non-infected and a patient known to have had SARS-CoV-2 infection. The positive predictive value of this assay is very strong.

[0043] Figure 7 depicts SARS-CoV-2 T-cell Assay (TNF-a & IL-2 Production in CD4+

Cells: Pfizer Vaccinated) in accordance with various embodiments of the present invention. Vaccine responses assessed 2 months after Pfizer vaccine trial participation. We surmised that both received the vaccine. This was later confirmed.

[0044] Figure 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.

[0045] 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.

[0046] Figures 10A-10B show the monitoring of CD4 and CD8 T cell immunity against

Spike protein of CoV-2 and variants. 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.

[0047] Figures 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.

[0048] Figures 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. The correlation of Nucleocapsid- specific IgG titers with CD4+ T-cell immune responses to one or more of 5 major SARS-CoV-2 peptides in 25 patients. 12D. 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%).

[0049] 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.

[0050] Figure 14A shows SARS-CoV-2 Spike-Specific CD4+/CD8+ T-cell Responses in

Normal Individuals and Transplant Patients Post-Pfizer Vaccination. This compares the immune response to SARS-CoV-2 Spike peptides in normal individuals’ pre-vaccination and post vaccination compared to immune response in immunocompromised transplant recipients 1 month post vaccination. This data that demonstrates the paucity of SARS-CoV-2 Spike-specific T-cell responses in renal transplant patients on immunosuppression compared to non- immunosuppressed individuals.

[0051] Figure 14B shows a summary of SARS-CoV-2 Spike-Specific immunity after

Pfizer vaccination in normal individuals and immunocompromised transplant recipients. This shows the lack of SARS-CoV-2 Spike IgG immune responses in this immunologically compromised population. 88% normal individuals develop CD4+/CD8+ T-cell responses to SARS-CoV-2 spike peptides after Pfizer vaccination, but only 11% showed T-cell responses and 35% antibody response in immunocompromised transplant recipients. The differences in immune responses are dramatic and concerning.

[0052] Figure 15 depicts a representative schema for revaccination in immunocompromised patients.

[0053] Figure 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. CD4+/CD8+ Spike- specific T cells from infected and vaccinated healthy individ als were assessed for reactivity to ancestral, Alpha variant and Delta variant Spike peptides. Each dot represents one individual. *P = .001.

[0054] Figure 17A depicts detectable CD4 ⁺ /CD8 ⁺ T-cell immune responses in vaccinated patients against SARS-CoV-2 spike peptides. Flow cytometry diagrams from 7 patients (P1-P7) assessed after 2 doses of PFIZER or MODERNA mRNA vaccines. 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 ⁺ ) and CD8 ⁺ T cells (IFNy'TNFa ¹ ) are shown in the upper right-hand comer of each flow box.

[0055] Figure 17B shows immune responses to SARS-CoV-2 spike peptides in 2 patients who obtained external revaccination with the Johnson & Johnson vaccine. As in (17A), 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. IFNy, interferon g; IL, interleukin; P, patient; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNFa, tumor necrosis factor a. [0056] Figures 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.

[0057] Figures 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

DESCRIPTION OF THE INVENTION

[0058] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton el al., Dictionary of Microbiology and Molecular Biology 3 ^rd ed. , Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 ^th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 ^th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0059] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0060] As used herein 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. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, 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.

[0061] “SARS-CoV-2” as used herein, and unless otherwise further defined, 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.

[0062] “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.

[0063] “SARS-CoV-2 variant” as used herein 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. Examples of 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) and its subvariants/sublineages (BA.l, BA.1.1, BA.2, BA.3, BA.4, BA.5).

[0064] “Washington coronavirus isolate” as used herein 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.

[0065] “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. With respect to 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.

[0066] As a non-limiting example, for BNT162b2 the full dosing regimen was two doses, while subsequent booster dose(s) have since been recommended by the CDC. Thus, if a test is to be performed 30 days after the full dosing regimen, 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. As another non-limiting example, for Ad26.CoV2.S, the full dosing regimen was one dose, while a subsequent booster dose has since been recommended. Thus, if a test is to be performed 30 days after the full dosing regimen, 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. With respect to administering additional booster doses, for example, BNT162b2, the previously used full dosing regimen is administered over two doses, 21 days apart. Thus, in embodiments wherein a 3 ^rd dose is administered, the time indicated is counted from the day the 2 ^nd dose is administered; in embodiments wherein a 4 ^th dose is administered, the time indicated is counted from the day the 3 ^nd dose is administered.

[0067] In analyzing the composition of sterilizing immunity, both 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.

[0068] 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.

[0069] 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. Thus, the present invention includes guiding decisions about vaccine development or need for re-vaccination. [0070] Described herein, we monitor the CoV-2-T and B cell activity in different groups of patients including those who are post-vaccination. The-CoV-2-specific T and B cells are quantified, and their function and phenotypes analyzed. The results obtained from this study are compared to other immunological parameters such as anti-CoV-2 antibody, and clinical characteristics such as symptoms, disease severity, duration and recovery. It will greatly help us to understand the persisting pathologic features of CoV-2 infection and provide information on the possible need for re-vaccination for immune compromised individuals. Data from our studies are described in more detail herein. Briefly, we can see the 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. However, we now know that T-cell immunity against SARS-CoV-2 is more diverse and cross-reactive with peptides expressed on other Coronaviruses. These observations suggest that robust T-cell responses are an important and essential element of long-term immunity to SARS-CoV-2. However, assessments of T-cell immunity to SARS-CoV-2 are not readily available. In this regard, herein, we present data from a flow cytometry-based assay detecting dual cytokine-producing, SARS-CoV-2-antigen-specific memory T-cells which demonstrates specificity and accuracy for detection of CD4+/CD8+ T-cell responses to SARS-CoV-2 peptides and differentiates infected and vaccinated individuals from those not exposed to SARS-CoV-2.

[0072] Finally, analysis of T-cell responses to an important variant of concern (VOC)

(B.1.1.7) showed that exposure to SARS-CoV-2 infection or BNT162b2 vaccine (Pfizer vaccine) elicited equivalent T-cell responses. Recent observations suggest that IgG responses to SARS-CoV- 2 infection did not reduce viremia in patients infected with the B.1.1.7 variant however, SARS- CoV-2 T-cell responses were not explored in that study. Long term analysis of immune responses will be important since memory responses differ greatly from acute responses, especially at the antibody level. Here, dormancy of memory T-cells, B-cells, and plasma cells that can rapidly be activated upon re-exposure to SARS-CoV-2 exposure are likely to have an important role in preventing SARS-CoV-2 infection and possibly infection from current and emerging VOC.

[0073] As such, various embodiments of the present invention are based, at least in part, on these findings.

Methods of detecting SARS CoV-2-specific T-cells

[0074] 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.

[0075] In various embodiments, the method comprises detecting a quantity of both SARS

CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject.

[0076] In various embodiments, 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.

[0077] 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.

[0078] In various embodiments, the method comprises measuring a quantity of both

SARS CoV-2-specific CD4+ T-cells and CD8+ T-cells in a subject.

[0079] In various embodiments, 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.

[0080] In various embodiments, 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. In various embodiments, 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. In various embodiments, it is performed 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. In various embodiments, it is performed 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. In various embodiments, it is performed 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. In various embodiments, it is performed 180 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 180 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, or 180 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, 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.

[0081] 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. In various embodiments, 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. In various embodiments, it is performed 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. In various embodiments, it is performed about 21-42 days after the subject received a full dosing regimen, about 21-42 days after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or about 21-42 days after the subject has had a SARS CoV-2 infection.

[0082] In various embodiments, 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.

[0083] In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0084] 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 0.05% 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.

[0085] In various embodiments, the SARS CoV-2 peptide fragments are fragments of

SARS CoV-2 spike protein. In various embodiments, the SARS CoV-2 peptide fragments are fragments of SARS CoV-2 variant spike protein. For example, the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence. In various embodiments, 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). Examples of nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6. Examples of assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10. [0086] In various embodiments, the SARS CoV-2 is a natural isolate SARS-CoV-2. As such, the peptide fragments are from the natural isolate SARS-CoV-2.

[0087] In various embodiments, the SARS CoV-2 is Washington isolate of SARS-CoV-

2. 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.

[0088] In various embodiments, the SARS CoV-2 is a SARS CoV-2 variant. As such, the peptide fragments are from the variant SARS-CoV-2. Examples of 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.

[0089] In various embodiments, the period of time in which the composition comprising

SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours. In various embodiments, period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In various embodiments, period of time is about 4-6 hours. In various embodiments, period of time is about 6-8 hours. In various embodiments, period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.

[0090] In various embodiments, 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.

[0091] In various embodiments, 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, and anti-CD8 antibodies.

[0092] In various embodiments, 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 two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies. In various embodiments, 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.

[0093] In various embodiments, the method comprises measuring the quantities of both

SARS CoV-2-specific CD4+ T-cells and SARS CoV-2-specific CD8+ T-cells. [0094] In various embodiments, 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.

[0095] In various embodiments, 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 using flow cytometry. [0096] In various embodiments, 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. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label. In various embodiments, 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, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7. Additional examples include (PerCP Cy5.5, V450, V500, and PE-CF594.

[0097] In various embodiments, the biological sample comprises T-cells. In various embodiments, 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. [0098] In various embodiments, 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.

[0099] In various embodiments, the subject has received an organ or tissue transplant.

For example, 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. In various embodiments, the subject has received a kidney transplant.

[0100] In various embodiments, the subject is immunocompromised. Examples of 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.

[0101] In various embodiments, the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.

[0102] In various embodiments, the subject has not been known to be infected with

SARS CoV-2 and has not received a vaccine for SARS CoV-2. In these instances, 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.

[0103] In various embodiments, 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. In various embodiments, 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.

Methods of detecting protective immunity

[0104] 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. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0105] In various embodiments, 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.

[0106] In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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.

[0107] 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 0.05% 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.

[0108] In various embodiments, the method further comprising re-vaccinating the subject against SARS CoV-2. For example, the subject is given a booster dose of the SARS CoV-2 vaccine.

[0109] In various embodiments, 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. In various embodiments, it is performed 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 SARS CoV-2 vaccine, or 45 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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 SARS CoV-2 vaccine, or 60 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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 SARS CoV-2 vaccine, or 90 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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 SARS CoV-2 vaccine, or 120 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 180 days or more after the subject received a full dosing regimen of a SARS CoV-2 vaccine, 180 days or more after the subject received a booster dose SARS CoV-2 vaccine, or 180 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, 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 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 SARS CoV-2 vaccine, or 1 year or more after the subject has had a SARS CoV-2 infection.

[0110] 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. In various embodiments, 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. In various embodiments, it is performed about 21-56 days after the subject received a full dosing regimen, about 21-56 days after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or about 21-56 days after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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. In various embodiments, 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.

[0111] In various embodiments, the SARS CoV-2 peptide fragments are fragments of

SARS CoV-2 spike protein. For example, the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence. In various embodiments, the SARS CoV-2 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). Examples of nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6. Examples of assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.

[0112] In various embodiments, the SARS CoV-2 is a natural isolate SARS-CoV-2. As such, the peptide fragments are from the natural isolate SARS-CoV-2.

[0113] In various embodiments, the SARS CoV-2 is Washington isolate of SARS-CoV-

2. 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.

[0114] In various embodiments, the SARS CoV-2 is a SARS CoV-2 variant. As such, the peptide fragments are from the variant SARS-CoV-2. Examples of 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.

[0115] In various embodiments, the period of time in which the composition comprising

SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours. In various embodiments, period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In various embodiments, period of time is about 4-6 hours. In various embodiments, period of time is about 6-8 hours. In various embodiments, period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.

[0116] In various embodiments, 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.

[0117] In various embodiments, 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, and anti-CD8 antibodies.

[0118] In various embodiments, 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 two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies. In various embodiments, 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.

[0119] In various embodiments, 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.

[0120] In various embodiments, 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 using flow cytometry. [0121] In various embodiments, 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. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label. In various embodiments, 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, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7. Additional examples include (PerCP Cy5.5, V450, V500, and PE-CF594.

[0122] In various embodiments, the biological sample comprises T-cells. In various embodiments, 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.

[0123] In various embodiments, 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.

[0124] In various embodiments, the subject has received an organ or tissue transplant.

For example, 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. In various embodiments, the subject has received a kidney transplant.

[0125] In various embodiments, the subject is immunocompromised. Examples of 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.

[0126] In various embodiments, the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.

[0127] In various embodiments, the subject has not been known to be infected with

SARS CoV-2 and has not received a vaccine for SARS CoV-2. In these instances, 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.

[0128] In various embodiments, 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. In various embodiments, 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.

Methods of determining likelihood of contracting COVID-19

[0129] 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. In still other examples, 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. In these instances, the test would utilize a composition comprising SARS CoV-2 variant peptide fragment(s).

[0130] Thus, in these embodiments, 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. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0131] In various embodiments, 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.

[0132] In various embodiments, 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.

[0133] 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 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. 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 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. 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 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. 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 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. 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 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. [0134] 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 0.02% 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. 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 0.15% 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. 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 0.25% compared to a control.

[0135] In various embodiments, 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. 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 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. In various embodiments, it is performed 45 days or more after the subject received a full dosing regimen, 45 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or 45 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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. In various embodiments, it is performed 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. In various embodiments, it is performed 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. 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. 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. In various embodiments, it is performed 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. In various embodiments, it is performed 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. In various embodiments, it is performed 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. In various embodiments, 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.

[0136] In various embodiments, the SARS CoV-2 peptide fragments are fragments of

SARS CoV-2 spike protein. For example, the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence. In various embodiments, the SARS CoV-2 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). Examples of nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6. Examples of assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10. [0137] In various embodiments, the SARS CoV-2 is a natural isolate SARS-CoV-2. As such, the peptide fragments are from the natural isolate SARS-CoV-2.

[0138] In various embodiments, the SARS CoV-2 is Washington isolate of SARS-CoV-

2. 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.

[0139] In various embodiments, the SARS CoV-2 is a SARS CoV-2 variant. As such, the peptide fragments are from the variant SARS-CoV-2. Examples of 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.

[0140] In various embodiments, the period of time in which the composition comprising

SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours. In various embodiments, period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In various embodiments, period of time is about 4-6 hours. In various embodiments, period of time is about 6-8 hours. In various embodiments, period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.

[0141] In various embodiments, 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.

[0142] In various embodiments, 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, and anti-CD8 antibodies.

[0143] In various embodiments, 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 two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies. In various embodiments, 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.

[0144] In various embodiments, 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.

[0145] In various embodiments, 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 using flow cytometry. [0146] In various embodiments, 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. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label. In various embodiments, 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, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7. Additional examples include (PerCP Cy5.5, V450, V500, and PE-CF594.

[0147] In various embodiments, the biological sample comprises T-cells. In various embodiments, 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. [0148] In various embodiments, 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.

[0149] In various embodiments, the subject has received an organ or tissue transplant.

For example, 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. In various embodiments, the subject has received a kidney transplant.

[0150] In various embodiments, the subject is immunocompromised. Examples of 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.

[0151] In various embodiments, the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.

[0152] In various embodiments, the subject has not been known to be infected with

SARS CoV-2 and has not received a vaccine for SARS CoV-2. In these instances, 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.

[0153] In various embodiments, 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. In various embodiments, 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.

Methods of Vaccinating

[0154] 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 cells are less than 0.05% compared to a control; and administering to the subject a vaccine against SARS CoV-2. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0155] In various embodiments, 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

[0156] In various embodiments, 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.

[0157] 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 to the subject a vaccine against SARS CoV-2. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0158] In various embodiments, 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.

[0159] In various embodiments, 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.

[0160] 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 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. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0161] In various embodiments, 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.

[0162] In various embodiments, 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.

[0163] 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 0.05% compared to a control; and administering to the subject a vaccine against SARS CoV-2. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0164] In various embodiments, 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.

[0165] In various embodiments, 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. [0166] 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 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. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0167] In various embodiments, 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.

[0168] In various embodiments, 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.

[0169] 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 CD4+ T cells and SARS CoV-2-specific CD8+ T cells are less than 0.05% compared to a control. In various embodiments, the percent positives are after deducting the background levels in blood only conditions.

[0170] In various embodiments, 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. [0171] In various embodiments, 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.

[0172] In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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.

[0173] 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 0.02% 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. 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 0.15% 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. 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 0.25% or greater as compared to a control.

[0174] In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. [0175] 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 0.02% 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 0.1% 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. 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 0.2% 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.

[0176] 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 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. 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 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. 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 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. 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 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. 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 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.

[0177] 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 0.02% 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. 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 0.15% 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. 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 0.25% compared to a control.

[0178] In various embodiments, 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. In various embodiments, 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. In various embodiments, it is performed 45 days or more after the subject received a full dosing regimen, 45 days or more after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or 45 days or more after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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. In various embodiments, it is performed 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. In various embodiments, it is performed 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. 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. 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. In various embodiments, it is performed 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. In various embodiments, it is performed about 21-56 days after the subject received a full dosing regimen, about 21-56 days after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or about 21-56 days after the subject has had a SARS CoV-2 infection. In various embodiments, it is performed 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. In various embodiments, it is performed 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, 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, about 60-120 days after the subject received a booster dose of a SARS CoV-2 vaccine, of a SARS CoV-2 vaccine or about 60-120 days after the subject has had a SARS CoV-2 infection. [0179] In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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. In various embodiments, 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.

[0180] Examples of 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.

[0181] In various embodiments, the SARS CoV-2 peptide fragments are fragments of

SARS CoV-2 spike protein. For example, the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence. In various embodiments, the SARS CoV-2 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). Examples of nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6. Examples of assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.

[0182] In various embodiments, 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-

2. 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.

[0184] In various embodiments, the SARS CoV-2 is a SARS CoV-2 variant. As such, the peptide fragments are from the variant SARS-CoV-2. Examples of 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.

[0185] In various embodiments, the period of time in which the composition comprising

SARS CoV-2 peptide fragments is in contact with the biological sample is about 8 to 10 hours. In various embodiments, period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In various embodiments, period of time is about 4-6 hours. In various embodiments, period of time is about 6-8 hours. In various embodiments, period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours.

[0186] In various embodiments, 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.

[0187] In various embodiments, 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, and anti-CD8 antibodies.

[0188] In various embodiments, 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 two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies. In various embodiments, 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.

[0189] In various embodiments, 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.

[0190] In various embodiments, 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 using flow cytometry. [0191] In various embodiments, 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. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label. In various embodiments, 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, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7. Additional examples include (PerCP Cy5.5, V450, V500, and PE-CF594.

[0192] In various embodiments, the biological sample comprises T-cells. In various embodiments, 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.

[0193] In various embodiments, 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.

[0194] In various embodiments, the subject has received an organ or tissue transplant.

For example, 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. In various embodiments, the subject has received a kidney transplant.

[0195] In various embodiments, the subject is immunocompromised. Examples of 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.

[0196] In various embodiments, the subject has received a vaccine for SARS CoV-2. In various embodiments, the subject has been infected with SARS CoV-2.

[0197] In various embodiments, the subject has not been known to be infected with

SARS CoV-2 and has not received a vaccine for SARS CoV-2. In these instances, 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.

[0198] In various embodiments, 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. In various embodiments, 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.

Methods of testing vaccines

[0199] 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. [0200] In various embodiments, 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.

[0201] In various embodiments, 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.

[0202] 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.

[0203] In various embodiments, 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. [0204] In various embodiments, 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.

[0205] In various embodiments, the SARS CoV-2 variant peptide fragments are fragments of SARS CoV-2 spike protein. For example, the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence. In various embodiments, 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). Examples of nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6. Examples of assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.

[0206] Examples of SARS CoV-2 variants 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.

[0207] In various embodiments, the period of time in which the composition comprising

SARS CoV-2 variant peptide fragments is in contact with the biological sample is about 8 to 10 hours. In various embodiments, period of time is about 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. In various embodiments, period of time is about 4-6 hours. In various embodiments, period of time is about 6-8 hours. In various embodiments, period of time is about 9-11 hours. In various embodiments, the period of time is about 9 hours. [0208] In various embodiments, 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.

[0209] In various embodiments, 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, and anti-CD8 antibodies.

[0210] In various embodiments, 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 two or more of anti-IL-2 antibodies, anti-TNF-a antibodies, anti-y-IFN antibodies, anti-CD4 antibodies, and anti-CD8 antibodies. In various embodiments, 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.

[0211] In various embodiments, 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.

[0212] In various embodiments, 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 using flow cytometry. [0213] In various embodiments, 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. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label. In various embodiments, 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, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7. Additional examples include (PerCP Cy5.5, V450, V500, and PE-CF594.

[0214] In various embodiments, the biological sample comprises T-cells. In various embodiments, 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.

[0215] In various embodiments, 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.

[0216] In various embodiments, the subject has received an organ or tissue transplant.

For example, 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. In various embodiments, the subject has received a kidney transplant.

[0217] In various embodiments, the subject is immunocompromised. Examples of 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.

[0218] In various embodiments, the subject has received a vaccine for SARS CoV-2 that is not specifically developed for the SARS CoV-2 variant. In various embodiments, the subject has been infected with SARS CoV-2 variant.

[0219] In various embodiments, 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. In various embodiments, 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

[0220] Various embodiments of the present invention provide for a kit 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. In various embodiments, 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.

[0221] In various embodiments, the kit further comprises brefeldin A, phytohemagglutinin (PHA), anti-CD28 antibodies, or anti-CD49d antibodies, or combinations thereof.

[0222] In various embodiments, 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.

[0223] In various embodiments, 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. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the peptides can be labeled with a detectable tag which can be detected using an antibody specific to the label. In various embodiments, 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, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7. Additional examples include (PerCP Cy5.5, V450, V500, and PE-CF594.

[0224] In various embodiments, the SARS CoV-2 peptide fragments are fragments of

SARS CoV-2 spike protein. For example, the composition comprises overlapping fragments of the spike protein that together contains the entire spike protein sequence. In various embodiments, the SARS CoV-2 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). Examples of nonstructural proteins include Nspl, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, NsplO, Nspll, Nspl2, Nspl3, Nspl4, Nspl5, and Nspl6. Examples of assessor proteins include 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10.

[0225] In various embodiments, the SARS CoV-2 is a natural isolate SARS-CoV-2. As such, the peptide fragments are from the natural isolate SARS-CoV-2.

[0226] In various embodiments, the SARS CoV-2 is Washington isolate of SARS-CoV-

2. 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.

[0227] In various embodiments, the SARS CoV-2 is a SARS CoV-2 variant. As such, the peptide fragments are from the variant SARS-CoV-2. Examples of 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.

[0228] In various embodiments, 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. In various embodiments, 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.

[0229] In various embodiments, 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.

EXAMPLES

[0230] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

[0231] 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. We have seen stark examples of this in our preliminary work with kidney transplant recipients who received vaccines but have shown no IgG anti-spike protein antibodies nor spike-protein reactive T-cells. This is a disturbing revelation, indicating that these patients are not protected by the vaccine and will need other strategies to help prevent severe SARS-CoV-2 infection.

[0232] 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.20 variants. This is very encouraging since this assay can potentially be used to determine the degree of T-cell reactivity to these and other emerging variants and if there is a need for creation of new vaccines to address them. We are very encouraged to see this cross-reactivity in the T-cell compartment which may be lacking or dissipate rapidly in the antibody compartment. [0233] 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. With the emergence of COVID-19 variants, specifically the CAL.20C stain in Southern California and Oxford (UK) strain which is the most common variant in the US. It is critical to develop assays to monitor the efficacy of vaccination against the variant. Our data show that in vaccinated patients and patients who have recovered from SARS-CoV-2 infection, T-cell immune responses to SARS-CoV-2 and variants are equivalent.

Example 2

Understanding the Composition, Specificity and Permanence of Adaptive Immune Responses to SARS-CoV-2 in Infected & Vaccinated Individuals [0234] We measure the SARS-CoV-2 (CoV-2)-T cell (CD8+ & CD4+) activity, memory

B cells (CD19+, CD27+) and 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. To assess vaccine efficacy, the CoV-2 T & B cell immunity in volunteers before and after vaccination are monitored. We also monitor patients at different times post-infection or vaccination for the permanence of T and B cell immunity to CoV-2. 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. With the emergence of COVID-19 variants, specifically the CAL.20C strain in Southern California, it is critical to develop assays to monitor the efficacy of vaccination against the variant. Here, we examine the cross-reactive immunity with CoV-2 which likely would render protection.

[0235] We determine whether pre-existing CoV-2-reactive T cell clones exist in unexposed individuals and healthy volunteers before vaccination. It is still debatable whether some healthy individuals possess cross-reactive T cells against CoV-2 antigens. We will confirm the percentage of these T cells in unexposed population through the CoV-2 T cell assay of the present invention. The phenotypes of COVID-19-specific T cells are further analyzed for memory subsets, activation, and functional markers. The in-depth analysis of pre-existing CoV-2-reactive T cells will help to explain the age discrepancy in COVID-19 infection and likely identify individuals at less risk for severe CoV-2.

[0236] We determine whether Cov-2 T and B cell immunity is generated and persist in recovered patients. Monitoring COVID-19-specific T and B cell immunity in patients recently infected or recovered is done. Time points for monitoring are 2 months, 6 months, and 1 year after infection. T & B-cell immunity will be compared to clinical parameters such as age, symptom severity, duration, cytokines profiles, recovery, and other underlining disease. These data provide invaluable information regarding lasting immunity against reinfection.

[0237] We determine whether the CoV-2 T and B cells immunity develops post vaccination and recognize variant stains. The current vaccines induce T and B cell immunity against the Spike protein of CoV-2. The duration of anti-spike T and B cell immunity is monitored 2 months, 6 months and 1 year after vaccination. The vaccine efficacy and induced T cell immunity are analyzed in different patient populations to include normal individuals and transplant patients on immunosuppression. Moreover, the specific T cell assay developed tests whether vaccinated individuals have immunity against COVID-19 variant such as CAL.20C and Oxford variant.

[0238] We determine whether pre-existing T and B cell immunity and CoV-2-T- and B cells induced post- vaccination provide protection against CoV-2 infection. The correlation of CoV-2-T cells with CoV-2-specific antibodies or other parameters such as patients’ demographics and clinical complications are analyzed using multivariate analysis. Preliminary data from vaccinated patients (normals and transplant patients) those recovered from CoV-2 infection are shown.

[0239] Blood from recovered COVID-19 patients are obtained. This is important in understanding the potential role of persistent immune activation in mediating the “long haul” syndrome related to COVID-19 and/or determining immunity in symptomatic patients not confirmed by viral PCR or antibody testing. CoV-2 T-cell testing will clearly define their exposure. Blood from healthy volunteers pre- and post-vaccination are also obtained. This will allow us to determine the breadth and depth of immune responses to COVID-19 in normal and compare with immunocompromised individuals. This approach provides valuable information on the ability of vaccines to establish immunity in immune compromised patients.

[0240] We monitor the 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.

Example 3

Participants and Sample Collection

[0241] Informed consent was obtained prior to study initiation. This study was approved by the institutional review board at Cedars-Sinai Medical Center (protocol IRB numbers: 000 42267 and 000 00621). The study was conducted in accordance with the ethical guideline based on federal regulations and the common rule. The manuscript was composed and written by the authors without outside assistance or payment.

[0242] Nineteen healthy controls, 151 patients with confirmed COVID-19 infection (of which, 18 received vaccination), and 38 vaccinated healthy individuals without history of SARS- CoV-2 infection were enrolled in the study with similar age and gender composition. The time of blood draw from previously infected patients ranged from day 11 to day 368 after reported SARS-CoV-2 infection by viral PCR and/or antibody test. The blood from vaccinated individuals was drawn 1 -month post 2 ^nd dose of Pfizer BNT162b2 mRNA vaccine. Fresh whole blood was collected in sodium heparinized tubes and stimulated with SARS-CoV-2 peptides overnight. Plasma obtained was stored at -80°C for SARS-CoV-2 Spike IgG analysis.

T-cell Stimulation with SARS-CoV-2 and VOC Peptides

[0243] Whole blood was incubated with 1 pg/mL SARS CoV-2 Spike glycoprotein (S), or variant B.1.1.7 Spike (B.1.1.7). For patients with previous infection, additional peptides were tested. These included virus membrane protein (VME), nucleoprotein (NCAP), protein 3 A (AP3A), and non-structural protein 7A (NS7A) (JPT Peptide Technologies GmbH, Berlin, Germany). For all conditions, Brefeldin A and anti-CD28/CD49d (BD Biosciences, San Jose, CA) 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).

Cytokine Flow Cytometry Analysis

[0244] Fresh whole blood was incubated with SARS-CoV-2-specific peptides for 9 hours and immune cells were stained for surface markers. Cells were stained with fluorochrome conjugated antibodies to CD3+ (FITC), CD4+ (PerCP Cy5.5), CD8+ (V450), CD45+ (V500) and CD56+ (PE-CF594) (BD Bioscience, CA). After erythrocytes were lysed by permeabilization, intracellular cytokines were stained with fluorochrome conjugated antibodies to IL-2 (APC), IFN-g (PE), and TNF-a (PE-Cy7) (BD Bioscience, CA). The CD4+ (IL-2/TNF-a) ⁺ cells and CD8+ (TNF-a/IFN-Y) ⁺ stimulated with S, B.1.1.7 S, VME, NCAP, AP3A, NS7A were enumerated and defined as CoV-2-specific T-cells after deducting the background levels in blood only conditions. Dual cytokines % in CD4+ or CD8+ cells > 0.05% were considered positive. Measurement of Nucleocapsid-Specific IgG Levels

[0245] Nucleocapsid-specific IgG titer in serum of patients was analyzed using SARS-

CoV-2 IgG assay (Abbott Core Laboratory) on the Architect instrument according to the manufacturer instructions.

Measurement of SARS-CoV-2-Spike-specific IgG in Plasma

[0246] We next determined the levels of SARS-CoV-2 Spike IgG using CoVSl-RBD

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.

Statistical analysis

[0247] Data were congregated in Excel and Graphpad Prism for statistical analysis.

Student t-test was used for analyzing the statistical difference between two groups. Correlation between T-cell immune response and IgG titer was analyzed by Pearson’s correlation coefficient test. P- value less than 0.05 was considered significant.

SARS-CoV-2-specific T-cell Detection in Whole Blood

[0248] 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. After incubating whole blood with SARS-CoV-2 Spike peptide pool, we were able to discern Spike-reactive T-cells by dual cytokine gating (Figure 11A-11B). Here, healthy individuals with no history of SARS-CoV-2 infection demonstrated no significant T-cell responses to SARS-CoV-2 spike peptide. However, T-cells from SARS-CoV-2-infected or vaccinated individuals showed substantial spike-specific CD4+ and CD8+ T-cells.

T-cell Immunity in Infected Individuals

[0249] To explore the breadth and depth of memory T-cell immunity against SARS-

CoV-2, we examined responses in 38 patients with documented SARS-CoV-2 infection (Figure 12A). T-cell immune responses to peptide pools of 5 major SARS-CoV-2 proteins (Spike, VME, NCAP, AP3A, and NS7A) were analyzed. In healthy control individuals, no significant CD4+ T- cell responses to the 5 SARS-CoV2 proteins were seen (Figure 12B, IL-2+TNF-a+(%) in CD4+ <0.05%, mean = 0.01%). However, 20% of healthy individuals showed heterogenous TNF- a+/rFN-y+ CD8+ T-cell (>0.05%) responses to the 5 SARS-CoV-2 proteins, which could represent cross-reaction of CD8+ T-cells generated from previous endemic coronavirus infection (Figure 12B). Based on the background level of CD4+ T-cell response in healthy controls, we set 0.05% of dual-positive CD4+ and CD8+ T-cells as the cutoff level determining positive T-cell immunity against SARS-CoV-2. Overall, we observed 84% (32 of 38) infected patients had either positive CD4+ or CD8+ T-cell immunity to one or more of 5 CoV-2 peptides. Most patients showed positive CD4+ T-cell immunity (82%, 31 of 38), and 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.

T-cell Immunity in Vaccinated Individuals

[0250] Next, we analyzed Spike-specific CD4+/CD8+ immune responses to the Pfizer

BNT162b2 vaccine. We 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.

Association of T-cell Immunity with IgG Serology

[0251] T-cell immunity to SARS-CoV-2 is usually associated with Spike-specific IgG responses. Serum from SARS-CoV-2 patients were submitted for clinical nucleoprotein IgG titer. In 25 patients with nucleocapsid IgG positivity, we analyzed the association with CD4+ T-cell immunity to the 5 SARS-CoV-2 peptides (the highest response was compared). Although the T- cell immunity was not nucleocapsid-specific, there was a significant association between CD4+ T-cell immunity and nucleocapsid IgG titers (p=0.022; R= 0.457; Figure 12C).

[0252] We then examined Spike-specific IgG levels in 80 SARS-CoV2 infected patients and compared them to Spike-specific T-cell immunity. Here, 72.58% of patients with positive Spike-specific CD4+ T-cells had positive Spike-specific IgG levels. For patients with negative Spike-specific T-cell immunity, 61.11% also showed negative Spike-specific IgG serology. In 13 patients with high Spike-specific CD4+ T-cell immunity (IL-2+TNF-a+(%) in CD4+ > 0.3%), we also observed a strong correlation between T-cell immunity and level of Spike-specific IgG (p=0.0316; R=0.5288; Figure 2D). Therefore, 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.

UK Variant and Immune Evasion

[0253] The B.1.1.7 variant contains the E484K mutation which renders resistance to serologic responses in infected individuals. To determine if this VOC evaded T-cell immunity, we analyzed 20 infected/recovered patients and 18 vaccinated individuals for CD4+/CD8+ T-cell responses against B.1.1.7 variant Spike protein. As shown in Figure 13, there is no significant reduction in CD4+/CD8+ T-cell responses to the variant B.1.1.7 Spike peptides as compared to the original Wuhan Spike peptides (mean of infected patients: 0.23% original to 0.17% variant; mean of vaccinated individuals: 0.16% original to 0.14% variant). Five of 20 infected patients and 6 of 18 vaccinated individuals had no detectable CD8+ T-cells against the original or variant Spike peptides (data not shown). The other 15 infected and 12 vaccinated individuals demonstrated nearly identical CD8+ responses to the original Wuhan Spike and variant Spike (Figure 13B). When the individual T-cell responses to the original and variant spikes were compared, those with higher CD4+ T-cell immunity tend to lose some reactivity to the variant peptide, but not at a significant level (Figure 3C and 3D). In summary, T-cell memory induced by SARS-CoV-2 infection or vaccination establishes effective immune response against the B.1.1.7 variant. This would suggest protective immunity against B.l.1.7 infection and possibly other VOC.

Example 4

[0254] For these immunocompromised or organ transplant recipient patients we identify those who lack both SARS-CoV-2 spike IgG responses and SARS-CoV-2 spike-specific CD4+/CD8+ T-cell responses. Patients are offered revaccination with the BNT162b2 with monitoring of immune responses at 30 days post-booster vaccine. If positive, patients are monitored at 6M and 12M post-booster. This offers the patients the best opportunity for robust protection from SARS-CoV-2 infection. The schema is outlined in figure 15.

Example 5

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. Herein, we report on 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).

[0256] Spike-specific IgG receptor-binding domain (RBD) antibodies are evanescent and do not reflect important memory compo- nents. Thus, additional analysis of CD4+/CD8+ T cell responses to SARS-CoV-2 spike peptides and VOCs could broaden our understanding of SARS- CoV-2-specific T cell immunity.

[0257] 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.

[0258] We developed a whole-blood assay to detect SARS-CoV-2- specific T cells.

Analysis of cytokine production in stimulated T cells confirmed that 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. After incubating whole blood with a SARS-CoV-2 Spike peptide pool, we were able to identify Spike- reactive T cells by dual-cytokine gating. In this assay, healthy individuals with no history of SARS-CoV-2 infection demonstrated no significant T cell response to the SARS- CoV-2 spike peptide. However, T cells from SARS-CoV-2-infected or vaccinated individuals showed substantial spike-specific CD4+ and CD8+ T cell populations.

[0259] Next, we examined memory T cell immunity against SARS-CoV-2 in 134 patients with documented SARS-CoV-2 infection. T cell immune responses to peptide pools of 5 major SARS-CoV-2 proteins (Spike, VME, NCAP, AP3A, and NS7A) were analyzed. For healthy control individuals, no significant CD4+ T cell responses to the 5 SARS- CoV-2 proteins were seen (Fig. 16A, IL-2+/TNF-a+ (%) in CD4+ <0.05%, mean = 0.01%). However, 20% of healthy individuals showed heterogeneous TNF-a+/IFN-Y+CD8+ T cell (>0.05%) responses to the 5 SARS-CoV-2 proteins, which could represent cross-reactivity among CD8+ T cells generated during previous endemic coronavirus infection (Fig. 16B). Based on the background level of the CD4+ T cell response in healthy controls, we set 0.05% dual -positive CD4+ and CD8+ T cells as the cutoff level for identifying positive T cell immunity against SARS-CoV-2. Overall, we observed that 88% (30 of 34) of infected patients had either positive CD4+ or CD8+ T cell immunity to one or more of the 5 SARS-CoV-2 proteins. Most patients showed positive CD4+ T cell immunity (85%, 29 of 34), and CD4+ T cells demonstrated immunodominant responses to Spike peptides (Fig. 16A). CD8+ T cells showed similar responses to the 5 proteins. [0260] Next, we analyzed Spike-specific CD4+/CD8+ immune responses induced by the

Pfizer BNT162b2 vaccine. We 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.

[0261] Serum from SARS-CoV-2 patients was submitted for clinical nucleoprotein IgG titering. In 25 patients with nucleocapsid IgG positivity, we analyzed the association with CD4+ T cell immunity to the 5 SARS-CoV-2 peptides (the highest response was compared). Although T cell immunity was not nucleocapsid-specific, there was a significant association between CD4+ T cell immunity and nucleocapsid IgG titers (p = 0.022; R = 0.457; Fig. 16C).

[0262] We then examined Spike-specific IgG levels in 80 SARS-CoV-2- infected patients and compared them to Spike-specific T cell immunity results. In total, 72.58% of patients with detectable Spike-specific CD4+ T cells were positive for Spike-specific IgG. Among patients without detectable Spike-specific T cell immunity, 61.11% also were negative for Spike- specific IgG by serology. In 13 patients with high Spike-specific CD4+ T cell immunity (IL- 2+/TNF-a+ (%) in CD4+ >0.3%), we also observed a strong correlation between T cell immunity and the level of Spike-specific IgG (p = 0.0316; R = 0.5288; Fig. 16D).

[0263] The Alpha (B.1.1.7) variant contains the E484K mutation, which establishes resistance to serologic responses in infected individuals. To determine whether this VOC evades T cell immunity, we analyzed 19 infected/recovered patients and 18 healthy vaccinated individuals for CD4+/CD8+ T cell responses against the Alpha variant Spike protein. As shown in 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). In addition, 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).

[0264] In summary, 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. Herein, we present data identifying memory T cells with specificity and accuracy for the detection of CD4+/CD8+ T cell responses to SARS-CoV-2 peptides that differentiate infected and vaccinated individuals from those not exposed to SARS-CoV-2. In addition, 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.

[0265] The development of dormancy in memory T cells, B cells, and plasma cells is 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. However, 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.

Example 6

Divergent Immune Responses to SARS-CoV-2 Vaccines in Immunocompromised Patients [0266] Understanding the composition and duration of immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is critical for prevention of infection. The most important elements of immunity to SARS-CoV-2 are neutralizing antibody and 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. These issues become more prescient in determining immunity to SARS-CoV-2 in immunocompromised individuals where a majority show no IgG responses to vaccines and the recent emergence of the Delta variant with reports of >74% vaccine breakthrough cases.

[0267] Here, we report on patients demonstrating divergent immune responses to SARS-

CoV-2 vaccination. Seven patients were identified from a cohort of 70 immunocompromised patients, with all demonstrating spike-specific IgG unresponsiveness 2-4 mo postvaccination. All had received treatment with B-cell modifying agents. We subsequently examined CD4 ⁺ /CD8 ⁺ T- cell-spike-specific immune responses in all patients and repeat examination after revaccination in 2 patients. CD4 ⁺ /CD8 ⁺ T-cell responses are shown in Figure 17A-17B and demonstrate robust CD4 ⁺ T-cell (0.94 ± 1.2%, Normal > 0.05%) and CD8 ⁺ T-cell (0.89 ± 1.2%, Normal > 0.05%) immune responses to spike peptides. Two patients receiving Johnson & Johnson booster vaccines demonstrated increased T-cell responses but remained spike IgG negative. This suggests that T- cell immune responses to SARS- CoV-2 vaccines are primal and retentive and that B-cell depletion before vaccine exposure prevents the cascade of progression of B-cell activation necessary for SARS- CoV-2 spike IgG production.

[0268] Absence of IgG responses to SARS-CoV-2 suggests patients be considered

“unvaccinated.” However, we show that patients treated with B-cell-modifying agents develop robust T-cell immune responses to SARS-CoV-2 without generating IgG responses. These observations should be considered in light of data presented by Peng et al showing robust CD4 ⁺ /CD8 ⁺ T-cell responses to SARS-CoV-2 after infection. A critical observation was the diversity of T-cell responses that likely extend beyond the persistence of spike antibody. IgG is necessary for sterilizing immunity and T-cells cannot prevent infection as antigen presentation is required, but T-cell immunity can be at the ready for viral elimination. This may provide an inside track for rapid deployment of SARS-CoV-2 immunity, and although not preventing infection, could alter the severity and duration of SARS-CoV-2 disease. Thus, detection of 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.

Example 7

Assessment of humoral and cellular immune responses to SARS CoV-2 vaccination (BNT162b2) in immunocompromised renal allograft recipients

Study participants

[0269] Kidney transplant recipients who were greater than 1 month post-second dose of the Pfizer BNT162b2 mRNA vaccine had determinations of Spike-receptor binding domain (RBD)-specific IgG levels and analysis of Spike-specific CD4 /CD8 T-cell immune responses. Responses were compared to healthy individuals (non-immunocompromised) enrolled as controls. Fresh whole blood was collected in sodium heparinized tubes for T-cell stimulation assay. Plasma obtained was stored at -80 C for SARS-CoV-2 Spike- RBD-IgG analysis.

SARS-CoV-2 Spike-specific T-cell assay

[0270] The 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).

CMV-specific T-cell assay

[0271] 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.

Cytokine flow cytometry analysis

[0272] Cultured cells were stained with fluorochrome-conjugated antibodies to CD3+

(FITC), CD4 (PerCP Cy5.5), CD 8 (V450), CD45 (V500), and CD56 (PE-CF594) (BD Biosciences). After erythrocytes were lysed by permeabilization, intracellular cytokines were stained with fluorochrome-conjugated antibodies to IL-2 (APC), IFN-y (PE), and TNF-a (PE- Cy7) (BD Biosciences).

Measurement of SARS-CoV-2Spike-RBD-specific IgG in plasma

[0273] The levels of SARS-CoV-2 Spike IgG were measured by using CoVSl-RBD

ELISA kit (Ray Biotech, GA, USA) as per the manufacture’s manual.

[0274] 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, Horse Radish Peroxidase (HRP)-conjugated strep- tavidin was added, and Spike-specific IgG was quantitated by Optical Density (OD) 450 nm reading.

Statistical analysis

[0275] Data were congregated in GraphPad Prism for statistical analysis. Mann-Whitney

U test and Fisher’s exact test were used for analyzing the statistical difference between two groups. p-Value less than .05 was considered significant. Evaluation of SARS-CoV-2 Spike-specific T-cell responses

[0276] To determine the impact of immunosuppression on vaccine responses, we analyzed Spike-specific T-cell immunity in 16 pre-vaccinated healthy individuals, 41 vaccinated healthy controls (1 month post- second dose of vaccine), and 61 vaccinated kidney transplant recipients (49 at 1 month and 12 at 2-3 months post-second dose of vaccine). T- cell immune responses to SARS-CoV-2 Spike peptides were not determined prior to vaccination in kidney transplant patients. Data are summarized in Figure 18A-18B. Briefly, no healthy controls showed CD4+ T-cell reactivity to Spike proteins prior to vaccination (pre-vaccination). However, there was a significant response to vaccination detected at 1 month post-vaccination. This contrasted with poor SARS-CoV-2 Spike-specific CD4+ T-cell responses seen in transplant recipients 1 month post-second dose of the BNT162b2 vaccination, 88% (36 of 41) positive in healthy controls versus 37% (18 of 49) positive in Tx recipients (p .0001). Repeat analysis performed 2-3 months post-second vaccination demonstrated that ~42% of transplant recipients developed positive CD4 T-cell responses (five of 12, 1 month). CD8 Spike-specific T cells were detected in 56% (23 of 41) healthy controls and 37% (18 of 49) kidney transplant recipients 1 month post- second second dose of the BNT162b2 vaccination (p=NS) (Figure 18B).

[0277] CoV-2 specific CD8 Spike-specific T-cell responses remained low in transplant recip- ients when analyzed 2-3 months post-vaccination (33%, four of 12). Our data suggest that CD4 /CD8 T-cell responses to Spike proteins in transplant recipients was significantly lower than those seen in non- immunocompromised individuals.

Impact of immunosuppressive agents on T-cell immune responses after BNT162b2 vaccination

[0278] 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). Our analysis of SARS-CoV-2 Spike specific CD4+ T-cell responses in the Tac versus Bela groups, showed no significant differences, but better CD8+ T-cell response in Bela group (p = .022, power = 0.33). These observations suggest that selected patients receiving belatacept can develop de novo immune responses to Spike peptides at the T-cell level. This contrasts with previous reports of poor Spike IgG and T-cell responses to vaccination in patients treated with belatacept therapy. Reasons for this are not readily apparent except for the possible impact of assay differences used.

Analysis of CMV-specific T-cell immune responses in BNT162b2 vaccinated immunocompromised patients [0279] CMV is the most common viral infection in transplant patients and CMV-specific

T-cell immune responses could be dampened by immunosuppression post transplantation. To better understand T-cell immunity in immunocompromised kidney transplant recipients, we compared the SARS-CoV-2-specific T-cell responses (CoV-2T) to CMV-Tc. Here, CMV-Tc represents a memory response and all CMV-Tc+ patients were also CMV-IgG+. For the CMV-Tc negative patients ( n = 10), all but one was CMV-IgG negative. This is consistent with no previous exposure to CMV. However, from our previous experience, patients who were CMV-IgG+ and failed to demonstrate +CMV-Tc responses were intensely immunosuppressed and more likely to develop opportunistic infections. We have previously shown that CMV-Tc responses were detectable in belatacept treated patients and were not affected by high doses of belatacept. This is consistent with the observation that memory T cells do not depend on CD28 signaling for recall responses and are primarily CD8+. There were no significant differences in T-cell immune responses to CMV or SARS-CoV-2 by immunosuppressive regimens.

[0280] Here, we saw that patients on both types of immunosuppression generate vigorous

CMV-Tc responses, suggesting memory responses are conserved and are resistant to inhibition by immunosuppression at levels maintained in our patients. We did not see a correlation with SARS- CoV-2 vaccine-induced T-cell responses and CMV-Tc responses, the latter being present in all but one CMV-IgG+ individual.

Humoral immune responses to SARS-CoV-2vaccination (BNT162b2)

[0281] 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. Here, we found 93% of healthy vaccinated individuals demonstrated positive IgG responses, which contrasted with impaired positive IgG responses (21% in total, 33% for belatacept recipients and 10% for tacrolimus recipients) in transplant recipients 1 month post- second dose of Pfizer BNT162b2 vaccine (Figure 19A). Further stratification of Spike-RBDIgG and SARS-CoV-2 Spike-specific T-cell responses in transplant recipients showed that 16% expressed both positive T cells (either CD4+ or CD8+) and IgG responses while 45% failed to demonstrate IgG and T-cell responses. Importantly, 35% of transplant patients who failed to show Spike-RBDIgG responses demonstrated positive T-cell response (either CD4+ or CD8+). Again, we saw no significant differences in Spike-RBD specific immune responses related to type of immunosuppression.

[0282] These observations are important and expand on the analysis of immunity developed after BNT162b2 vaccination that would not be apparent by analysis of Spike-RBD IgG responses alone. Discussion

[0283] Current assessments of immunity to SARS-CoV-2 depend on detection of antibodies to SARS-CoV-2 Spike-RBD. However, this represents a limited and often-unreliable method since IgG responses are transient in nature and do not reflect the likely presence of memory B cells, T cells, and plasma cells. Assays of T-cell responses (CD4+/CD8+) are emerging and may aid in identifying a more durable immunity aimed at eliminating infected cells (CD8+) and initiating CD4+ T cells which are critical to coordinating adaptive immunity toward the virus and generating long-lasting immunologic memory.

[0284] Recent reports have demonstrated poor IgG and T-cell immunity in patients receiving belatacept-based immunosuppression. However, our analysis of Spike-RBD-IgG and CD4+/CD8+ Spike specific T-cell responses in renal transplant patients vaccinated with BNT162b2 showed no significant differences in either Spike-RBD-IgG or CD4+/CD8+ T-cell responses. In this regard, we were suspicious of the effect of MMF in both groups. Our analysis of Spike-RBD-IgG showed only 21% with positive responses in transplant patients.

[0285] However, analysis of CD4+/CD8+ Spike-specific T cells showed that 35% of patients had a positive T-cell response (CD4+ and/or CD8+). Cucchiari et al. reported that 70% of kidney transplant recipients had no IgG/IgM seroconversion after mRNA-1273 SARS-CoV-2 vaccination, in which 50% showed positive T cells against Spike proteins.

[0286] This indicates that despite the absence of SARS-CoV-2 Spike-RBD-IgG, T-cell immunity is present in one-third of those vaccinated. We have recently reported on divergent immune responses to SARS-CoV-2 vaccines in immunocompromised patients receiving B-cell depletion.

[0287] Here, we saw no SARS-CoV-2 Spike-RBD-IgG, but were able to detect vigorous

CD4+/CD8+ T-cell responses in all patients. Revaccination in two patients resulted in a significant expansion of T cells specific for SARS-CoV-2 Spike peptides, but Spike-RBD-IgG remained negative.

[0288] Reports of the efficacy of a third BNT162b2 booster vaccine resulted in FDA recommendations for the broad application of booster vaccinations in immunocompromised individuals. However, these findings contrast with data from Chavarot et al. who examined Spike IgG responses in transplant patients maintained on belatacept therapy. Here, only 6.4% of patients showed Spike IgG after the third BNT162b2 vaccine. Of importance, 12 patients developed COVID 19 infections with 50% mortality. These authors also identified poor T-cell immunity to SARS-CoV-2 peptides in belatacept treated patients. There were no confirmed cases of COVID 19 infection in our transplant patients studied here. However, there were two patient deaths from SARS-CoV-2 in vaccinated (x2 BNT162b2) patients in whom we did not have the opportunity to assess Spike IgG or T-cell immunity.

[0289] Although our study is limited by low statistical power primarily due to low sample size, we feel the observations are still important. Overall, our study showed that 45% of our kidney transplant patient cohort failed to demonstrate Spike IgG and CD4+/CD8+ T-cell responses.

[0290] 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.

[0291] The essential question here regards the importance and possible protective capacity of CD4+/CD8+ T cells in patients who fail to demonstrate SARS-CoV-2 Spike-RBD- IgG responses. Recent data have elucidated this question in patients who recovered from SARS- CoV-2 infection. Peng et al.24 demonstrated robust CD4+/CD8+ T-cell responses to SARS-CoV- 2 after infection which also included a demonstration of diverse T-cell responses that likely extended beyond the persistence of Spike-IgG. Here, understanding the function of both humoral and cellular responses to SARS-CoV-2 and vaccines is critical in defining the composition of an effective immune response.

[0292] Spike IgG responses are essential for mediation of sterilizing immunity. Here,

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. With this, 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. Thus, detection of CD4+/CD8+ T-cell immunity to SARS-CoV-2 in patients failing to generate Spike-IgG likely infers an important component of protective immunity allowing us to no longer consider these patients “unvaccinated” based on assessment of Spike IgG alone.

[0293] Another important aspect of analysis of T-cell immune responses is the ability to detect immune responses to VOCs. We have recently reported on CD4+/CD8+ T-cells reactivity with SARS-CoV-2 (ancestral Spike) and VOCs. Here, equivalent reactivity to alpha and delta Spike peptides was seen. Thus T-cell immunity confers a diverse and broadly reactive immune responses to ancestral and emerging VOCs.

[0294] In summary, our study demonstrates that vaccination with BNT162b2 vaccination can result in SARS-CoV-2-specific T-cell immunity in immunosuppressive patients who show no SARS-CoV-2 Spike IgG responses. Although T-cell responses alone are not able to prevent SARS-CoV-2 infection, they would likely emerge rapidly killing infected cells and result in decreased length and severity of illness.

[0295] Various embodiments of the invention are described above in the Detailed

Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0296] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0297] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

[0298] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g ” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0299] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0300] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.