Compositions and methods for detecting cell-mediated immunity to epstein-BARR virus

Abstract

Compositions and methods are provided for the detection of cell-mediated immunity to Epstein-Barr virus (EBV), including a method for detecting or confirming an EBV infection (e.g., a latent EBV infection) by contacting, in vitro, whole blood samples from subjects suspected to have an EBV infection with compositions comprising combinations of T-cell epitope-containing peptides derived from EBNA-1, EBNA-3A, and EBNA-3B proteins, and indirectly detecting EBV-specific activated T-cells by determining production of a T-cell response indication (e.g., interferon-γ) in response to stimulation by the peptides. Also disclosed are sample collection vessels comprising the peptide compositions as well as kits comprising the peptide composition for use in detecting an EBV infection (e.g., a latent EBV infection) in a subject. Further disclosed are vaccines comprising T-cell epitope-containing peptides derived from EBNA-3A, and EBNA-3B proteins and methods for stimulating T cell response using such vaccines.

Description

[0001] COMPOSITIONS AND METHODS FOR DETECTING CELL-MEDIATED IMMUNITY TO EPSTEIN-BARR VIRUS

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (830109_430WO_SL.xml; Size: 84,049 bytes; and Date of Creation: October 31, 2024) is herein incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] Technical Field

[0006] The present disclosure relates to compositions and methods for detecting cell-mediated immunity to Epstein-Barr virus (EBV). More specifically, there are provided compositions comprising combinations of T-cell epitope-containing peptides derived from EBNA-1, EBNA-3A, and EBNA-3B proteins of EBV that are expressed during the latent phase of EBV infection for use in an in vitro method of detecting EBV-specific T-cell immune responsiveness. The present disclosure also relates to vaccines comprising T-cell epitope-containing peptides derived from EBNA-3A and / or EBNA-3B.

[0007] Description of the Related Art

[0008] Epstein-Barr Virus is a member of the herpes virus family, also known as human herpesvirus 4 (HHV-4). Approximately 90-95% of people have been infected with the virus by the time they reach adulthood. EBV is predominantly spread through bodily fluids, particularly saliva, and produces both acute and latent stages of infection in humans. If acquired during childhood, EBV typically results in a clinically asymptomatic infection. In contrast, infection in adulthood often results in a symptomatic and acute viral syndrome termed “acute infectious mononucleosis.”

[0009] Re-activation of EBV during latency is typically well-controlled by the immune system in healthy individuals. However, EBV can have a pronounced effect on individuals that are immunocompromised or have weakened immune systems. For example, in the transplant setting, post-transplant lymphoproliferative disorders (PTLD) can arise as a consequence of immunosuppressive therapies used to prevent transplant rejection (Singavi et al. 2015 Cancer Treat Res. 165:305-27). Such immunosuppressive therapies cause a loss of T cells that typically control EBV reactivation. Detection of elevated and rising EBV viral loads may indicate patients at risk for PTLD, a cause of morbidity and mortality in transplant patients (Shahid S et al. 2021 Cancer Drug Resist. 4(3):646-664). Monitoring EBV-specific T cell responses may aid in the detection of patients with an increased risk of reactivated EBV.

[0010] SUMMARY OF DISCLOSURE

[0011] The present disclosure provides compositions for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV), methods for detecting a T- cell-mediated immune response to EBV, sample collecting vessels comprising a composition for stimulating a T-cell-mediated immune response, and kits for detecting a latent EBV infection in a subject.

[0012] In one aspect, the present disclosure provides a composition for stimulating a T-cell-mediated immune response to Epstein-Barr virus (EBV), comprising:

[0013] (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 isolated CD4 EBNA-1 peptides, each of which comprises:

[0014] (a) a CD4+ T cell epitope, and

[0015] (b) a peptide selected from the group consisting of EBNA-1 peptides having the amino acid sequences set forth in SEQ ID NOS: 1-40 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40; and / or

[0016] (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 isolated CD8 EBNA-1 peptides each of which comprises

[0017] (a) a CD8+ T cell epitope, and

[0018] (b) a peptide selected from the group consisting of EBNA-1 peptides having the amino acid sequences set forth in SEQ ID NOS :41-66 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS :41-66; and / or

[0019] (iii) 1, 2, or 3 isolated CD8 EBNA-3A peptides each of which

[0020] (a) a CD8+ T cell epitope, and (b) a peptide selected from the group consisting of EBNA-3A peptides having the amino acid sequences set forth in SEQ ID NOS:67-69 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and / or

[0021] (iv) 1, 2, 3, or 4 isolated CD8 EBNA-3B peptides that each of which comprises

[0022] (a) a CD8+ T cell epitope, and

[0023] (b) a peptide selected from the group consisting of EBNA-3B peptides having the amino acid sequences set forth in SEQ ID NOS:70-73 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

[0024] In another aspect, the present disclosure provides a method for detecting a T-cell-mediated immune response to EBV, comprising:

[0025] (A) contacting in vitro (1) a biological sample obtained from a subject suspected of being infected with EBV, wherein the biological sample comprises T-cells and antigen-presenting cells, and (2) a peptide composition for stimulating a T- cell-mediated immune response to EBV, to obtain a test incubation mixture;

[0026] (B) incubating the test incubation mixture under conditions and for a time sufficient for specific recognition by said T-cells of an EBV T-cell epitope that is present in said peptide composition to stimulate generation of a T-cell immune response indicator; and

[0027] (C) detecting a level of the T-cell immune response indicator in the test incubation mixture, wherein presence of an EBV infection in the subject is indicated by detection in (C) of said level of the T-cell immune response indicator that is increased relative to a control level of the T-cell immune response indicator obtained by incubating the biological sample in a control incubation mixture without the peptide composition for stimulating a T-cell-mediated immune response to EBV, or relative to a reference level of the T-cell immune response indicator obtained by incubating a reference incubation mixture of a reference biological sample obtained from a subject known to have not been infected with EB V and the peptide composition for stimulating T-cell-mediated immune response to EBV, and wherein the peptide composition for stimulating the T-cell-mediated immune response to EBV is a composition disclosed herein.

[0028] In another aspect, the present disclosure provides a sample collection vessel comprising a composition for stimulating the T-cell-mediated immune response to EBV disclosed herein.

[0029] In a further aspect, the present disclosure provides a kit for detecting a latent EBV infection in a subject, comprising: (i) a composition for stimulating the T- cell-mediated immune response to EBV disclosed herein, and (ii) one or more reagents for performing an ELISA assay.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 shows soluble IFN-y levels in plasma after in vitro T-cell activation with Epstein-Barr virus (EBV) peptides derived from one or more of EBNA- 1, EBNA-3A, and EBNA-3B. One (1) mL of whole blood from EBV EBNA-1 IgG seropositive donors (n=10) was incubated at 37°C ± 1°C with different combinations of EBV peptides (SEQ ID NOS: 1-73), as indicated, after which plasma was isolated for quantitation of IFN-y by QUANTIFERON® human IFN-y ELISA (QIAGEN) according to the manufacturer’s instructions. Results are shown as individual data points with median and interquartile ranges (IQR) reported in international units per milliliter (lU / mL) adjusted to the baseline interferon gamma level (Nil).

[0032] FIG. 2 shows soluble IFN-y levels in plasma after in vitro T-cell activation with Epstein-Barr virus (EBV) peptides derived from one or more of EBNA- 1, EBNA-3A, and EBNA-3B. One (1) mL of whole blood from EBV EBNA-1 IgG seropositive donors (n=l 1) was incubated at 37°C ± 1°C with different combinations of EBV peptides (SEQ ID NOS: 1-73), as indicated, after which plasma was isolated for quantitation of IFN-y by QUANTIFERON® human IFN-y ELISA (QIAGEN) according to the manufacturer’s instructions. ELISA results are presented as Ag-Nil (lU / mL) for each donor across the different combinations of EBV peptides. By using an arbitrary cut off set to 0.20 lU / mL, antigens sufficient for detection are noted at the bottom of the table. FIG. 3A and 3B show soluble IFN-y levels in plasma after in vitro T-cell activation with Epstein-Barr virus (EBV) peptides derived from one or more of EBNA- 1, EBNA-3A, and EBNA-3B. Each peptide is at 0.5 mg / tube for Pools A to H and Img / tube for Subpools 1 to 27. One (1) mL of whole blood from EBV EBNA-1 and EBV VC A IgG seropositive donors (n=10) or seronegative donors (n=l to 2) was incubated at 37°C ± 1°C with different combinations of EBV peptides (SEQ ID NOS: 1- 73) as indicated, after which plasma was isolated for quantitation of IFN-y by QUANTIFERON® human IFN-y ELISA (QIAGEN) according to the manufacturer’s instructions. ELISA results are presented as Ag-Nil (lU / mL) for each donor across the different combinations of EBV peptides. By using an arbitrary cut off set to >0.10 lU / mL, the number of EBV EBNA-1 and EBV VC A IgG positive donors responding are noted at the right of the table.

[0033] FIG. 4 shows the fraction of donors responding based on soluble IFN-y levels in plasma after in vitro T-cell activation with Epstein-Barr virus (EBV) peptides derived from one or more of EBNA-1, EBNA-3A, and EBNA-3B. Each peptide is at 0.5 mg / tube for Pools A to H and Img / tube for Subpools 1 to 27. One (1) mL of whole blood from EBV EBNA-1 IgG and EBV VCA IgG seropositive donors (n=10) was incubated at 37°C ± 1°C with different combinations of EBV peptides (SEQ ID NOS: 1- 73) as indicated, after which plasma was isolated for quantitation of IFN-y by QUANTIFERON® human IFN-y ELISA (QIAGEN) according to the manufacturer’s instructions. Results are shown as percent donors responding [number of ELISA positive donors (with >0.1 IU / ml) / total numb of EBV EBNA-1 IgG and EBV VCA IgG seropositive donors xlOO] across the different antigen pools and subpools.

[0034] DETAILED DESCRIPTION

[0035] In one aspect, the present disclosure provides compositions, methods, sample collection vessels, and kits for detecting cell-mediated immunity to Epstein-Barr virus (EBV). More specifically, there are provided compositions comprising, consisting essentially of, or consisting of combinations of T-cell epitope-containing peptides derived from EBNA-1 (SEQ ID NOS: 1-66), EBNA-3A (SEQ ID NOS:67-69), and EBNA-3B (SEQ ID NOS:70-73) proteins, and variants thereof for use in an in vitro method of detecting EBV-specific T-cell immune responsiveness. Also provided are sample collection vessels comprising the peptide compositions as well as kits comprising the peptide compositions for use in detecting an EBV infection e.g., a latent EBV infection) in a subject.

[0036] EBV-specific T cell responses have been reported to differ considerably in breadth, magnitude, and immunodominance. There remains a need for improved diagnostic assessment of cell-mediated responses to latent EBV antigens, for example for monitoring cell-mediated immunity in immunosuppressed patients such as transplant recipients. The presently disclosed embodiments address these needs by providing CD4+ T cell-specific peptides, CD8+ T cell-specific peptides, or combinations of CD4+ and CD8+ T cell-specific peptides to elicit T-cell-mediated immune response. In certain embodiments, the present disclosure provides combining both CD4+ T cell- and CD8+ T cell-specific peptides to elicit a surprisingly synergistic response. Disclosed herein are compositions comprising CD4+ T cell-specific EBNA-1 peptides, CD8+ T cell-specific EBNA-1 peptides, CD8+ T cell-specific EBNA-3A peptides, CD8+ T cell-specific EBNA-3B peptides, or combinations thereof that induce robust activation of T-cells from subjects known to have or suspected of being infected with EBV. The EBV-specific T cell response can be detected via T cell response indicators (e.g., interferon-y) using assays such as QUANTIFERON® human IFN-y ELISA (QIAGEN). The peptide compositions can be added directly to whole blood samples or be used to coat whole blood sample collection vessels for direct in vitro stimulation of a secondary EBV-specific antigen response.

[0037] As described below, in certain preferred embodiments, whole blood samples containing T cells from subjects (e.g., donors serologically positive for EBV) respond to a peptide cocktail containing CD4+ T cell-specific EBNA-1 peptides (e.g., one, multiple, or all of the peptides in Table 1), CD8+ T cell-specific EBNA-3A peptides (e.g., one, multiple, or all of the peptides in Table 3), and CD8+ T cell-specific EBNA-3B peptides (e.g., one, multiple, or all of the peptides in Table 4), optionally further containing CD8+ T cell-specific EBNA-1 peptides (e.g., one, multiple, or all of the peptides in Table 2) by eliciting specific T-cell mediated immune responses. Moreover, the combination of both CD4+ and CD8+ T cell-specific peptides (e.g., the combination of CD4+ T cell-specific EBNA-1 peptides with CD8+ T cell-specific EBNA-3 A peptides and CD8+ T cell-specific EBNA-3B peptides, and optionally further with CD8+ T cell-specific EBNA-1 peptides may induce a synergistic immune response. The compositions and methods provide a method for detecting T-cell mediated immune responses to EBV infections including a latent EBV infection.

[0038] In another aspect, the present disclosure provides vaccines comprising one or more CD8+ T cell-specific EBNA-3A / 3B peptides (e.g., one, multiple, or all of the peptides in Tables 3 and 4). Such vaccines are useful in stimulating T cell response in immunocompromised subjects who have been previously exposed to EBV, such as being administered to the subjects as booster vaccines.

[0039] I. EBV Latent Antigens

[0040] In one aspect, the present disclosure provides a composition for stimulating a T-cell-mediated immune response to Epstein-Barr virus (EBV), comprising, consisting essentially of, or consisting of:

[0041] (i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 isolated CD4 EBNA-1 peptides, each of which comprises:

[0042] (a) a CD4+ T cell epitope, and

[0043] (b) a peptide selected from the group consisting of EBNA-1 peptides having the amino acid sequences set forth in SEQ ID NOS: 1-40 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40; and / or

[0044] (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 isolated CD8 EBNA-1 peptides each of which comprises

[0045] (a) a CD8+ T cell epitope, and

[0046] (b) a peptide selected from the group consisting of EBNA-1 peptides having the amino acid sequences set forth in SEQ ID NOS :41-66 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS :41-66; and / or

[0047] (iii) 1, 2, or 3 isolated CD8 EBNA-3A peptides each of which

[0048] (a) a CD8+ T cell epitope, and

[0049] (b) a peptide selected from the group consisting of EBNA-3A peptides having the amino acid sequences set forth in SEQ ID NOS:67-69 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and / or (iv) 1, 2, 3, or 4 isolated CD8 EBNA-3B peptides that each of which comprises

[0050] (a) a CD8+ T cell epitope, and

[0051] (b) a peptide selected from the group consisting of EBNA-3B peptides having the amino acid sequences set forth in SEQ ID NOS:70-73 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

[0052] In certain embodiments, the composition comprises, consists essentially of, or consists of:

[0053] (1) one or more isolated CD4 EBNA-1 peptides of (i),

[0054] (2) one or more isolated CD4 EBNA-1 peptides of (i) and one or more isolated CD8 EBNA-1 peptides of (ii),

[0055] (3) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3A peptides of (iii),

[0056] (4) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3B peptides of (iv),

[0057] (5) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv),

[0058] (6) one or more isolated CD4 EBNA-1 peptides of (i) and one or more isolated CD8 EBNA-3 A peptides of (iii),

[0059] (7) one or more isolated CD4 EBNA-1 peptides of (i) and one or more isolated CD8 EBNA-3B peptides of (iv),

[0060] (8) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-3 A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv), (9) one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3 A peptides of (iii),

[0061] (10) one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3B peptides of (iv),

[0062] (11) one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3 A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv), or

[0063] (12) one or more isolated CD8 EBNA-3A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv).

[0064] In certain embodiments, the composition comprises, consists essentially of, or consists of: one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-3 A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv).

[0065] In certain embodiments, the composition comprises, consists essentially of, or consists of: one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3a peptides of (iii), and one or more isolated CD8 EBNA-3b peptides of (iv).

[0066] In certain embodiments, the composition comprises, consists essentially of, or consists of: one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3a peptides of (iii), and one or more isolated CD8 EBNA-3b peptides of (iv).

[0067] In certain embodiments, the composition comprises, consists essentially of, or consists of: at least 5, at least 10, at least 15, or at least 35 isolated CD4 EBNA-1 peptides. In certain embodiments, the composition further comprises at least 5 isolated CD8 EBNA-1 peptides.

[0068] In the embodiments disclosed herein, the one or more isolated CD4 EBNA-1 peptides of (i) comprise a sequence selected from the group consisting of SEQ ID NOS: 1-40, the one or more isolated CD8 EBNA-1 peptides of (ii) comprise a sequence selected from the group consisting of SEQ ID NOS :41-66, the one or more isolated CD8 EBNA-3 A peptides of (iii) comprise a sequence selected from the group consisting of SEQ ID NOS:67-69, and the one or more isolated CD4 EBNA-1 peptides of (iv) comprise a sequence selected from the group consisting of SEQ ID NOS:70-73.

[0069] In certain embodiments, the composition comprises, consists essentially of, or consists of:

[0070] (i) one isolated CD4 EBNA-1 peptide selected from SEQ ID NOS: 1-40; and

[0071] (ii) one isolated CD8 EBNA-1 peptide selected from SEQ ID NOS:41-66;

[0072] (iii) one isolated CD8 EBNA-3 A peptide selected from SEQ ID NOS:67-69; and

[0073] (iv) one isolated CD8 EBNA-3B peptide selected from SEQ ID NOS:70-73, wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

[0074] In certain embodiments, the composition comprises, consists essentially of, or consists of:

[0075] (i) one isolated CD4 EBNA-1 peptide selected from SEQ ID NOS: 1-40;

[0076] (ii) one isolated CD8 EBNA-3 A peptide selected from SEQ ID NOS:67-69; and

[0077] (iii) one isolated CD8 EBNA-3B peptides selected from SEQ ID NOS:70-73.

[0078] In certain embodiments, the composition comprises, consists essentially of, or consists of: (i) one isolated CD8 EBNA-1 peptide selected from SEQ ID NOS:41-66;

[0079] (ii) one isolated CD8 EBNA-3 A peptide selected from SEQ ID NOS:67-69; and

[0080] (iii) one isolated CD8 EBNA-3B peptides selected from SEQ ID NOS:70-73.

[0081] In certain embodiments, the composition for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV) comprises, consists essential of, or consists of:

[0082] (i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1-40, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40; and

[0083] (ii) 5 to 26 isolated CD8 EBNA-1 peptides as set forth in SEQ ID NOS :41-66, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS :41-66;

[0084] (iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and

[0085] (iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

[0086] In certain embodiments, the composition for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV) comprises, consists essential of, or consists of:

[0087] (i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID

[0088] NOS: 1-40, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40; (ii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and

[0089] (iii) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

[0090] In certain embodiments, the composition for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV) comprises, consists essential of, or consists of:

[0091] (ii) 5 to 26 isolated CD8 EBNA-1 peptides as set forth in SEQ ID NOS :41-66, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS :41-66;

[0092] (iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and

[0093] (iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

[0094] In certain embodiments, the composition for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV) comprises, consists essential of, or consists of:

[0095] (i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1-40; and

[0096] (ii) 5 to 26 isolated CD8 EBNA-1 peptides as set forth in SEQ ID NOS:41-66; (iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69; and

[0097] (iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73.

[0098] In certain embodiments, the composition for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV) comprises, consists essential of, or consists of:

[0099] (i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1-40;

[0100] (iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69; and

[0101] (iii) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73.

[0102] In certain embodiments, the composition for stimulating a T-cell- mediated immune response to Epstein-Barr virus (EBV) comprises, consists essential of, or consists of:

[0103] (ii) 5 to 26 isolated CD8 EBNA-1 peptides as set forth in SEQ ID NOS:41-66;

[0104] (iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69; and

[0105] (iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73.

[0106] In certain embodiments, the composition comprises, consists essentially of, or consists of:

[0107] (1) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-31, 35-36, and 38-40; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS :41-57 and 59-66; isolated CD8 EBNA-3 A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73; (2) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16-20, 21-25, and 26-30; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;

[0108] (3) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16-20 and 26-30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS: 57-62; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO:72;

[0109] (4) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16-30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:57-62; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;

[0110] (5) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16-20, 26-31, 35-36, and 38-40; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO: 67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;

[0111] (6) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16-20 and 26-30; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO:72;

[0112] (7) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS:26-30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:46-51; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;

[0113] (8) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS:26-30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:46-51; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NOS:70 and 72;

[0114] (9) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16-20 and 26-30; and isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO: 67;

[0115] (10) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-31, 35-36, and 38-40; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:41-57 and 59-66; (11) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-31, 35-36, and 38-40;

[0116] (12) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS:26-30;

[0117] (13) isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;

[0118] (14) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-40; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS :41-66; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;

[0119] (15) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-40; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;

[0120] (16) isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:41-66; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67- 69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;

[0121] (17) isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73; or

[0122] (18) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-40.

[0123] An EBNA-1, EBNA-3A, or EBNA-3B peptide that comprises an EBV T-cell epitope for use in certain embodiments contemplated herein may comprise the amino acid sequence set forth for an EBNA-1 peptide in any one of SEQ ID NOS: 1-66, for an EBNA-3A peptide in any one of SEQ ID NOS:67-69, and for an EBNA-3B peptide as set forth in SEQ ID NOS:70-73, and may in certain other embodiments comprise an EBV T-cell epitope-containing peptide variant comprising a peptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of such peptides of SEQ ID NOS: 1-73 and that is capable of being specifically recognized by a T-cell reactive with at least one EBV strain, which may include but need not be limited to B95-8, AG876, GD-1, Cao, and Raji, preferably B95-8.

[0124] An EBNA-1 peptide that comprises an EBV CD4+ T-cell specific epitope for use in certain embodiments contemplated herein may comprise the amino acid sequence set forth for an EBNA-1 peptide in any one of SEQ ID NOS: 1-40 (see Table 1), and may in certain embodiments comprise an EBV CD4+ T-cell specific epitope-containing peptide variant comprising a peptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of such peptides of SEQ ID NOS: 1-40 and that is capable of being specifically recognized by a T-cell that is reactive with at least one EBV strain, which may include but need not be limited to B95-8, AG876, GD-1, Cao, and Raji, preferably B95-8. An EBNA-1 peptide that comprises an EBV CD4+ T-cell specific epitope is referred to herein as a “CD4 EBNA- 1 peptide.”

[0125] An EBNA-1 peptide that comprises an EBV CD8+ T-cell specific epitope for use in certain embodiments contemplated herein may comprise the amino acid sequence set forth for an EBNA-1 peptide in any one of SEQ ID NOS :41-66 (see Table 2), and may in certain embodiments comprise an EBV CD8+ T-cell specific epitope-containing peptide variant comprising a peptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of such peptides of SEQ ID NOS :41-66 and that is capable of being specifically recognized by a T-cell that is reactive with at least one EBV strain, which may include but need not be limited to B95-8, AG876, GD-1, Cao, and Raji, preferably B95-8. An EBNA-1 peptide that comprises an EBV CD8+ T-cell specific epitope is referred to herein as a “CD8 EBNA- 1 peptide.”

[0126] An EBNA-3 A peptide that comprises an EBV CD8+ T-cell specific epitope for use in certain embodiments contemplated herein may comprise the amino acid sequence set forth for an EBNA-3A peptide in any one of SEQ ID NOS:67-69 (see Table 3), and may in certain embodiments comprise an EBV CD8+ T-cell specific epitope-containing peptide variant comprising a peptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of such peptides of SEQ ID NOS:67-69 and that is capable of being specifically recognized by a T-cell that is reactive with at least one EBV strain, which may include but need not be limited to B95-8, AG876, GD-1, Cao, and Raji, preferably B95-8. An EBNA-3A peptide that comprises an EBV CD8+ T-cell specific epitope is referred to herein as a “CD8 EBNA- 3 A peptide.”

[0127] An EBNA-3B peptide that comprises an EBV CD8+ T-cell specific epitope for use in certain embodiments contemplated herein may comprise the amino acid sequence set forth for an EBNA-3B peptide in any one of SEQ ID NOS:70-73 (see Table 4), and may in certain embodiments comprise an EBV CD8+ T-cell specific epitope-containing peptide variant comprising a peptide having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of such peptides of SEQ ID NOS:70-73 and that is capable of being specifically recognized by a T-cell that is reactive with at least one EBV strain, which may include but need not be limited to B95-8, AG876, GD-1, Cao, and Raji, preferably B95-8. An EBNA-3B peptide that comprises an EBV CD8+ T-cell specific epitope is referred to herein as a “CD8 EBNA- 3B peptide.”

[0128] A naturally occurring EBV nuclear antigen 1 (EBNA-1) is a multifunctional, dimeric viral protein associated with Epstein-Barr Virus. The functions of a naturally occurring EBNA-1 include (1) origin of replication (oriP)- dependent replication and maintenance of viral episomes during latency, (2) counteraction of the stabilization of host p53 / TP53 by host USP7, thereby decreasing apoptosis and increasing host cell survival, and (3) induction of degradation of host promyelocytic leukemia nuclear bodies through the ubiquitin-proteasome system, which promotes lytic reactivation and may impair the host cell DNA repair. An exemplary naturally occurring EBNA-1 is the EBNA-1 from Epstein-Barr virus strain B95-8 with the amino acid sequence according to UniProtKB - P03211 (SEQ ID NO:74).

[0129] “EBNA-1” or “EBNA-1 protein” as used herein refers to a naturally occurring EBNA-1 or a variant thereof (1) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the naturally occurring EBNA-1 protein and (2) retaining one, more or all of the functions of the naturally occurring EBNA-1 protein.

[0130] In certain embodiments, EBNA-1 is a protein with the amino acid sequence according to SEQ ID NO:74. In certain other embodiments, EBNA-1 is a variant of SEQ ID NO:74 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 74.

[0131] An “EBNA-1 peptide” as used herein refers to a fragment (e.g., a fragment that is 5 to 50 amino acids, 7 to 27 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 14 to 27 amino acids, 15 to 26 amino acids, 20 to 27 amino acids, or 21 to 26 amino acids in length) of an EBNA-1 protein or a variant of the EBNA-1 fragment having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the EBNA-1 fragment. An EBNA-1 peptide may be a CD4 EBNA-1 peptide that is 14 to 27 amino acids, 15 to 26 amino acids, preferably 20 to 27 amino acids, or more preferably 21 to 26 amino acids in length. An EBNA-1 peptide may be a CD8 EBNA-1 peptide that is 7 to 13 amino acids, 8 to 13 amino acids, preferably 8 to 12 amino acids, or more preferably 8 to 11 amino acids in length.

[0132] In certain embodiments, an EBNA-1 peptide is a fragment (e.g., a fragment that is 5 to 50 amino acids, 7 to 27 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 14 to 27 amino acids, 15 to 26 amino acids, 20 to 27 amino acids, or 21 to 26 amino acids in length) of the EBNA-1 as set forth in SEQ ID NO:74. An EBNA-1 peptide may be a CD4 EBNA-1 peptide that is 14 to 27 amino acids, 15 to 26 amino acids, preferably 20 to 27 amino acids, or more preferably 21 to 26 amino acids in length of the EBNA-1 as set forth in SEQ ID NO:74. An EBNA-1 peptide may be a CD8 EBNA-1 peptide that is 7 to 13 amino acids, 8 to 13 amino acids, preferably 8 to 12 amino acids, or more preferably 8 to 11 amino acids in length of the EBNA-1 as set forth in SEQ ID NO:74. In certain other embodiments, an EBNA-1 peptide is a variant of an EBNA-1 fragment having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a fragment of SEQ ID NO:74.

[0133] A naturally occurring Epstein-Barr nuclear antigen 3 A (EBNA-3 A) (also referred to as “EBNA-3” and “EBV nuclear antigen 3”) is a viral protein associated with Epstein-Barr Virus. The functions of EBNA-3A include but are not limited to: (1) activation and immortalization of human B-cells, (2) repression of transcription of viral promoters TP1 and Cp through interaction with host DNA-binding protein RBPJ, and (3) inhibition of EBNA2-mediated activation of TP1 and Cp promoters. An exemplary naturally occurring EBNA-3 A is the EBNA-3 A from Epstein-Barr virus strain B95-8 with the amino acid sequence according to UniProtKB - P12977 (SEQ ID NO:75).

[0134] “EBNA-3 A,” “EBNA-3a,” or “EBNA-3A protein” as used herein refers to a naturally occurring EBNA-3 A or a variant thereof (1) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the naturally occurring EBNA-3 A protein and (2) retaining one, more, or all of the functions of the naturally occurring EBNA-3 A protein.

[0135] In certain embodiments, EBNA-3 A is a protein with the amino acid sequence according to SEQ ID NO:75. In certain other embodiments, EBNA-3A is a variant of SEQ ID NO:75 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 75.

[0136] An “EBNA-3 A peptide” as used herein refers to a fragment (e.g., a fragment that is 5 to 50 amino acids, 7 to 27 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 14 to 27 amino acids, 15 to 26 amino acids, 20 to 27 amino acids, or 21 to 26 amino acids in length) of an EBNA-3 A protein or a variant of the EBNA-3 A fragment having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the EBNA-3A fragment. An EBNA-3A peptide may be a CD8 EBNA-3A peptide that is 7 to 13 amino acids, 8 to 13 amino acids, preferably 8 to 12 amino acids, more preferably 8 to 11 amino acids, 8 to 10 amino acids, or 9 to 10 amino acids in length.

[0137] In certain embodiments, an EBNA-3A peptide is a fragment (e.g., a fragment that is 5 to 50 amino acids, 7 to 27 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 8 to 10 amino acids, 9 to 10 amino acids, 14 to 27 amino acids, 15 to 26 amino acids, 20 to 27 amino acids, or 21 to 26 amino acids in length) of the EBNA-3A as set forth in SEQ ID NO:75. In certain other embodiments, an EBNA-3 A peptide is a variant of an EBNA-3 A fragment having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a fragment of SEQ ID NO: 75. An EBNA-3 A peptide may be a CD8 EBNA-3 A peptide that is 7 to 13 amino acids, 8 to 13 amino acids, preferably 8 to 12 amino acids, more preferably 8 to 11 amino acids, 8 to 10 amino acids, or 9 to 10 amino acids in length of the EBNA-3 A as set forth in SEQ ID NO:75.

[0138] A naturally occurring Epstein-Barr nuclear antigen 3B (EBNA-3B) (also referred to as “EBNA-4” or “EBV nuclear antigen 4”) is a viral protein associated with Epstein-Barr Virus. The functions of EBNA-3B include transcriptional regulation of both viral and host cellular genes by interacting with the host DNA-binding protein RBPJ. An exemplary naturally occurring EBNA-3B is the EBNA-3B from Epstein- Barr virus strain B95-8 with the amino acid sequence according to UniProtKB - P03203 (SEQ ID NO:76).

[0139] “EBNA-3B,” “EBNA-3b,” or “EBNA-3B protein” as used herein refers to a naturally occurring EBNA-3B or a variant thereof (1) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the naturally occurring EBNA-3B protein and (2) retaining one, more, or all of the functions of the naturally occurring EBNA-3B protein.

[0140] In certain embodiments, EBNA-3B is a protein with the amino acid sequence according to SEQ ID NO:76. In certain other embodiments, EBNA-1 is a variant of SEQ ID NO:76 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 76.

[0141] An “EBNA-3B peptide” as used herein refers to a fragment (e.g., a fragment that is 5 to 50 amino acids, 7 to 27 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 8 to 10 amino acids, 14 to 27 amino acids, 15 to 26 amino acids, 20 to 27 amino acids, or 21 to 26 amino acids in length) of an EBNA-3B protein or a variant of the EBNA-3B fragment having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of the EBNA-3B fragment. An EBNA-3B peptide may be a CD8 EBNA-3B peptide that is 7 to 13 amino acids, 8 to 13 amino acids, preferably 8 to 12 amino acids, more preferably 8 to 11 amino acids, or 8 to 10 amino acids in length.

[0142] In certain embodiments, an EBNA-3B peptide is a fragment (e.g., a fragment that is 5 to 50 amino acids, 7 to 27 amino acids, 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 8 to 10 amino acids, 14 to 27 amino acids, 15 to 26 amino acids, 20 to 27 amino acids, or 21 to 26 amino acids in length) of the EBNA-3B as set forth in SEQ ID NO:76. In certain other embodiments, an EBNA-3B peptide is a variant of an EBNA-3B fragment having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a fragment of SEQ ID NO:76. An EBNA-3B peptide may be a CD8 EBNA-3B peptide that 7 to 13 amino acids, 8 to 13 amino acids, preferably 8 to 12 amino acids, more preferably 8 to 11 amino acids, 8 to 10 amino acids in length of the EBNA-3B as set forth in SEQ ID NO:76.

[0143] Variants of EBV T cell-epitope containing peptides such as any of the peptides set forth in SEQ ID NOS: 1-73 may contain one or more amino acid substitutions, additions, deletions, and / or insertions relative to the native EBV T cell- epitope-containing peptide sequence set forth in SEQ ID NOS: 1-73 (e.g. wildtype, or predominant or naturally occurring allelic forms). Variants preferably exhibit at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% amino acid sequence identity and more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to a corresponding portion of a native EBV T cell-epitope-containing peptide sequence region.

[0144] For determining sequence identity, the WU-BLAST-2 program (Altschul et al., Methods in Enzymology 266:460-480, 1996) is used. This program uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=l 1. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A percent sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

[0145] Furthermore, computer algorithms are available in the art that enable the skilled artisan to predict the three-dimensional structure of a protein or peptide to ascertain functional variants of a particular polypeptide. For instance, variants can be identified wherein all or a portion of the three-dimensional structure is not substantially altered by one or more modification, substitution, addition, deletion and / or insertion. (See, for example, Seemayer et al., 2014 Bioinformat. 30:3128; Raman et al., 2010 ScienceExpress 4 February 2010 10.1126 / science.1183649; Gribenko et al., 2009 Proc. Nat. Acad. Sci. USA 106:2601; Bradley et al., Science 309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USN 103: 1244 (2006); Dodson et al., Nature 450: 176 (2007); Qian et al., Nature 450:259 (2007); Baker, 2014 Biochem. Soc. Trans. 42:225; Correia et al., 2014 Nature 507:201; King et al., 2014 Proc. Nat. Acad. Set. USA 111 :8577; Roche et al., 2012 PLoS One 7(5):e38219; Zhang et al., 2013 Meths. Enzymol. 523:21; Khoury et al., 2014 Trends Biotechnol. 32:99; O’Meara et al., 2015 J. Chem. Theory Comput. 11 :609; Park et al., 2015 Structure 23: 1123; Bale et al., 2015 Protein Sci. doi: 10.1002 / pro.2748 Epub PMID 26174163; Park et al., 2015 Proteins doi: 10.1002 / prot.24862 Epub PMID 26205421; Lin et al., 2015 Proc. Nat. Acad. Sci. USA pii :201509508 Epub PMID 26396255). In this way, one of skill in the art can readily determine whether a particular EBV T-cell epitope-containing peptide variant, or a functional fragment thereof, retains sufficient epitope structure to be capable of being specifically recognized by a T-cell that is reactive with at least one pathogenic EBV strain.

[0146] An EBV T-cell epitope-containing peptide may be a peptide of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous amino acids and in certain embodiments may typically be not more than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 amino acids in length. In certain preferred embodiments the EBV T-cell epitope-containing peptide may be a peptide of 7 to 27, 8 to 26, 7 to 14, 7 to 13, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 10, 14 to 27, 15 to 26, 20 to 27, or 21 to 26 contiguous amino acids in length, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous amino acids in length.

[0147] It is understood that a T-cell epitope refers to a structural region of an antigen that can be specifically recognized by a T-cell receptor for antigen (“T-cell receptor”), typically in the context of an appropriate major histocompatibility complex (MHC) class I or class II molecule that presents the epitope to the T-cell receptor. T- cell epitope specifically recognized by a CD4+ T cell receptor is referred to herein as “CD4+ T cell epitope” or “CD4+ T cell specific epitope,” while T-cell epitope specifically recognized by a CD8+ T cell receptor is referred to herein as “CD8+ T cell epitope” or “CD8+ T cell epitope.” T-cell epitope-containing peptides of about 7 to about 14 amino acids in length (e.g., 8, 9, 10, 11, 12, or 13 amino acids in length) are typically presented to CD8+ T-cell receptors by class I MHC molecules; such peptides are referred to herein as “CD8+ T cell epitope-containing peptides” or “CD8+ T cell specific epitope-containing peptides.” T-cell epitope-containing peptides of about 14- 27 amino acids in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acids) are typically presented to CD4+ T-cell receptors by class II MHC molecules; such peptides are referred to herein as “CD4+ T cell epitope-containing peptides” or “CD4+ T cell epitope-containing peptides.” T-cell receptors are not absolute in their specificity but are instead regarded as promiscuous; that is to say, a given T-cell receptor may be capable of specifically recognizing a particular T-cell epitope structure and also a range of closely related epitope structures. Specific recognition of an appropriately presented T-cell epitope by a T-cell may be detectable as stimulation of the generation of a T-cell immune response indicator such as those described herein (e.g., cytokine release by T-cells, such as IFN-y release). Hence an EBV T-cell epitopecontaining peptide may refer to a peptide antigen that is capable, in a secondary in vitro immune response, of stimulating a T-cell that has been primed (e.g., activated) to recognize an EBV antigen, including situations where the EBV T-cell epitopecontaining peptide is not identical to the EBV antigen with which the T-cell may have been primed in vivo.

[0148] In certain embodiments, an EBNA-1 peptide comprising a CD4+ T cell epitope (also referred to as “CD4 EBNA-1 peptide”) is 14 to 27 amino acids, 15 to 26 amino acids, preferably 20 to 27 amino acids, or 21 to 26 amino acids in length.

[0149] In certain embodiments, an EBNA-1 peptide, an EBNA-3A, and / or an EBNA-3B peptide comprising a CD8+ T cell epitope (also to as “CD8 EBNA-1 peptide,” “CD8 EBNA-3 A peptide,” and “CD8 EBNA-3B peptide,” respectively) is 7 to 14 amino acids, 8 to 13 amino acids, preferably 7 to 13 amino acids, 8 to 13 amino acids, 8 to 12 amino acids, 8 to 11 amino acids, 8 to 10 amino acids, or 9 to 10 amino acids in length.

[0150] Methodologies for the design, production and testing of EBV T-cell epitope-containing peptides and variants functional fragments thereof as provided herein are all available by minor modification to existing knowledge in the art, for example, using conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques, which are explained fully in the literature. See, e.g, Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

[0151] The terms "protein," “polypeptide,” and "peptide" as used herein do not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation, formylation, and addition or deletion of signal sequences. Such terms mean one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds.

[0152] The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polypeptide or nucleic acid present in a living animal is not isolated, but the same polypeptide or nucleic acid, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and / or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.

[0153] The terms "isolated protein," “isolated polypeptide” and “isolated peptide” referred to herein means that a subject protein, polypeptide or peptides that (1) is free of at least some other proteins, polypeptides or peptides with which it would typically be found in nature, (2) is essentially free of other proteins, polypeptides or peptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein or polypeptide with which the isolated protein, isolated peptide or isolated polypeptide may be associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein, polypeptide or peptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin according to any of a number of well known chemistries for artificial peptide and protein synthesis, or any combination thereof. In certain embodiments, the isolated protein, polypeptide or peptide is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0154] A “protein fragment” refers to a peptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally-occurring or recombinantly-produced polypeptide. As used herein, “contiguous amino acids” refers to covalently linked amino acids corresponding to an uninterrupted linear portion of a disclosed amino acid sequence. In certain embodiments, a protein fragment comprises an amino acid chain that is 5 to 100 amino acids long, such as 5 to 50, 7 to 27, 8 to 26, 8 to 13, 14 to 27, or 15 to 26 amino acids long.

[0155] Certain preferred embodiments contemplate wholly artificial chemical synthesis of the herein described peptides (e.g., an EBV T-cell epitope-containing peptide) according to any of established methodologies, such as those described in Amino Acid and Peptide Synthesis (Jones, J., 2002 Oxford Univ. Press USA, New York), Ramakers et al. (2014 Chem. Soc. Rev. 43:2743), Verzele et al. (2013 Chembiochem. 14: 1032), Chandrudu et al. (2013 Molecules 18:4373), and / or Made et al. (2004 Beilstein J. Org. Chem. 10: 1197). For example, manual or preferably automated solid-phase peptide synthesis based on the Merrifield method or other solidphase peptide synthetic techniques and subsequent improvements (e.g., Merrifield, 1963 J. Am. Chem. Soc. 85:2149; Mitchell et al., 1978 J. Org. Chem. 43:2485; Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press; Nilsson et al., 2005 Annu. Rev. Biophys. Biomol. Struct. 34; Schnolzer et al., Int. J. Peptide Res. Therap. 13 (1-2): 31; Li et al. 2013 Molecules 18:9797) are routine in the peptide synthesis art and may be employed to chemically synthesize the herein described peptides.

[0156] Systems for recombinant expression of peptides, polypeptides and proteins are known in the art and may in certain embodiments be used to produce the herein described peptides. For example, certain bacterial expression systems such as E. coli recombinant protein expression systems yield polypeptide products having N- terminal formylated methionine. In some situations, a recombinantly produced peptide may therefore comprise an N-terminal methionine residue (which may be unmodified methionine or formyl-methionine or another methionine analog, variant, mimetic or derivative as provided herein), sometimes referred to as initiator methionine, immediately preceding the desired peptide sequence (e.g., the EBV T-cell epitopecontaining peptide). Thus also contemplated are embodiments in which one or more of the herein described EBV T-cell epitope-containing peptides are generated containing N-terminal methionine (e.g., as methionine or N-formylmethionine) and may be recombinantly expressed according to art-accepted practices in a host cell that also expresses methionine aminopeptidase (MAP), an enzyme that is capable of cleaving the N-terminal methionine to remove it from the nascent polypeptide product. See, e.g., Natarajan et al., 2011 PLoS ONE 6(5): e20176; Shen et al., 1993 Proc. Natl. Acad. Sci. USA 90:8108; Shen et al., 1997 Prot. Eng. 10: 1085. Alternatively, the MAP enzyme itself may be produced recombinantly (e.g., Tsunasawa et al., 1997 J. Biochem. 122:843; Bradshaw et al., 1998 Trends Bioch. Sci. 23:263; Ben-Bassat et al., 1987 J. Bacteriol. 169:751) or obtained commercially (Sigma-Aldrich, St. Louis, MO, e.g., catalog number M6435) and used to remove N-terminal methionine from the present peptides post-synthesis.

[0157] According to certain preferred embodiments an EBV T-cell epitopecontaining peptide may comprise a peptide, polypeptide or peptidomimetic that includes, or that shares close sequence identity to or structural features with, a polypeptide of at least 5, 6, 7, or 8 amino acids and no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids, comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-73, wherein the peptide where the EBV T-cell epitope is present is capable of being specifically recognized by a T-cell reactive with at least one pathogenic EBV strain, preferably an EBV strain that is pathogenic in humans, which pathogenic EBV strains may include but need not be limited to B95-8, AG876, GD-1, Cao, and Raji, preferably B95-8. Assay methods for determining such T-cell reactivity are described herein and are also known generally in the art, except for the identities of EBV T-cell epitope-containing proteins and peptides which are disclosed herein for the first time.

[0158] "Natural or non-natural amino acid" includes any of the common naturally occurring amino acids which serve as building blocks for the biosynthesis of peptides, polypeptides and proteins (e.g., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine(Y)) and also includes modified, derivatized, enantiomeric, rare and / or unusual amino acids, whether naturally occurring or synthetic, for instance, N-formylmethionine, hydroxyproline, hydroxylysine, desmosine, isodesmosine, s-N-methyllysine, s-N-trimethyllysine, methylhistidine, dehydrobutyrine, dehydroalanine, a-aminobutyric acid, P-alanine, y- aminobutyric acid, homocysteine, homoserine, citrulline, ornithine and other amino acids that may be isolated from a natural source and / or that may be chemically synthesized, for instance, as may be found in Proteins, Peptides and Amino Acids Sourcebook (White, J.S. and White, D.C., 2002 Humana Press, Totowa, NJ) or in Amino Acid and Peptide Synthesis (Jones, J., 2002 Oxford Univ. Press USA, New York) or in Unnatural Amino Acids, ChemFiles Vol. 1, No. 5 (2001 Fluka Chemie GmbH; Sigma- Aldrich, St. Louis, MO) or in Unnatural Amino Acids II, ChemFiles Vol. 2, No. 4 (2002 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, MO). Additional descriptions of natural and / or non-natural amino acids may be found, for example, in Kotha, 2003 Acc. Chem. Res. 36:342; Maruoka et al., 2004 Proc. Nat. Acad. Sci. USA 101 :5824; Lundquist et al., 2001 Org. Lett. 3:781; Tang et al., 2002 J. Org. Chem. 67:7819; Rothman et al., 2003 J. Org. Chem. 68:6795; Krebs et al., 2004 Chemistry 10:544; Goodman et al., 2001 Biopolymers 60:229; Sabat et al., 2000 Org. Lett. 2: 1089; Fu et al., 2001 J. Org. Chem. 66:7118; and Hruby et al., 1994 Meths. Mol. Biol. 35:249. The standard three-letter abbreviations and one-letter symbols are used herein to designate natural and non-natural amino acids.

[0159] Peptides disclosed herein may in certain embodiments include L- and / or D- amino acids so long as the biological activity of the peptide is maintained e.g., the EBV T-cell epitope-containing peptide is capable of being recognized in a secondary in vitro immune response by T-cells from a subject infected with an EBV strain, as evidenced by stimulation of the generation of a T-cell immune response indicator as described herein). The peptides also may comprise in certain embodiments any of a variety of known natural and artificial post-translational or post-synthetic covalent chemical modifications by reactions that may include glycosylation (e.g., N-linked oligosaccharide addition at asparagine residues, O-linked oligosaccharide addition at serine or threonine residues, glycation, or the like), fatty acylation, acetylation, formylation, PEGylation, and phosphorylation. Any combination of the peptides of SEQ ID NOS: 1-73 may be employed in certain presently contemplated embodiments of the herein disclosed compositions. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more peptides selected from SEQ ID NOS: 1-40, one or more peptides selected from SEQ ID NOS:41-66, one or more peptides selected from SEQ ID NOS:67-69, one or more peptides selected from SEQ ID NOS:70-73, or combinations thereof. For example, in certain embodiments, the composition comprises, consists essentially of, or consists of at least 5 peptides (e.g., 5 to 40) selected from SEQ ID NOS: 1-40, at least 5 peptides (e.g., 5 to 16) selected from SEQ ID NOS :41-66, at least one (e.g., 1 to 3) selected from SEQ IDNO:67-69, and at least one (e.g., 1 to 4) selected from SEQ ID NOS:70-73. In certain other embodiments, all seventy-three (73) peptides of SEQ ID NOS: 1-73 are present in the composition, including the EBNA-1 CD4+ T cell-epitope-containing peptides of SEQ ID NOS: 1-40 (see Table 1), the EBNA-1 CD8+ T cell-epitope-containing peptides of SEQ ID NOS :41-66 (see Table 2), the EBNA-3A CD8+ T cell-epitope-containing peptides of SEQ ID NOS:67-69 (see Table 3), and the EBNA-3B CD8+ T cell-epitope-containing peptides of SEQ ID NOS:70-73 (see Table 4).

[0160] In certain embodiments, the composition comprises, consists essentially of, or consists of one or more peptides selected from SEQ ID NOS: 1-40, one or more peptides selected from SEQ ID NOS:67-69, and one or more peptides selected from SEQ ID NOS:70-73. For example, in certain embodiments, the composition comprises, consists essentially of, or consists of at least 5 (e.g, 5 to 40) peptides selected from SEQ ID NOS: 1-40, at least one (e.g, 1 to 3) peptides selected from SEQ ID NOS:67- 69, and at least 1 (e.g., 1 to 4) peptides selected from SEQ ID NOS:70-73. In certain other embodiments, the composition comprises, consists essentially of, or consists of all the EBNA-1 CD4+ T cell-epitope-containing peptides of SEQ ID NOS: 1-40 (see Table 1), the EBNA-3A CD8+ T cell-epitope-containing peptides of SEQ ID NOS:67-69 (see Table 3), and the EBNA-3B CD8+ T cell-epitope-containing peptides of SEQ ID NOS:70-73 (see Table 4). In certain embodiments, the composition comprises, consists essentially of, or consists of one or more peptides selected from SEQ ID NOS :41-66, one or more peptides selected from SEQ ID NOS:67-69, and one or more peptides selected from SEQ ID NOS:70-73. For example, in certain embodiments, the composition comprises, consists essentially of, or consists of 5 to 16 peptides selected from SEQ ID NOS:41-66, 1 to 3 peptides selected from SEQ ID NOS:67-69, and 1 to 4 peptides selected from SEQ ID NOS:70-73. In certain other embodiments, the composition comprises, consists essentially of, or consists of all the EBNA-1 CD8+ T cell-epitope-containing peptides of SEQ ID NOS :41-66 (see Table 2), the EBNA-3A CD8+ T cell-epitope-containing peptides of SEQ ID NOS:67-69 (see Table 3), and the EBNA-3B CD8+ T cell-epitope-containing peptides of SEQ ID NOS:70-73 (see Table 4).

[0161] The amounts of the several peptides relative to one another, and the absolute amounts of one or more of said peptides, may be varied according to the assay design and particular technique in which the composition is to be used, as will be familiar to the skilled artisan according to particular immunochemical, immunological and / or biochemical methodologies. By way of illustration and not limitation, in certain embodiments, the composition may comprise at least about one nanogram of each peptide and not more than about 100 nanograms of each peptide; in certain embodiments, the composition may comprise at least about 100, 200, 300, or 400 nanograms and not more than about 500 nanograms of each peptide; in certain embodiments, the composition may comprise at least about 500, 600, 700, 800, or 900 nanograms and not more than about 1000 nanograms of each peptide; in certain embodiments, the composition may comprise at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 micrograms and not more than about 2 micrograms of each peptide, and in certain embodiments, the composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9 micrograms and not more than about 10 micrograms of each peptide.

[0162] II. Methods for Detecting Cell-Mediated Immunity to Epstein-Barr Virus (EBV)

[0163] In another aspect, the present disclosure provides a method for detecting a T-cell-mediated immune response to EBV, comprising:

[0164] (A) contacting in vitro (1) a biological sample obtained from a subject suspected of being infected with EBV, wherein the biological sample comprises T-cells and antigen-presenting cells, and (2) a composition as described above in Section I. EBV Latent Antigens of this disclosure for stimulating a T-cell-mediated immune response to EBV, to obtain a test incubation mixture;

[0165] (B) incubating the test incubation mixture under conditions and for a time sufficient for specific recognition by said T-cells of an EBV T-cell epitope that is present in said peptide composition to stimulate generation of a T-cell immune response indicator; and

[0166] (C) detecting a level of the T-cell immune response indicator in the test incubation mixture, wherein presence of an EBV infection in the subject is indicated by detection in (C) of said level of the T-cell immune response indicator that is increased relative to a control level of the T-cell immune response indicator obtained by incubating the biological sample in a control incubation mixture without the peptide composition for stimulating a T-cell-mediated immune response to EBV or relative to a reference level of the T-cell immune response indicator obtained by incubating a reference incubation mixture of a reference biological sample obtained from a subject known to have not been infected with EBV and the peptide composition for stimulating T-cell-mediated immune response to EBV.

[0167] A “control incubation mixture” refers to an incubation mixture that is otherwise identical to the test incubation mixture of step (A) except that the control incubation mixture does not contain any of the isolated EBNA-1, EBNA-3A and EBNA-3B peptides in the test incubation mixture of step (A).

[0168] A “control level” refers to the level of a T-cell response indicator obtained by incubating a biological sample in a control incubation mixture without a peptide composition for stimulating a T-cell mediated immune response to EBV. The control level represents nonspecific reactivity produced by a biological sample.

[0169] A “reference incubation mixture” refers to an incubation mixture that is otherwise identical to the test incubation mixture of step (A) except that the biological sample (e.g., whole blood) obtained from a subject suspected of being infected with EBV is replaced with a reference biological sample of the same type (e.g., also whole blood) obtained from a subject known to have not been infected with EBV.

[0170] A “reference level” refers to the level of a T-cell response indicator obtained by incubating a reference biological sample obtained from a subject not infected with EBV (e.g., a seronegative subject) in a reference incubation mixture. In certain embodiments, the reference level may be the average level of a T-cell response indicator obtained by incubating reference biological samples obtained from multiple subjects not infected with EBV. In certain preferred embodiments, the EBV infection is a latent EBV infection.

[0171] In certain embodiments, the subject from which a biological sample is obtained is serologically positive for EBV, and the method provided herein is useful in confirming a latent EBV infection.

[0172] In certain embodiments, the subject from which a biological sample is obtained for testing is a subject whose immune system is compromised, such as by immunosuppressive drugs (e.g., treatments used in transplant recipients and autoimmune diseases), viral infections (e.g., HIV infections), or genetic changes. In certain embodiments, the subject from which a biological sample is obtained for testing is a transplant recipient or a subject who plans to receive transplant.

[0173] In certain embodiments, the method disclosed herein may further comprise administering an effective amount of an anti-viral or anti-EBV agent to treat EBV infection in the subject if T-cell-mediated immune response to EBV is detected, especially if the subject whose immune system is compromised. Such agents include: nucleoside analogues (e.g., acyclovir, valacyclovir, ganciclovir, and valgancicilovir), nucleotide analogues (e.g., cidofovir), interferon-alfa, interferon-gamma, pyrophosphate analogues (e.g., phosphonacetic acid), inhibitors of EBV protein kinase BGLF4 (e.g., maribavir), inhibitors of EBV DNA polymerase (e.g., 1-di oxolane thymidine analog, 1- [(2S,4S-2-(hydroxymethyl)-l,3-dioxolan-4-yl]5-vinylpyrimidine-2,4(lH,3H)-dione), thionucleoside derivatives (e.g., KAY-2-41 and KAH-39-149), methylenecyclopropane nucleoside analogues, inhibitors of EBNA1, and verdinexor (see e.g., Andrei et aL, Andrei, Molecules 24(5): 997, 2019).

[0174] “Treating EBV infection,” “EBV infection treatment” or the like refers to reduce EBV viral loads, delay or eliminate onset of one or more symptoms (e.g, fatigue, fever, inflamed throat, swollen lymph nodes in the neck, enlarged spleen, swollen liver, and rash) associated with an EBV infection or EBV-induced malignancies (e.g, B-cell lymphomas, nasopharyngeal carcinoma, central nervous system lymphomas, and post-transplant lymphorproliferative disorder) at a statistically significant manner in a subject compared to the subject when not receiving such a treatment. For use in the methods described herein, a biological sample may be obtained from a subject for determining the presence and level of a T-cell immune response indicator as presently disclosed. A suitable biological sample may comprise, for instance, a whole blood sample (optionally with an anticoagulant), a cellular fraction of whole blood, isolated peripheral blood white cells, or isolated peripheral blood mononuclear cells, any of which may be obtained from a subject (e.g., a human or animal subject and, in preferred embodiments, a human) known or suspected of being infected with EBV. In some embodiments, biological samples may be obtained from a subject with an unknown EBV serological status. In preferred embodiments, biological samples may be obtained from a subject serologically positive for EBV.

[0175] In vitro antigen-specific T-cell responses are typically determined by comparisons of observed T-cell responses according to any of a number of described measurable T-cell functional parameters (e.g., proliferation, cytokine expression, cytokine biosynthesis, cytokine release, altered cell surface marker phenotype, etc. that may be made between T-cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T-cells, when presented by immunocompatible antigen-presenting cells) and T-cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen. A response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.

[0176] The level of a secondary in vitro EBV-specific immune response may be determined by any one of numerous immunological methods described herein and / or routinely practiced in the art. For example, as described in Example 1, an exemplary composition for detection of cell-mediated immunity to EBV comprising EBV T-cell epitope-containing peptides, SEQ ID NOS: 1-73, was shown to be capable of stimulating detectable generation of a T-cell immune response indicator in a test incubation in vitro with biological samples comprising T-cells and antigen-presenting cells from EBV seropositive donors.

[0177] An increased level of a T-cell immune response indicator as provided herein thus may, in certain embodiments, take the form of a statistically significant increase in the level of the indicator that is detectable following incubation of T-cells and antigen presenting cells with the herein described EBV T-cell epitope-containing peptide composition under conditions and for a time sufficient for specific antigen recognition by the T-cells, relative to the level of the indicator that is detectable under appropriate control conditions (e.g., without the EBV peptide composition present) or relative to the level of the indicator detectable following incubation of T-cells and antigen presenting cells of a sample from a reference subject known to have not had any EBV infection with the herein described EBV T-cell epitope-containing peptide composition under conditions and for a time sufficient for specific antigen recognition by the T-cells.

[0178] By way of illustration and not limitation, in preferred embodiments, the T-cell immune response indicator that is generated by stimulation of T-cells with the EBV T-cell epitope-containing peptide composition is at least one T-cell cytokine that is induced, expressed and / or released by the T-cells following incubation in vitro. Preferably, the T-cell cytokine is selected from IL-la, IL-ip, IL-2, IL-10, IL-12, IL-17, TNF-a, TNF-P, and IFN-y. In certain preferred embodiments, released IFN-y is the T- cell immune response indicator that is generated by stimulation of T-cells with the EBV T-cell epitope-containing peptide composition. The contemplated embodiments are not, however, intended to be so limited, and therefore may include any of a wide variety of methodologies for assessing a biological sample as provided herein for its ability to mount a secondary in vitro antigen-specific response to the herein described EBV T-cell epitope-containing peptide composition. Various assay configurations and techniques are known in the art (e.g., Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009)) and may be adapted to the present methods based on the instant disclosure. Levels of cytokines thus may be determined according to methods described herein and practiced in the art, including for example, ELISA (e.g., QUANTIFERON®-ELISA (QIAGEN N.V., Venlo, The Netherlands)), ELISPOT, intracellular cytokine staining, and / or flow cytometry.

[0179] In a preferred embodiment the T-cell immune response indicator is IFN- y released by T-cells of the biological sample during the step of incubating the sample with the herein described EBV T-cell epitope-containing peptide composition, and the level of released IFN-y is determined immunochemically using any of a number of in vitro techniques by which IFN-y is detected by determining detectable specific binding of a binding agent to the T-cell cytokine. The binding agent may thus comprise at least one antibody that binds specifically to the cytokine, which antibody may comprise at least one monoclonal antibody or which may instead comprise a polyclonal antibody. According to certain embodiments, the T-cell immune response indicator is a cytokine that is released by T-cells and is detected by binding to an antibody that is immobilized on a solid phase.

[0180] An exemplary immunometric assay for IFN-y is the QUANTIFERON®- ELISA (QIAGEN N.V., Venlo, The Netherlands), which is established as a gold standard for IFN-y determination in a widely used test for tuberculosis (e.g., Pai et al., 2004 Lancet Infect Dis 4:761) and from which the quantitative detection of IFN-y can be adapted for use with the herein described EBV antigens instead of with tuberculosis antigens. (See also, e.g., Yun et al., 2014 J. Clin. Microbiol. 52:90; Belknap et al., 2014 Clin. Lab. Med. 34:337; Ferguson et al., 2015 Transplantation 99: 1084; Ruan et al. 2014 Clin. Rheumatol. Epub PMID 25376466.)

[0181] A binding partner or an antibody is said to be “immunospecific,” “specific for” or to “specifically bind” an antigen of interest (e.g., a cytokine that is being assayed as a detectable T-cell immune response indicator, for instance, IFN-y) if the antibody reacts at a detectable level with the antigen, preferably with an affinity constant, Ka, of greater than or equal to about 104M’1, or greater than or equal to about 105M’1, greater than or equal to about 106M’1, greater than or equal to about 107M’1, or greater than or equal to 108M’1. Affinity of an antibody for its cognate antigen is also commonly expressed as a dissociation constant KD, and an antibody specifically binds to the antigen of interest if it binds with a KD of less than or equal to 10'4M, less than or equal to about IO'5M, less than or equal to about 10'6M, less than or equal to 10'7M, or less than or equal to 10'8M.

[0182] Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51 :660 (1949)) and by surface plasmon resonance (SPR; BIAcore™, Biosensor, Piscataway, NJ). For surface plasmon resonance, target molecules are immobilized on a solid phase and exposed to a binding partner (or ligand) in a mobile phase running along a flow cell. If ligand binding to the immobilized target occurs, the local refractive index changes, leading to a change in SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rates of change of the SPR signal can be analyzed to yield apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al., Cancer Res. 53:2560-2565 (1993)).

[0183] As used herein, the term “polyclonal antibody” refers to an antibody obtained from a population of antigen-specific antibodies that recognize more than one epitope of the specific antigen. “Antigen” or “immunogen” refers to a peptide, lipid, polysaccharide or polynucleotide which is recognized by the adaptive immune system. Antigens may be self or non-self molecules. Examples of antigens include, but are not limited to, bacterial cell wall components, pollen, and rh factor. The region of an antigen that is specifically recognized by a specific antibody, or by a specific T-cell receptor, is an “epitope” or “antigenic determinant.” A single antigen may have multiple epitopes.

[0184] The term “monoclonal antibody” (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol, 222:581-597 (1991), for example. III. Sample Collection Vessels and EBV Detection Kits

[0185] In another aspect, the present disclosure provides sample collection vessels comprising the compositions for stimulating a T-cell-mediated immune response to EBV as disclosed herein. Preferably, the compositions are coated onto the interior surface of the sample collection vessels.

[0186] The amount of the compositions in the sample collection vessels are sufficient for the intended use of the compositions, that is, to stimulate a T-cell- mediated immune response to EBV. The sample collection vessels may be used to collect samples (e.g., whole blood) of 1 to 10 ml in volume.

[0187] The amounts of isolated EBNA peptides relative to one another, and the absolute amounts of one or more of said peptides in the sample collection vessels may be varied according to the assay design and particular technique. By way of illustration and not limitation, in certain embodiments, the composition in a sample collection vessel may comprise at least about one nanogram of each peptide and not more than about 100 nanograms of each peptide; in certain embodiments the composition may comprise at least about 100, 200, 300, or 400 nanograms and not more than about 500 nanograms of each peptide; in certain embodiments the composition may comprise at least about 500, 600, 700, 800, or 900 nanograms and not more than about 1000 nanograms of each peptide; in certain embodiments the composition may comprise at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 micrograms and not more than about 2 micrograms of each peptide, and in certain embodiments the composition may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9 micrograms and not more than about 10 micrograms of each peptide.

[0188] In a related aspect, the present disclosure provides a kit for detecting an EBV infection (e.g., a latent EBV infection) in a subject that comprises a composition for stimulating a T-cell-mediated immune response to EBV as disclosed herein. In certain embodiments, the composition is stored in, for example coated onto the interior surface of, a sample collection vessel.

[0189] The kit may further comprise one or more of reagents for detecting a level of a T-cell immune response indicator (e.g., IFN-y) via various techniques (e.g., ELISA). Exemplary reagents include a plate or strip coated with an anti-T-cell immune response indicator (e.g., IFN-y) capture antibody, an enzyme (e.g., horse radish peroxidase or alkaline phosphatase)-linked anti-T-cell immune response indicator (e.g., IFN-y) detection antibody for detecting and / or quantifying the T-cell immune response indicator, the T-cell immune response indicator standard (e.g., in lyophilized form), an enzyme substrate solution (e.g., TMB (3,3',5,5'-Tetramethylbenzidine) or ABTS (2-2'- azino-di-(3-ethylbenzthiazoline sulfonic acid)), an enzyme stopping solution that stop enzymatic reaction, and wash buffer.

[0190] The kit may further comprise a positive control, such as a mitogen, to ensure that the detection assay is properly performed, such as using a sufficient amount of the biological sample (e.g., a blood sample) and proper handling of the sample. Exemplary mitogens include phytohemagglutinin (PHA), concanavalin A (conA), and pokeweed mitogen (PWM).

[0191] IV. Vaccines and Their Uses

[0192] In another aspect, the present disclosure provides vaccines that comprise the peptides disclosed herein and methods for stimulating a T cell response in an immunocompromised subject using such vaccines.

[0193] In certain embodiments, the vaccine comprises, consists essentially of, or consists of:

[0194] (i) (A) 1, 2, or 3 isolated CD8 EBNA-3 A peptides each of which comprises:

[0195] (a) a CD8+ T cell epitope, and

[0196] (b) a peptide selected from the group consisting of EBNA-3A peptides having the amino acid sequences set forth in SEQ ID NOS:67-69 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and

[0197] (B) 1, 2, 3, or 4 isolated CD8 EBNA-3B peptides that each of which comprises:

[0198] (a) a CD8+ T cell epitope, and

[0199] (b) a peptide selected from the group consisting of EBNA-3B peptides having the amino acid sequences set forth in SEQ ID NOS:70-73 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; and (ii) one or more pharmaceutically acceptable adjuvants or excipients.

[0200] In certain embodiments, the vaccine comprises, consists essentially of, or consists of:

[0201] (i) (A) 1, 2, or 3 isolated CD8 EBNA-3 A peptides each of which comprises:

[0202] (a) a CD8+ T cell epitope, and

[0203] (b) a peptide selected from the group consisting of EBNA-3A peptides having the amino acid sequences set forth in SEQ ID NOS:67-69 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and

[0204] (ii) one or more pharmaceutically acceptable adjuvants or excipients.

[0205] In certain embodiments, the vaccine comprises, consists essentially of, or consists of:

[0206] (i) (B) 1, 2, 3, or 4 isolated CD8 EBNA-3B peptides that each of which comprises:

[0207] (a) a CD8+ T cell epitope, and

[0208] (b) a peptide selected from the group consisting of EBNA-3B peptides having the amino acid sequences set forth in SEQ ID NOS:70-73 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; and

[0209] (ii) one or more pharmaceutically acceptable adjuvants or excipients.

[0210] In certain embodiments, the vaccine comprises 1, 2, 3, 4, 5, 6, or 7 peptides selected from SEQ ID NOS:67-73.

[0211] In a related aspect, the present disclosure provides a method for stimulating T cell response in an immunocompromised subject (preferably a human) with previous exposure to EBV, comprising administering to the subject an effective amount of the vaccine disclosed herein.

[0212] In certain embodiments, the subject is or was subject to an immunosuppressive therapy. In certain embodiments the subject is a transplant patient that is or was subject to an immunosuppressive therapy.

[0213] In certain embodiments, the vaccine comprises, in addition to one or more peptides disclosed herein, one or more pharmaceutically acceptable excipients, which are inert substances used in vaccines that aid the manufacturing process, or protect, support or enhance stability (e.g., preservatives, stabilizers, and buffers). In an embodiment, the vaccine is buffered at, or adjusted to, between pH 7.0 and 8.0.

[0214] The vaccine is optionally lyophilized in the presence of a stabilizing agent.

[0215] Optionally, the vaccine contains an amount of an adjuvant sufficient to enhance the immune response to the immunogen. As used herein, the term “adjuvant” refers to a chemical substance that improves the immune response to the peptide(s) disclosed herein in the vaccine. Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875.

[0216] Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York).

[0217] As with all immunogenic compositions or vaccines, the immunologically effective amounts of the immunogens must be determined empirically. Factors to be considered include the immunogenicity, whether the immunogen will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, route of administrations and the number of immunizing dosages to be administered.

[0218] The active agent, peptide(s) disclosed herein, can be present in varying concentrations in the vaccines of the present disclosure. Typically, the minimum concentration of the active agent is an amount necessary to achieve its intended use, while the maximum concentration is the maximum amount that will remain in solution or homogeneously suspended within the initial mixture. Generally, it is expected that each dose may comprise l-100mg of peptide antigen(s), optionally 5-50mg or 5-25mg. The vaccine preparations of the present disclosure may be used to stimulate a T cell response in a subject (preferably a human patient), especially in an immunocompromised subject (e.g., a transplant patient) with prior exposure to EBV (e.g., a subject who has been previously infected with EBV). An effective amount of the vaccine may be administered to the subject systemically, including injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral / alimentary, respiratory, genitourinary tracts.

[0219] An “immunocompromised subject” refers to a subject having a weakened immune system due to a medical condition or immunosuppressive medications or treatments. Examples of medical conditions or treatment that may result in immunocompromise include but are not limited to Active treatment for solid tumor and hematologic malignancies, hematologic malignancies, receipt of solid-organ transplant or an islet transplant and taking immunosuppressive therapy, receipt of chimeric antigen receptor (CAR)-T-cell therapy or hematopoietic stem cell transplant, moderate or severe primary immunodeficiency (e.g., common variable immunodeficiency disease, severe combined immunodeficiency, Di George syndrome, Wiskott-Aldrich syndrome), advanced or untreated HIV infection (people with HIV and CD4 cell counts less than 200 / mm3, history of an AIDS-defining illness without immune reconstitution, or clinical manifestations of symptomatic HIV), active treatment with high-dose corticosteroids (z.e., 20 or more mg of prednisone or equivalent per day when administered for 2 or more weeks), alkylating agents, antimetabolites, transplant-related immunosuppressive drugs, cancer chemotherapeutic agents classified as severely immunosuppressive, tumor necrosis factor (TNF) blockers, and other biologic agents that are immunosuppressive or immunomodulatory. Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to”. By “consisting of’ is meant including, and typically limited to, whatever follows the phrase “consisting of.” By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are required and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0220] In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%.

[0221] SEQUENCES

[0222] SEQ ID NO:1 (peptide 1 - CD4 EBNA-1) MSDEGPGTGPGNGLGEKGDTSGPEG

[0223] SEQ ID NO:2 (peptide 2 - CD4 EBNA-1) GNGLGEKGDTSGPEGSGGSGPQRRG

[0224] SEQ ID NO:3 (peptide 3 - CD4 EBNA-1) SGPEGSGGSGPQRRGGDNHGRGRGR

[0225] SEQ ID NO:4 (peptide 4 - CD4 EBNA-1) PQRRGGDNHGRGRGRGRGRGGGRPG

[0226] SEQ ID NO:5 (peptide 5 - CD4 EBNA-1) RGRGRGRGRGGGRPGAPGGSGSGPR

[0227] SEQ ID NO:6 (peptide 6 - CD4 EBNA-1) GGRPGAPGGSGSGPRHRDGVRRPQK

[0228] SEQ ID NO:7 (peptide 7 - CD4 EBNA-1) GSGPRHRDGVRRPQKRPSCIGCKGT

[0229] SEQ ID NO:8 (peptide 8 - CD4 EBNA-1) RRPQKRPSCIGCKGTHGGTGAGAGA

[0230] SEQ ID NO:9 (peptide 9 - CD4 EBNA-1) GCKGTHGGTGAGAGAGGAGAGGAGA

[0231] SEQ ID NQ:10 (peptide 10 - CD4 EBNA-1) GAGAGGGGRGRGGSGGRGRGGSGGR

[0232] SEQ ID NO:11 (peptide 11 - CD4 EBNA-1) RGGSGGRGRGGSGGRGRGGSGGRRG

[0233] SEQ ID NO:12 (peptide 12 - CD4 EBNA-1)

[0234] GSGGRGRGGSGGRRGRGRERARGGS

[0235] SEQ ID NO:13 (peptide 13 - CD4 EBNA-1) GGRRGRGRERARGGSRERARGRGRG SEQ ID NO:14 (peptide 14 - CD4 EBNA-1) ARGGSRERARGRGRGRGEKRPRSPS

[0236] SEQ ID NO:15 (peptide 15 - CD4 EBNA-1) GRGRGRGEKRPRSPSSQSSSSGSPP

[0237] SEQ ID NO:16 (peptide 16 - CD4 EBNA-1) PRSPSSQSSSSGSPPRRPPPGRRPF

[0238] SEQ ID NO:17 (peptide 17 - CD4 EBNA-1) SGSPPRRPPPGRRPFFHPVGEADYF

[0239] SEQ ID NO:18 (peptide 18 - CD4 EBNA-1) GRRPFFHPVGEADYFEYHQEGGPDG

[0240] SEQ ID NO:19 (peptide 19 - CD4 EBNA-1) EADYFEYHQEGGPDGEPDVPPGAIE

[0241] SEQ ID NQ:20 (peptide 20 - CD4 EBNA-1) GGPDGEPDVPPGAIEQGPADDPGEG

[0242] SEQ ID NO:21 (peptide 21 - CD4 EBNA-1) PGAIEQGPADDPGEGPSTGPRGQGD

[0243] SEQ ID NO:22 (peptide 22 - CD4 EBNA-1)

[0244] DPGEGPSTGPRGQGDGGRRKKGGWF

[0245] SEQ ID NO:23 (peptide 23 - CD4 EBNA-1)

[0246] RGQGDGGRRKKGGWFGKHRGQGGSN

[0247] SEQ ID NO:24 (peptide 24 - CD4 EBNA-1)

[0248] KGGWFGKHRGQGGSNPKFENIAEGL

[0249] SEQ ID NO:25 (peptide 25 - CD4 EBNA-1)

[0250] GQGGSNPKFENIAEGLRALLARSHVE

[0251] SEQ ID NO:26 (peptide 26 - CD4 EBNA-1)

[0252] IAEGLRALLARSHVERTTDEGTWVA

[0253] SEQ ID NO:27 (peptide 27 - CD4 EBNA-1)

[0254] RSHVERTTDEGTWVAGVFVYGGSKT

[0255] SEQ ID NO:28 (peptide 28 - CD4 EBNA-1)

[0256] GTWVAGVFVYGGSKTSLYNLRRGTA

[0257] SEQ ID NO:29 (peptide 29 - CD4 EBNA-1)

[0258] GGSKTSLYNLRRGTALAIPQCRLTP SEQ ID NQ:30 (peptide 30 - CD4 EBNA-1)

[0259] RRGTALAIPQCRLTPLSRLPFGMAP

[0260] SEQ ID NO:31 (peptide 31 - CD4 EBNA-1)

[0261] CRLTPLSRLPFGMAPGPGPQPGPLR

[0262] SEQ ID NO:32 (peptide 32 - CD4 EBNA-1)

[0263] FGMAPGPGPQPGPLRESIVCYFMVF

[0264] SEQ ID NO:33 (peptide 33 - CD4 EBNA-1)

[0265] PGPLRESIVCYFMVFLQTHIFAEVL

[0266] SEQ ID NO:34 (peptide 34 - CD4 EBNA-1)

[0267] YFMVFLQTHIFAEVLKDAIKDLVMT

[0268] SEQ ID NO:35 (peptide 35 - CD4 EBNA-1)

[0269] FAEVLKDAIKDLVMTKPAPTCNIRV

[0270] SEQ ID NO:36 (peptide 36 - CD4 EBNA-1)

[0271] DLVMTKPAPTCNIRVTVCSFDDGVD

[0272] SEQ ID NO:37 (peptide 37 - CD4 EBNA-1)

[0273] CNIRVT VC SFDDGVDLPPWFPPMVE

[0274] SEQ ID NO:38 (peptide 38 - CD4 EBNA-1)

[0275] DDGVDLPPWFPPMVEGAAAEGDDGD

[0276] SEQ ID NO:39 (peptide 39 - CD4 EBNA-1) PPMVEGAAAEGDDGDDGDEGGDGDE

[0277] SEQ ID NQ:40 (peptide 40 - CD4 EBNA-1)

[0278] GDDGDDGDEGGDGDEGEEGQE

[0279] SEQ ID NO:41 (peptide 41 - CD8 EBNA-1) MSDEGPGTG

[0280] SEQ ID NO:42 (peptide 42 - CD8 EBNA-1) GPGTGPGNGL

[0281] SEQ ID NO:43 (peptide 43 - CD8 EBNA-1) RPQKRPSCI SEQ ID NO:44 (peptide 44 - CD8 EBNA-1) RPRSPSSQS

[0282] SEQ ID NO:45 (peptide 45 - CD8 EBNA-1) RPPPGRRPF

[0283] SEQ ID NO:46 (peptide 46 - CD8 EBNA-1) HPVGEADYF

[0284] SEQ ID NO:47 (peptide 47 - CD8 EBNA-1) RRKKGGWFGK

[0285] SEQ ID NO:48 (peptide 48 - CD8 EBNA-1) FENIAEGL

[0286] SEQ ID NO:49 (peptide 49 - CD8 EBNA-1) TTDEGTWVA

[0287] SEQ ID NQ:50 (peptide 50 - CD8 EBNA-1) AGVFVYGGSK

[0288] SEQ ID NO:51 (peptide 51 - CD8 EBNA-1) FVYGGSKTSLY

[0289] SEQ ID NO:52 (peptide 52 - CD8 EBNA-1) KTSLYNLRR

[0290] SEQ ID NO:53 (peptide 53 - CD8 EBNA-1) YNLRRGTAL

[0291] SEQ ID NO:54 (peptide 54 - CD8 EBNA-1) IPQCRLTPL

[0292] SEQ ID NO:55 (peptide 55 - CD8 EBNA-1) CRLTPLSRL

[0293] SEQ ID NO:56 (peptide 56 - CD8 EBNA-1) GPGPQPGPL

[0294] SEQ ID NO:57 (peptide 57 - CD8 EBNA-1) RESIVCYFM

[0295] SEQ ID NO:58 (peptide 58 - CD8 EBNA-1) SIVCYFMVF

[0296] SEQ ID NO:59 (peptide 59 - CD8 EBNA-1) YFMVFLQTHIF

[0297] SEQ ID NQ:60 (peptide 60 - CD8 EBNA-1) FMVFLQTHI

[0298] SEQ ID NO:61 (peptide 61 - CD8 EBNA-1) FLQTHIFAEV

[0299] SEQ ID NO:62 (peptide 62 - CD8 EBNA-1) VLKDAIKDL

[0300] SEQ ID NO:63 (peptide 63 - CD8 EBNA-1) AIKDLVMTK

[0301] SEQ ID NO:64 (peptide 64 - CD8 EBNA-1) CNIRVTVCSF SEQ ID NO:65 (peptide 65 - CD8 EBNA-1) DLPPWFPPM

[0302] SEQ ID NO:66 (peptide 66 - CD8 EBNA-1) FPPMVEGAAA

[0303] SEQ ID NO:67 (peptide 67 - CD8 EBNA-3A) FLRGRAYGL

[0304] SEQ ID NO:68 (peptide 68 - CD8 EBNA-3A) RPPIFIRRL

[0305] SEQ ID NO:69 (peptide 69 - CD8 EBNA-3A) QAKWRLQTL

[0306] SEQ ID NQ:70 (peptide 70 - CD8 EBNA-3B) AVFDRKSDAK

[0307] SEQ ID NO:71 (peptide 71 - CD8 EBNA-3B) IVTDFSVIK

[0308] SEQ ID NO:72 (peptide 72 - CD8 EBNA-3B) VSFIEFVGW

[0309] SEQ ID NO:73 (peptide 73 - CD8 EBNA-3B) HRCQAIRK

[0310] SEQ ID NO:74 (EBNA1 (Epstein-Barr virus strain B95-8; UniProtKB P03211))

[0311] MSDEGPGTGPGNGLGEKGDTSGPEGSGGSGPQRRGGDNHGRGRGRGRGRGG

[0312] GRPGAPGGSGSGPRHRDGVRRPQKRPSCIGCKGTHGGTGAGAGAGGAGAGGA

[0313] GAGGGAGAGGGAGGAGGAGGAGAGGGAGAGGGAGGAGGAGAGGGAGAG

[0314] GGAGGAGAGGGAGGAGGAGAGGGAGAGGGAGGAGAGGGAGGAGGAGAG

[0315] GGAGAGGAGGAGGAGAGGAGAGGGAGGAGGAGAGGAGAGGAGAGGAGA

[0316] GGAGGAGAGGAGGAGAGGAGGAGAGGGAGGAGAGGGAGGAGAGGAGGA

[0317] GAGGAGGAGAGGAGGAGAGGGAGAGGAGAGGGGRGRGGSGGRGRGGSGG

[0318] RGRGGSGGRRGRGRERARGGSRERARGRGRGRGEKRPRSPSSQSSSSGSPPRRP

[0319] PPGRRPFFHPVGEADYFEYHQEGGPDGEPDVPPGAIEQGPADDPGEGPSTGPRG

[0320] QGDGGRRKKGGWFGKHRGQGGSNPKFENIAEGLRALLARSHVERTTDEGTW

[0321] VAGVFVYGGSKTSLYNLRRGTALAIPQCRLTPLSRLPFGMAPGPGPQPGPLRESI

[0322] VCYFMVFLQTHIFAEVLKDAIKDLVMTKPAPTCNIRVTVCSFDDGVDLPPWFPP MVEGAAAEGDDGDDGDEGGDGDEGEEGQE

[0323] SEQ ID NO:75 (EBNA3A (Epstein-Barr virus strain B95-8; UniProtKB P12977))

[0324] MDKDRPGPPALDDNMEEEVPSTSVVQEQVSAGDWENVLIELSDSSSEKEAEDA HLEPAQKGTKRKRVDHDAGGSAPARPMLPPQPDLPGREAILRRFPLDLRTLLQ AIGAAATRIDTRAIDQFFGSQISNTEMYIMYAMAIRQAIRDRRRNPASRRDQAK WRLQTLAAGWPMGYQAYSSWMYSYTDHQTTPTFVHLQATLGCTGGRRCHVT FSAGTFKLPRCTPGDRQWLYVQSSVGNIVQSCNPRYSIFFDYMAIHRSLTKIWE EVLTPDQRVSFMEFLGFLQRTDLSYIKSFVSDALGTTSIQTPWIDDNPSTETAQA WNAGFLRGRAYGIDLLRTEGEHVEGATGETREESEDTESDGDDEDLPCIVSRG GPKVKRPPIFIRRLHRLLLMRAGKRTEQGKEVLEKARGSTYGTPRPPVPKPRPE

[0325] VPQSDETATSHGSAQVPEPPTIHLAAQGMAYPLHEQHGMAPCPVAQAPPTPLPP VSPGDQLPGVFSDGRVACAPVPAPAGPIVRPWEPSLTQAAGQAFAPVRPQHMP VEPVPVPTVALERPVYPKPVRPAPPKIAMQGPGETSGIRRARERWRPAPWTPNP PRSPSQMSVRDRLARLRAEAQVKQASVEVQPPQLTQVSPQQPMEGPLVPEQQ

[0326] MFPGAPFSQVADVVRAPGVPAMQPQYFDLPLIQPISQGAPVAPLRASMGPVPPV PATQPQYFDIPLTEPINQGASAAHFLPQQPMEGPLVPEQWMFPGAALSQSVRPG VAQSQYFDLPLTQPINHGAPAAHFLHQPPMEGPWVPEQWMFQGAPPSQGTDV VQHQLDALGYTLHGLNHPGVPVSPAVNQYHLSQAAFGLPIDEDESGEGSDTSE PCEALDLSIHGRPCPQAPEWPVQEEGGQDATEVLDLSIHGRPRPRTPEWPVQGE GGQNVTGPETRRVVVSAVVHMCQDDEFPDLQDPPDEA

[0327] SEQ ID NO:76 (EBNA3B (Epstein-Barr virus strain B95-8; UniProtKB P03203))

[0328] MKKAWLSRAQQADAGGASGSEDPPDYGDQGNVTQVGSEPISPEIGPFELSAAS EDDPQSGPVEENLDAAAREEEEPHEQEHNGGDDPLDVHTRQPRFVDVNPTQAP VIQLVHAVYDSMLQSDLRPLGSLFLEQNLNIEEFIWMCMTVRHRCQAIRKKPLP IVKQRRWKLLSSCRSWRMGYRTHNLKVNSFESGGDNVHPVLVTATLGCDEGT RHATTYSAGIVQIPRISDQNQKIETAFLMARRARSLSAERYTLFFDLVSSGNTLY AIWIGLGTKNRVSFIEFVGWLCKKDHTHIREWFRQCTGRPKAAKPWLRAHPVA IPYDDPLTNEEIDLAYARGQAMNIEAPRLPDDPIIVEDDDESEEIEAESDEEEDKS GMESLKNIPQTLPYNPTVYGRPAVFDRKSDAKSTKKCRAIVTDFSVIKAIEEEHR KKKAARTEQPRATPESQAPTVVLQRPPTQQEPGPVGPLSVQARLEPWQPLPGP QVTAVLLHEESMQGVQVHGSMLDLLEKDDEVMEQRVMATLLPPVPQQPRAG RRGPCVFTGDLGIESDEPASTEPVHDQLLPAPGPDPLEIQPLTSPTTSQLSSSAPS CAQTPWPVVQPSQTPDDPTKQSRPPETAAPRQWPMPLRPIPMRPLRMQPIPFNH PVGPTPHQTPQVEITPYKPTWAQIGHIPYQPTPTGPATMLLRQWAPATMQTPPR APTPMSPPEVPPVPRQRPRGAPTPTPPPQVPPVPRQRPRGAPTPTPPPQVLPTPM

[0329] QLALRAPAGQQGPTKQILRQLLTGGVKKGRPSLKLQAALERQAAAGWQPSPG SGTSDKIVQAPIFYPPVLQPIQVMGQGGSPTAMAASAVTQAPTEYTRERRGVGP MPPTDIPPSKRAKIEAYTEPEMPHGGASHSPVVILENVGQGQQQTLECGGTAKQ ERDMLGLGDIAVSSPSSSETSNDE

[0330] EXAMPLES

[0331] EXAMPLE 1 : IDENTIFICATION AND TESTING OF EBNA-1, EBNA-3 A, AND EBNA- 3B PEPTIDES

[0332] Epstein-Barr virus (EBV) is a gamma herpes virus that produces both acute and latent stages of infection in humans. During EBV latency, only a portion of viral genes are expressed, including EBV nuclear antigens EBNA-1, EBNA-3 A, and EBNA-3B. The EBNA-1 protein is the most prevalent protein in latent infection and the only protein expressed in all EBV-related malignancies. CD8+ T-cells targeting EBNA- 3A / 3B represent the dominant immune response in latent infection whereas EBNA-1- specific CD8+ T cell responses are sub-dominant. The present inventors used CD4+ T cell-specific EBNA-1 peptides, CD8+ T cell-specific EBNA-1 peptides, CD8+ T cellspecific EBNA-3 A peptides, CD8+ T cell-specific EBNA-3B peptides, or combination thereof to induce robust activation of T-cells from subjects known to have or suspected of being infected with EBV.

[0333] Described here is the identification and testing of seventy-three (73) CD4+ T cell-specific and CD8+ T cell-specific EBV peptides spanning latent proteins EBNA-1, EBNA-3 A, and EBNA-3B, derived from the EBV strain, B95-8, which is highly prevalent worldwide in latent EBV infections. The peptides may be added to blood collection tubes or dried on the walls of blood collection tubes. Once whole blood is added to the peptide-spiked or peptide-coated tubes, the peptides elicit a cell-mediated immune response to the EBV antigens.

[0334] Forty (40) overlapping CD4+ T cell-specific peptides spanning EBNA-1 (with the exception of the GAr region) were designed using the PEPscreen Library design tool from (Sigma Aldrich, St. Louis, MO). Each consists of a 25-mer (with the exceptions of peptide-40, which is 21 amino acids in length, and peptide-25, which is 26 amino acids in length) with a 15 amino acid overlap (see Table 1).

[0335] Table 1. EBNA-1 CD4+ T cell-epitope-containing peptides Twenty-six (26) CD8+ T cell-specific peptides spanning EBNA-1, ranging in length between 8-11 amino acids, were identified using the binding prediction tool NetMHC 4.1 (see Table 2). Additional investigation was carried out to determine potential epitopes that could be used for targeting cell-mediated immune responses to EBV, including literature searches and the binding prediction tool NetMHC 4.1. It was determined that CD8+ T cell-specific peptides could be generated from EBNA-3 A and EBNA-3B. The resulting sequences are shown in Tables 3 and 4.

[0336] Table 2. EBNA-1 CD8+ T cell-epitope-containing peptides Table 3. CD8+ T cell-specific EBNA-3 A peptides

[0337] Table 4. CD8+ T cell-specific EBNA-3B peptides

[0338] The peptides were tested for their ability to elicit a T-cell-mediated response by measuring IFN-y in plasma after secondary in vitro T-cell activation with Epstein-Barr virus (EBV) peptides derived from one or more of EBNA-1, EBNA-3A, and EBNA- 3B. Whole blood (1 mL) from 10 EBV seropositive donors in study 1 (Figure 1) or 11 EBV seropositive donors in study 2 (Figure 2) was incubated with different combinations of EBV peptides (SEQ ID NOS: 1-73) at 37°C ± 1°C after which plasma was isolated for quantitation of IFN-y by QU ANTIFERON® -ELISA (QIAGEN N.V., Venlo, The Netherlands) according to the manufacturer’s instructions.

[0339] Figure 1 showed that the CD4 EBNA-1 and CD8 EBNA-1, -3A and -3B antigen peptides elicited specific T-cell mediated immune responses, and that the combination of both CD4 and CD8 antigen peptides provided a synergistic response across a population of donors. Specifically, CD4 EBNA-1 and CD8 EBNA-1, -3A and -3B antigen peptides combined elicited a larger median response than either CD4 EBNA-1 or CD8 peptides alone.

[0340] Figure 2 shows how each donor responded to different combinations of EBV peptides. In this figure, “CD4 EBNA-1” refers to the combination of the 40 peptides shown in Table 1, “CD8 EBNA-1” refers to the combination of the 26 CD8 EBNA-1 peptides shown in Table 2, “CD8 EBNA-3a” refers to the combination of the 3 CD8 EBNA-3a peptides shown in Table 3, and “CD8 EBNA-3b” refers to the combination of the 4 CD8 EBNA-3b peptides shown in Table 4. Combining different peptide pools (Pools A-F) provided a synergistic response not only across a population of donors as shown in Figure 1 but within donors (see Donors 1-11, Figure 2). For example, referring to the results for donor 1, when the CD4 EBNA-1 antigen peptides (Pool C, Donor 1, IU / mL=0.57) were combined with the CD8 EBNA-1, -3A and -3B antigen peptides (Pool F, Donor 1, IU / mL-0.70), the resulting T-cell mediated response was greater as indicated by greater IFN-g levels elicited (Pool A, Donor 1, IU / mL=1.77). Peptide pools from individual antigens that were sufficient for detection in a donor (using a cut off of 0.20 lU / mL) were identified at the bottom of the table in Figure 2. The cut off value of 0.20 lU / mL was selected as a conservative estimate that was above the average responses from seronegative donors. In this set of donors, 6 out of 11 had a response above cut off (z.e., >0.20 lU / mL) to CD4 EBNA-1 alone (Pool C highlighted in gray); 3 out of 11 had a measurable response to CD8 EBNA-1 alone (Pool D highlighted in gray); 8 out of 11 had a measurable response to CD8 EBNA-3 A / B alone (Pool E highlighted in gray). 11 out of 11 (all donors) responded to the CD4 EBNA-1 and CD8 EBNA-1, -3A and -3B antigen peptide combination (Pool A highlighted in gray). Thus, by combining peptide pools from different antigens, preferably including both CD4+ and CD8+ peptides, a measurable EBV specific T-cell mediated response was detected in every serologically positive EBNA-1 IgG donor (see Pools A, B and F).

[0341] EXAMPLE 2: ADDITIONAL TESTING OF EBNA-1, EBNA-3 A, AND EBNA-3B PEPTIDES

[0342] In this Example, experiments were performed as described in Example 1 with the following differences:

[0343] (1) Each peptide is at Img / tube for Subpools 1-27 instead of 0.5 mg / tube used in Example 1. However, for the ‘parent’ Pools A-H, the 0.5 mg / tube was used.

[0344] (2) The positive donors used in these experiments were chosen if they were positive for both EBV EBNA-1 and EBV VC A IgG instead of only by EBV EBNA-1 IgG positivity in Example 1.

[0345] (3) The arbitrary cut-off used to determine positivity in Figures 3 and 4 is >0.10 lU / mL instead of a cut-off of >0.20 lU / mL used in Figures 1 and 2. These data show that not all antigens as disclosed were needed for a positive result; the use of subsets of these antigens was sufficient. For example, in Subpools 21, 22, and 23, only peptides having SEQ ID NOS:67 and 72 were used as peptides of a CD8 EBNA-3 A peptide and a CD8 EBNA-3B peptide, respectively, to achieve 100% in detecting EBV positive donors. In addition, in Pool A and Subpools 21, 22, and 23, only subsets of peptides having SEQ ID NOS: 1-40 were used as CD4 EBNA-1 peptides, and in Pool A and Subpools 22 and 23, only subsets of peptides having SEQ ID NOS:41-66 were used as CD8 EBNA-1 peptides.

[0346] The data also show that for a positive result, antigens belonging to all four different groups (z.e., isolated CD4 EBNA-1 peptides, isolated CD8 EBNA-1 peptides, isolated CD8 EBNA-3 A peptides, and isolated CD8 EBNA-3B peptides) were not necessarily needed for a positive result. For example, in Subpool 21 (which achieved 100% detection of EBV positive donors), only subsets of CD4 EBNA-1 peptides, CD8 EBNA-3 A peptides, and CD8 EBNA-3B peptides were used.

[0347] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications, including U.S. Provisional Patent Application No. 63 / 596,860, filed November 7, 2023, referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0348] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMSWhat is claimed is:

1. A composition for stimulating a T-cell-mediated immune response to Epstein-Barr virus (EBV), comprising:(i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 isolated CD4 EBNA-1 peptides, each of which comprises:(a) a CD4+ T cell epitope, and(b) a peptide selected from the group consisting of EBNA-1 peptides having the amino acid sequences set forth in SEQ ID NOS: 1-40 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40; and / or(ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 isolated CD8 EBNA-1 peptides each of which comprises(a) a CD8+ T cell epitope, and(b) a peptide selected from the group consisting of EBNA-1 peptides having the amino acid sequences set forth in SEQ ID NOS :41-66 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS :41-66; and / or(iii) 1, 2, or 3 isolated CD8 EBNA-3A peptides each of which(a) a CD8+ T cell epitope, and(b) a peptide selected from the group consisting of EBNA-3A peptides having the amino acid sequences set forth in SEQ ID NOS:67-69 and variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and / or(iv) 1, 2, 3, or 4 isolated CD8 EBNA-3B peptides that each of which comprises(a) a CD8+ T cell epitope, and(b) a peptide selected from the group consisting of EBNA-3B peptides having the amino acid sequences set forth in SEQ ID NOS:70-73 and variantsthereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73; wherein the composition, after being contacted with whole blood obtained from a subject known to have been infected with EBV, is capable of eliciting an in vitro immune response by T-cells.

2. The composition of claim 1, comprising:(1) one or more isolated CD4 EBNA-1 peptides of (i),(2) one or more isolated CD4 EBNA-1 peptides of (i) and one or more isolated CD8 EBNA-1 peptides of (ii),(3) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3A peptides of(iii),(4) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3B peptides of(iv),(5) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv),(6) one or more isolated CD4 EBNA-1 peptides of (i) and one or more isolated CD8 EBNA-3 A peptides of (iii),(7) one or more isolated CD4 EBNA-1 peptides of (i) and one or more isolated CD8 EBNA-3B peptides of (iv),(8) one or more isolated CD4 EBNA-1 peptides of (i), one or more isolated CD8 EBNA-3 A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv),(9) one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3 A peptides of (iii),(10) one or more isolated CD8 EBNA-1 peptides of (ii), and one or more isolated CD8 EBNA-3B peptides of (iv),(11) one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3 A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv), or(12) one or more isolated CD8 EBNA-3A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv).

3. The composition of claim 1, comprising: one or more isolated CD4 EBNA-1 peptides of (i), optionally one or more isolated CD8 EBNA-1 peptides of (ii), one or more isolated CD8 EBNA-3 A peptides of (iii), and one or more isolated CD8 EBNA-3B peptides of (iv).

4. The composition of any one of claims 1 to 3, wherein the one or more isolated CD4 EBNA-1 peptides of (i) comprise a sequence selected from the group consisting of SEQ ID NOS: 1-40, the one or more isolated CD8 EBNA-1 peptides of (ii) comprise a sequence selected from the group consisting of SEQ ID NOS:41-66, the one or more isolated CD8 EBNA-3 A peptides of (iii) comprise a sequence selected from the group consisting of SEQ ID NOS:67-69, and the one or more isolated CD4 EBNA-1 peptides of (iv) comprise a sequence selected from the group consisting of SEQ ID NOS:70-73.

5. The composition of any one of claims 1 to 4, wherein the number of the one or more isolated CD4 EBNA-1 peptides of (i), if any is present, is at least 5, preferably at least 10 or at least 15, and the number of the one or more isolated CD8 EBNA-1 peptides of (ii), if any is present, is at least 5.

6. The composition of any one of claims 2 to 5, comprising at least 30 or at least 35 isolated CD4 EBNA-1 peptides of (i).

7. The composition of claim 1, comprising:(i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1-40, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40, and(ii) 5 to 26 isolated CD8 EBNA-1 peptides as set forth in SEQ ID NOS :41-66, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS :41-66;(iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and(iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73.

8. The composition of claim 1, comprising:(i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1-40, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS: 1-40;(iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:67-69; and(iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73, or variants thereof having at least 80% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NOS:70-73.

8. The composition of claim 1, comprising:(i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1- 40,(ii) 5 to 26 isolated CD8 EBNA-1 peptides as set forth in SEQ ID NOS:41-66,(iii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69, and(iv) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73.

9. The composition of claim 1, comprising:(i) 5 to 40 isolated CD4 EBNA-1 peptides as set forth in SEQ ID NOS: 1-(ii) 1 to 3 isolated CD8 EBNA-3 A peptides as set forth in SEQ ID NOS:67-69; and(iii) 1 to 4 isolated CD8 EBNA-3B peptides as set forth in SEQ ID NOS:70-73.

10. The composition of claim 1, comprising, consisting essentially of, or consisting of:(1) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1- 31, 35-36, and 38-40; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:41- 57 and 59-66; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;(2) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16- 20, 21-25, and 26-30; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO:72;(3) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16- 20 and 26-30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS: 57-62; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;(4) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16- 30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:57-62; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;(5) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16- 20, 26-31, 35-36, and 38-40; isolated CD8 EBNA-3 A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO:72;(6) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16- 20 and 26-30; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO:72;(7) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS:26- 30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:46-51; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NO: 72;(8) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS:26- 30; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:46-51; isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67, and isolated CD8 EBNA-3B peptide of (iv) as set forth in SEQ ID NOS:70 and 72;(9) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 16- 20 and 26-30; and isolated CD8 EBNA-3A peptide of (iii) as set forth in SEQ ID NO:67;(10) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1- 31, 35-36, and 38-40; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:41- 57 and 59-66;(11) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1- 31, 35-36, and 38-40;(12) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS:26- 30;(13) isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;(14) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1- 40; isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:41-66; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;(15) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1- 40; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;(16) isolated CD8 EBNA-1 peptide of (ii) as set forth in SEQ ID NOS:41- 66; isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73;(17) isolated CD8 EBNA-3A peptides of (iii) as set forth in SEQ ID NOS:67-69, and isolated CD8 EBNA-3B peptides of (iv) as set forth in SEQ ID NOS:70-73; or(18) isolated CD4 EBNA-1 peptides of (i) as set forth in SEQ ID NOS: 1-40.

11. The composition of any one of claims 1 to 10, wherein the isolatedCD4 EBNA-1 peptide of (i), if any is present, is 14 to 27 amino acids in length, preferably 15 to 26 amino acids in length.

12. The composition of any one of claims 1 to 11, wherein the isolated CD8 EBNA-1 peptide of (ii), the isolated CD8 EBNA-3A peptide of (iii), and / or the isolated CD8 EBNA-3B peptide of (iv), if any is present, is 7 to 14 amino acids, preferably 8 to 13 amino acids in length.

13. The composition of any one of claims 1 to 12, wherein at least one of:(a) the composition comprises at least about one nanogram of each peptide and not more than about 100 nanograms of each peptide,(b) the composition comprises at least about 100, 200, 300, or 400 nanograms and not more than about 500 nanograms of each peptide,(c) the composition comprises at least about 500, 600, 700, 800, or 900 nanograms and not more than about 1000 nanograms of each peptide,(d) the composition comprises at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 micrograms and not more than about 2 micrograms of each peptide, or(e) the composition comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9 micrograms and not more than about 10 micrograms of each peptide.

14. A method for detecting a T-cell-mediated immune response to EBV, comprising:(A) contacting in vitro (1) a biological sample obtained from a subject suspected of being infected with EBV, wherein the biological sample comprises T-cells and antigen-presenting cells, and (2) a peptide composition for stimulating a T-cell-mediated immune response to EBV, to obtain a test incubation mixture;(B) incubating the test incubation mixture under conditions and for a time sufficient for specific recognition by said T-cells of an EBV T-cell epitope that is present in said peptide composition to stimulate generation of a T-cell immune response indicator; and(C) detecting a level of the T-cell immune response indicator in the test incubation mixture, wherein presence of an EBV infection in the subject is indicated by detection in (C) of said level of the T-cell immune response indicator that is increasedrelative to a control level of the T-cell immune response indicator obtained by incubating the biological sample in a control incubation mixture without the peptide composition for stimulating a T-cell-mediated immune response to EBV, or relative to a reference level of the T-cell immune response indicator obtained by incubating a reference incubation mixture of a reference biological sample obtained from a subject known to have not been infected with EBV and the peptide composition for stimulating T-cell-mediated immune response to EBV, and wherein the peptide composition for stimulating the T-cell-mediated immune response to EBV is the composition of any one of claims 1 to 13.

15. The method of claim 14, wherein the EBV infection is a latent EBV infection.

16. The method of claim 14 or claim 15, wherein the biological sample comprises whole blood, a cellular fraction of whole blood, isolated peripheral blood white cells, or isolated peripheral blood mononuclear cells.

17. The method of claim 16, wherein the biological sample comprises whole blood.

18. The method of any one of claims 14 to 17, wherein the T-cell immune response indicator is interferon-gamma (IFN-y).

19. The method of claim 18, wherein the IFN-y is detected as soluble IFN- y released by the T-cells.

20. The method of any one of claims 14-17, wherein the T-cell immune response indicator is a T-cell cytokine.

21. The method of claim 20, wherein the T-cell cytokine is TNF-a, IL-2, IL- la, IL-ip, IL- 10, IL- 12, IL- 17, or TNF-p.

22. The method of claim 20 or claim 21, wherein the T-cell cytokine is detected as a soluble T-cell cytokine released by the T-cells.

23. The method of any one of claims 14-22, wherein the T-cell immune response indicator is detected by determining detectable specific binding of a binding agent to the T-cell immune response indicator.

24. The method of claim 23, wherein the binding agent comprises at least one antibody that binds specifically to the T-cell immune response indicator.

25. The method of claim 24, wherein the at least one antibody is a monoclonal antibody or a polyclonal antibody.

26. The method of claim 24 or claim 25, wherein the at least one antibody is immobilized on a solid phase.

27. The method of any one of claims 14-26, wherein the T-cell immune response indicator is detected by enzyme-linked immunoassay (ELISA) or enzyme-linked immunosorbent spot (ELISPOT).

28. The method of any one of claims 14-26, wherein the T-cell immune response indicator is detected by intracellular cytokine staining, and / or flow cytometry.

29. The method of any one of claims 14-28, further comprising:(D) administering to the subject an effective amount of anti-viral agent.

30. A sample collection vessel comprising the composition of any one of claims 1 to 13.

31. The sample collection vessel of claim 30, wherein the composition is coated on a portion of the interior surface of the vessel.

32. A kit for detecting a latent EBV infection in a subject, comprising:(i) the composition of any one of claims 1 to 13, and(ii) one or more reagents for performing an ELISA assay.

33. The kit of claim 32, wherein the one or more reagents comprise:(i) a plate or strip coated with an anti-ZFN-y capture antibody, and(ii) an anti-interferon-y detection antibody for detecting and / or quantifyingIFN-y.

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