JP2002255997A - Cd8+ cytotoxic t lymphocyte epitope peptide and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a CD8<+> cytotoxic T lymphocyte epitope peptide specific to an Epstein-Barr virus or human cytomegalovirus. SOLUTION: This CD8<+> cytotoxic T lymphocyte epitope peptide is specific to an Epstein-Barr virus or human cytomegalovirus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to Epstein-Barr virus (hereinafter referred to as EBV) or human cytomegalovirus (EBV).
virus, hereinafter referred to as HCMV) specific CD8 ⁺ cytotoxic T lymphocyte (hereinafter referred to as CTL) epitope peptide, EBV using the peptide
Or a vaccine for treating or preventing HCMV infection, EBV or H
The present invention relates to a passive immunotherapeutic agent for CMV and a method for quantifying CD8 ⁺ CTL specific for EBV or HCMV.

[0002]

2. Description of the Related Art EBV is closely related to the development of Burkitt's lymphoma and nasopharyngeal carcinoma, and has attracted attention for its association with Hodgkin's disease, gastric cancer and the like. In addition, it is important as a major causative virus of congenital immunodeficiency, opportunistic lymphoma, which is a problem in AIDS patients and after organ transplantation, and its suppression greatly affects the prognosis of patients.

[0003] On the other hand, HCMV is the most important pathogen as a causative virus of opportunistic infections, which has become a problem in AIDS patients and after organ transplantation. In addition, with the expansion of the bone marrow bank and the cord blood bank, hematopoietic stem cell transplantation between unrelated persons and using cord blood has increased remarkably in recent years. Since such transplantation provides stronger immunosuppression than usual for preventing rejection, opportunistic infections typified by HCMV frequently occur, and not only severe cases but also deaths are not rare.

In such medical circumstances and social background,
New methods for safely and effectively controlling EBV and HCMV are strongly desired. The main immunocompetent cell that regulates the activity of EBV and HCMV infected cells is CD8 ⁺ CTL. CD8 ⁺ CTL
When EBV and HCMV infected cells are discovered, they have the ability to destroy them.
It is likely to lead to the development of new diagnostics and treatments for V and CMV related diseases.

[0005] When CTL recognizes virus-infected cells, it has the following features. (1) CTL cannot recognize the virus particle itself. (2) CTL recognizes a peptide consisting of 9 to 10 amino acids (hereinafter referred to as epitope peptide) at a specific site in a viral protein and destroys infected cells. (3) The specific 9-10 amino acids are the human leukocyte antigen (human
leucocyte antigen (hereinafter referred to as HLA)
Presented to L. (4) HLA differs between races and individuals, and different HLAs have different epitope peptides even in the same virus.

[0006] As is apparent from the above (1) to (4), determining the amino acids which can be epitope peptides specific to EBV and HCMV without excess or deficiency is necessary for these viral infections. It can be an indispensable matter in diagnosing whether there is effective immunity and further in administering immunotherapy. However, for the HLA type possessed by many Japanese, there are very few reports of these EBV or HCMV-specific CD8 ⁺ CTL epitope peptides, and epitope peptides useful for treating or preventing EBV or HCMV infection are disclosed. Development is required.

[0007]

SUMMARY OF THE INVENTION The present invention relates to EBV and HCM
V-specific CD8 ⁺ CTL epitope peptide, EBV or HCMV
Vaccine to treat or prevent infection, passive immunotherapy against EBV or HCMV, and CD8 ⁺ C specific for EBV or HCMV
The purpose is to provide a method for quantifying TL.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found a plurality of epitope peptides that can be recognized by CD8 ⁺ CTL that regulates EBV and HCMV. It was completed. That is, the present invention is a CD8 ⁺ CTL epitope peptide specific to EBV and HCMV.

Further, the present invention relates to a EBV or HCMV-specific C
A vaccine for treating or preventing EBV or HCMV infection using a D8 ⁺ CTL epitope peptide. Furthermore, the present invention is a passive immunotherapy for EBV or HCMV prepared using the peptide. The present invention also relates to a method for quantifying EBV or HCMV-specific CD8 ⁺ CTL.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail. 1. Epitope peptide A peptide as referred to in the present invention means a linear amino acid molecular chain which has physiological activity and is mutually linked by a peptide bond between an α-amino group and a carboxyl group of adjacent amino acid residues. Peptides are not meant to be of a particular length, but may be of various lengths. It may be in an uncharged or salt form, and in some cases, may be modified by glycosylation, amidation, phosphorylation, carboxylation, phosphorylation, or the like. Furthermore, one or several (for example, 1 to 10) amino acids may be used as long as they do not substantially alter the physiological and immunological activities of the epitope of the present invention and have no harmful activity when administered. Insert, append,
Peptides in which substitution or the like has occurred are also included in the present invention. For example,
Also included are those in which an additional amino acid sequence is interposed at the N-terminal or C-terminal of the peptide. Further, the peptide of the present invention can be used in the form of a complex to which a saccharide, polyethylene glycol, lipid, or the like is added, a derivative with a radioisotope, or a polymer.

[0011] The CD8 ⁺ CTL of the present invention is a CD8 ⁺ CTL expressing CD8 which is one of surface antigen molecules present on human lymphocytes.
D8 ⁺ means cytotoxic T lymphocytes. EBV or HC
CD8 ⁺ CTL epitope peptide specific for MV is CD8 ⁺ CTL
A peptide consisting of a 9 to 10 amino acid sequence at a specific site in an EBV or HCMV protein that can be recognized by EBV or HCMV, which immunologically binds to an antigen receptor of CD8 ⁺ CTL.
Structural part of MV, which means an antigen group that activates the cellular immune mechanism of CD8 ⁺ CTL, which can directly injure EBV or HCMV infected cells and eliminate virus. FIG. 1 shows the mechanism.

The EBV or HCMV-specific CD8 ⁺ CTL epitope peptide of the present invention is an epitope peptide consisting of 9 to 10 amino acids having a binding motif such as HLA-A24 or A26 with respect to the amino acid sequence of the EBV or HCMV protein. Collation media that can search for, for example, BioInformatics & Molecular Analysis Sectio published on the Internet
n (BIMAS) HLA Peptide Binding Predictions (htt
p: //bimas.dcrt.nih.gov/molbio/hla_bind/index.htm
1), and a peptide that can be an epitope peptide (hereinafter referred to as epitope candidate peptide) can be screened.

The peptide of the present invention having the amino acid sequence of SEQ ID NOs: 1 to 32 has been confirmed as a CD8 ⁺ CTL epitope peptide as a result of the above screening. That is, the epitope peptides represented by SEQ ID NOs: 1 to 32 of the present invention can be prepared by various conventional peptide synthesis methods. For example, an organic chemical synthesis method such as a solid phase peptide synthesis method, or encoding a peptide
DNA can also be prepared and prepared using recombinant DNA technology. Further, synthesis using a commercially available chemical synthesizer (for example, a peptide synthesizer manufactured by Applied Biosystems) is also possible.

The epitope candidate peptide can be obtained by ordinary chemical synthesis. For the obtained epitope candidate peptide, for example, it can be determined whether or not it is a CD8 ⁺ CTL epitope peptide specific to EBV or HCMV by any of the methods described below.

(1) Method for determining epitope peptide 1 Lymphocytes isolated from an adult infected with EBV or HCMV were suspended at a cell concentration of 2 × 10 ⁶ / ml in RPMI1640 medium containing 10% fetal bovine serum, and The spheres are mixed with 1 × 10 ⁵ / ml of EBV or HCMV-infected cells previously separated and cultured from the same person, and cultured in a carbon dioxide incubator at 37 ° C. for 10 days. culture
On the 10th day, interleukin 2 (hereinafter, referred to as IL-2) was added, and thereafter, EBV or HCMV-infected cells and stimulation with IL-2 were repeated once a week. CD
To induce 8 ⁺ CTL. Whether or not the epitope candidate peptide has the ability to stimulate CD8 ⁺ CTLs induced in this manner is determined by an ELISPOT assay or the like.
Elispot assay is described in Kuzushima K et al., The Journal
al of Clinical Investigation, 104: 163-171, 1999
Reported by year.

(2) Epitope peptide determination method 2 Lymphocytes isolated from an adult infected with EBV or HCMV were suspended at a cell concentration of 2 × 10 ⁶ / ml in RPMI1640 medium containing 10% fetal bovine serum, and Any one of the epitope candidate peptides is added at a concentration of 1 μg / ml. Incubate at 37 ° C for 10 days in a carbon dioxide thermostat. On day 10, IL-2 is added, and thereafter, stimulation with the peptide and IL-2 is repeated once a week to induce specific CD8 ⁺ CTL. Whether or not the epitope candidate peptide stimulates EBV or HCMV-specific CD8 ⁺ CTLs induced in this way is determined by an ELISPOT assay or the like.

(3) Epitope peptide determination method 3 A peptide library consisting of about 20 amino acids covering the entire EBV or HCMV protein is synthesized. With respect to the synthesized peptide, the peptide reacted with CD8 ⁺ CTL is sequentially shortened, and finally an epitope peptide consisting of 9 to 10 amino acids is obtained as the epitope peptide of the present invention.

2. Vaccine The CD8 ⁺ CTL epitope peptide of the present invention can be used as a vaccine in active immunopeptide vaccine therapy. That is, a vaccine comprising the CD8 ⁺ CTL epitope peptide of the present invention is administered to a patient, and EBV or HCMV is administered.
Grown in vivo specific CD8 ⁺ CTL in, can help prevention and treatment for the disease. The epitope peptide to be used may be used alone, or two or more peptides may be used in combination according to the intended use of the vaccine.

Also, antigen-presenting cells (eg, dendritic cells, B cells, macrophages, etc.) include those pulsed with the CD8 ⁺ CTL epitope peptide of the present invention (hereinafter referred to as CD8 ⁺ CTL epitope peptide pulsed cells). Vaccines can also be used. Here, antigen-presenting cells are
Among cells expressing on its surface HLA to which the peptide can bind, those having CD8 ⁺ CTL stimulating ability, pulsing means that the antigen-presenting cells and the peptide are allowed to stand for 30 minutes in a suitable culture medium. It means mixing for 1 hour.

The vaccine comprising the CD8 ⁺ CTL epitope peptide or the CD8 ⁺ CTL epitope peptide-pulsed cells of the present invention can be prepared using methods known in the art. For example, such vaccines include injections or solid preparations containing the CD8 ⁺ CTL epitope peptide of the present invention or CD8 ⁺ CTL epitope peptide pulsed cells as an active ingredient. Epitope peptides can be formulated in a neutral or salt form, for example, as pharmaceutically acceptable salts, inorganic salts such as hydrochloric acid, phosphoric acid, or
Organic acids such as acetic acid and tartaric acid can be mentioned. The epitope peptide or epitope peptide-pulsed cells of the present invention are pharmaceutically acceptable and excipients compatible with the activity of the peptide or the cells, for example, water, saline, dextrose, ethanol, glycerol, DMSO And other adjuvants or the like, or a combination thereof. Further, if necessary, auxiliary agents such as albumin, a wetting agent, and an emulsifier may be added.

The vaccine of the present invention can be administered by parenteral administration or oral administration, but parenteral administration is generally preferred. Parenteral administration includes injections such as subcutaneous injection, intramuscular injection, and intravenous injection, and suppositories. Oral administration includes starch, mannitol, lactose,
It can be prepared as a mixture with excipients such as magnesium stearate and cellulose.

The vaccine of the present invention is administered in a therapeutically effective amount. The amount administered depends on the subject being treated, the immune system,
The required dosage is determined by the judgment of the clinician. Usually, the appropriate dosage will, per patient, in epitope peptide 1 -100 mg, the epitope peptide pulsed cells to 10 ⁶ to 10 ⁹ pieces of content. Further, the administration interval can be set depending on the subject and the purpose.

3. Passive immunotherapeutic agent The CD8 ⁺ CTL epitope peptide of the present invention can be used for preparing a passive immunotherapeutic agent for EBV or HCMV. That is, 1) CD8 ⁺ CTL obtained by stimulating peripheral blood lymphocytes with the peptide, 2) major histocompatibility complex (major histocompa) prepared from peripheral blood lymphocytes prepared from the peptide
tibility complex (hereinafter referred to as MHC) or MHC-tetramer, and react with MHC or MHC-tetramer as CD8 ⁺ CTL.
Is bound to form a conjugate, and CD8 ⁺ CTL obtained by isolation from the conjugate, or 3) reacting peripheral blood lymphocytes with MHC-labeled magnetic beads prepared from the peptide, and MHC-labeled magnetic beads To form a conjugate in which CD8 ⁺ CTL is bound,
A passive immunotherapeutic agent comprising CD8 ⁺ CTL obtained by isolation from the conjugate can be obtained.

MHC using CD8 ⁺ CTL epitope peptide
And MHC-tetramer can be prepared as follows, for example. Purified from E. coli for protein expression
MHC, a complex of the HLA heavy chain, β2 microglobulin and the CD8 ⁺ CTL epitope peptide of the invention, is formed in the buffer. A biotin binding site is previously added to the C-terminal of the recombinant HLA heavy chain protein, and biotin is added to this site after MHC formation. An MHC-tetramer is prepared by mixing a commercially available labeled dye streptavidin and biotinylated MHC at a molar ratio of 1: 4. 2 and 3 show the obtained MHC-tetramer and its reaction mechanism, respectively.
In each step, it is preferable to perform protein purification by gel filtration. EB in passive immunotherapy
CD8 ⁺ CTL specific to V or HCMV can be obtained by the following preparation method.

(1) Preparation method of CD8 ⁺ CTL 1 Lymphocytes are separated from peripheral blood and the like,
React with C or MHC-tetramer at 37 ° C for 15 minutes. CD8 specific for EBV or HCMV bound to MHC or MHC-tetramer
^{Since +} CTL is stained with a labeling dye, only the stained CD8 ⁺ CTL is isolated using a flow cytometer, a microscope, or the like. Alternatively, it can be reacted with MHC and / or MHC-tetramer previously immobilized on a sterile plate or the like. In order to isolate EBV or HCMV-specific CD8 ⁺ CTL bound to MHC and / or MHC-tetramer immobilized on the plate, after washing other cells that have not been bound, Only the antigen-specific CD8 ⁺ CTL remaining above is suspended in a fresh culture. EB isolated in this way
V or HCMV-specific CD8 ⁺ CTLs include anti-CD3 antibodies, PHA, IL-
Stimulate and proliferate with a T cell stimulating agent such as 2 to secure the number of cells required for passive immunotherapy.

(2) CD8 ⁺ CTL Preparation Method 2 Biotinylated MHC prepared from a peptide as described above is bound to streptavidin-labeled magnetic beads to prepare a conjugate (hereinafter referred to as MHC-magnetic beads). MHC-magnetic beads are shown in FIG. Lymphocytes are separated from peripheral blood or the like, and the MHC-magnetic beads at an appropriate concentration are reacted at a lymphocyte: beads ratio of 1:10. E bound to MHC-magnetic beads
When a test tube containing CD8 ⁺ CTL specific for BV or HCMV is placed in a magnetic field, the CD8 ⁺ CTL bound to the beads is attracted to the inner wall of the test tube on the side with the magnet. The mechanism is shown in FIG. Thereafter, after washing out the other cells, the test tube is removed from the magnetic field, and only the antigen-specific CD8 ⁺ CTL remaining on the inner wall of the test tube is suspended in a new culture solution. EBV thus isolated
Alternatively, CD8 ⁺ CTLs specific for HCMV include anti-CD3 antibodies, PHA, IL-2
And stimulated proliferation with a T cell stimulating agent such as the above to secure the number of cells necessary for passive immunotherapy.

(3) CD8 ⁺ CTL preparation method 3 Lymphocytes isolated from peripheral blood are stimulated directly with the CD8 ⁺ CTL epitope peptide of the present invention, or antigen-presenting cells (eg, dendritic cells, (B cells, macrophages, etc.). CD induced by stimulation
Incubate 8 ⁺ CTL at 37 ° C for 7 to 10 days in a carbon dioxide thermostat.
Then, IL-2 was added, and CD8 ⁺ CTL epitope peptide and IL-
2, or the number of CD8 ⁺ CT cells required for passive immunotherapy by repeating stimulation with the antigen-presenting cells and IL-2 once a week
Secure L. The CD8 ⁺ CTL specific for EBV or HCMV obtained as described above can be suspended in human albumin-containing PBS or the like to be used as a passive immunotherapy for EBV or HCMV.

4. CD8 ⁺ Quantitative EBV and specific CD8 ⁺ CTL in HCMV of CTL is a human immune ability by the patient (for some reason the high-risk is reduced, congenital immunodeficiency, rejection prevention undergoing hematopoietic stem cell transplantation or solid organ transplantation Knowing whether it is present in the peripheral blood of patients receiving immunosuppressive drugs, patients with chronic viral infections, AIDS patients, the elderly, young children, pregnant women, etc.) It is important information in the management of these infectious diseases, including the proper use of immunosuppressants. Quantification of CD8 ⁺ CTL specific for EBV or HCMV can be performed by the following two methods using the CD8 ⁺ CTL epitope peptide of the present invention.

The first quantification method is to stimulate lymphocytes separated from peripheral blood with the CD8 ⁺ CTL epitope peptide of the present invention, and to produce CD8 ⁺ CTL-produced interferon gamma (IFNγ), interleukin, etc. This is a method for quantifying cytokines and / or chemokines. The method is specifically described below using IFNγ as an example.

(1) Method 1 for quantification of cytokines (quantification of intracellular IFNγ-producing cells): Lymphocytes separated from peripheral blood were suspended at a cell concentration of 2 × 10 ⁶ / ml in RPMI1640 medium containing 10% fetal bovine serum. The CD8 ⁺ CTL epitope peptide of the invention is added at a concentration of 1 μg / ml. Further, Brefeldin A, which is an inhibitor of intracellular protein transport, is added, and the cells are cultured at 37 ° C. for 5 to 6 hours in a carbon dioxide gas thermostat. After the culture, the cells are fixed, subjected to membrane permeation treatment, and reacted with a dye-labeled anti-IFNγ antibody and an anti-CD8 antibody. Using a flow cytometer or the like, the percentage of IFNγ-positive cells in CD8 ⁺ lymphocytes is quantified.

(2) Method 2 by cytokine quantification (Elispot assay); 96-well MultiScreen-HA plate (M
i11ipore) is coated with an anti-IFNγ monoclonal antibody overnight at 4 ° C., and each well is washed with PBS. Then, lymphocytes separated from peripheral blood are spread on each well. The epitope peptide is put into each well and cultured in a 5% CO ₂ incubator at 37 ° C. for 20 hours. The next day, after washing the plate with PBS supplemented with 0.05% Tween-20,
Anti-IFNγ rabbit serum, peroxidase-labeled anti-rabbit IgG
The reaction is performed for 90 minutes at room temperature in the order of goat serum. further
3-amino-9-ethylcarbasole (Sigma) and 0.1 M sodium acetate buffer (pH 5.0) containing 0.015% H ₂ O ₂ are added to each well, and reacted at room temperature for 40 minutes. Visualize IFNγ spots and count with a stereomicroscope.

(3) Method 3 for quantifying cytokines (method for quantifying IFNγ secreted in culture supernatant); lymphocytes separated from peripheral blood were RPMI1640 containing 10% fetal bovine serum.
Suspension at a cell concentration of 2 × 10 ⁶ / ml in the medium, the CD8 ⁺ C of the present invention
TL epitope peptide is added at a concentration of 1 μg / ml. Incubate at 37 ° C for 24 to 48 hours in a CO 2 constant temperature oven. After culturing, collect the supernatant and determine the concentration of IFNγ contained in the supernatant using a commercially available ELISA.
Kits (eg, HUMAN IFN gamma ELISA from ENDOGEN)
Quantify using

As a second quantification method, the CD8 ⁺ CT of the present invention is used.
Using MHC-tetramer prepared using L epitope peptide, CD8 ⁺ CTL specific for EBV and HCMV in peripheral blood
Can be determined. Preparation of the MHC-tetramer is as described above. The quantification can be performed, for example, as follows. Lymphocytes are separated from peripheral blood and the like, and reacted with an appropriate concentration of MHC-tetramer at 37 ° C. for 15 minutes. Since the CD8 ⁺ CTL bound to the tetramer is stained with a labeling dye, it is counted using a flow cytometer, a microscope, or the like.

[0034]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. [Example 1] specific CD8 ⁺ CTL epitope peptide EBV Screening invention specific CD8 ⁺ CTL epitope candidate peptides to EBV, the
Regarding the amino acid sequence of EBV protein, HLA-A24, A2
6. BioInformatics & Molecular Ana published on the Internet that can search epitope peptides consisting of 9 to 10 amino acids having each binding motif of B61
HLA Peptide Binding Predic of Lysis Section (BIMAS)
tions (http://bimas.dcrt.nih.gov/molbio/hla_bind/i
ndex.html.), screening a large number of antigen epitope candidate peptides consisting of 9 to 10 amino acids having the respective binding motifs of HLA-A24, A26, and B61.
A variety of epitope candidate peptides were synthesized.

Example 2 Identification of CD8 ⁺ CTL Epitope Peptide Specific to EBV Preparation of Various Materials (1) Obtaining Blood Lymphocytes were isolated from peripheral blood of healthy adults already infected with EBV. HLA type was measured using a serum method.

(2) EBV-infected B cell line Peripheral blood lymphocytes contain supernatant of B95-8 cell, which is an EBV-producing cell line
Infected EBV (including live EBV virus)
(Lymphoblastoid cell line, hereinafter referred to as EBV-infected LCL) was established.

(3) Cells for peptide presentation HLA-A24, A26 and B61 genes were introduced into 174CEM.T2 (hereinafter referred to as T2 cells) as cells for peptide presentation, respectively.
Cells displaying each binding peptide of A24, A26, and B61 (hereinafter, referred to as T2-A24, T2-A26, and T2-B61 cells, respectively) were obtained. Each T
2-A24, T2-A26, T2-B61 cells are Iscove's modified Dulbecco's medium (GIBCO) with 10% fetal calf serum, L-glutamine, penicillin, streptomycin, and geneticin
Was used for culturing.

2. Establishment of culture of CD8 ⁺ CTL specific for EBV (1) CD8 ⁺ CTL specific for EBV The peripheral blood lymphocytes obtained in 1. (1) above were previously established from the same person. The cells were mixed and cultured in a flask together with the LCL of 2). At this time, the LCL was irradiated with a cell lethal dose of radiation to prevent proliferation. If the blood donor is positive for HLA-A24, A26 or B61, some of the HLA-A24, A26 or B61 molecules on the LCL cell surface of that person will have 9-10 amino acids from the EBV protein In response, CD8 ⁺ CTLs started to proliferate. To aid the growth of CD8 ⁺ CTL, IL-2 was added to the culture. (2) Establishment of EBV-specific CD8 ⁺ CTL clone From the established EBV-specific CD8 ⁺ CTL strain, a CD8 ⁺ CTL clone was established using the limiting dilution method.

3. Elispot assay 96-well MultiScreen-HA plates (Mi11ipore) were coated overnight at 4 ° C. with anti-IFNγ monoclonal antibody (R & D Systems, Minneapolis, MN). After washing each well with PBS,
T2-A24, A26, and B61 cells were seeded in each well. Each epitope candidate peptide synthesized in Example 1 was placed in each well, and 30
After standing at room temperature for a minute, an appropriate number of EBV-specific CD8 ⁺ CTLs
Was added and the cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 20 hours. CD8 ⁺ CTLs that reacted with the epitope candidate peptide secreted IFNγ during this culture period. The next day, the plate was washed with PBS supplemented with 0.05% Tween-20, and then anti-IFNγ rabbit serum (Genzyme, C
ambridge, MA) and peroxidase-labeled anti-rabbit IgG goat serum in this order for 90 minutes at room temperature. 3-am
0 containing ino-9-ethylcarbasole (Sigma) 0.015% H ₂ O _2.
1M sodium acetate buffer (pH 5.0) was added to each well, and allowed to react at room temperature for 40 minutes to visualize IFNγ spots. Spots were counted with a stereomicroscope.

In order to confirm that the CD8 ⁺ CTL epitope peptide is processed endogenously, vaccinia viruses encoding EBV proteins were converted to HLA-A24, A2
6, B61-positive skin fibroblasts were infected, and an Elispot assay was performed using these cells as antigen-presenting cells. Here, endogenously processed means that newly produced viral proteins in infected cells are cleaved into antigen peptides of the correct length by the cell's own antigen processing mechanism, and HLA heavy chains and β2 Forms a complex with globulin,
It means the state presented on the infected cell surface.
According to the method described above, CD8 ⁺ CTL specific for EBV
The following SEQ ID NOS: 1 to 22 reacted with the CD8 ⁺ CTL clone
CD8 ⁺ CTL epitope peptide was obtained.

Arg Tyr Ser Ile Phe Phe Asp Tyr Met (SEQ ID NO: 1) Leu Tyr Ala Leu Ala Leu Leu Leu Leu (SEQ ID NO: 2) Ala Tyr Arg Arg Arg Trp Arg Arg Leu (SEQ ID NO: 3) Arg Tyr Cys Cys Tyr Tyr Cys Leu Thr Leu (SEQ ID NO: 4) Thr Tyr Pro Val Leu Glu Glu Met Phe (SEQ ID NO: 5) Ser Tyr Lys Thr Leu Arg Glu Phe Phe (SEQ ID NO: 6) Asp Tyr Asn Phe Val Lys Gln Leu Phe (SEQ ID NO: 6) 7) Thr Tyr Thr Ser Gly Glu Ala Cys Leu (SEQ ID NO: 8) Cys Tyr Glu Asn Asp Asn Pro Gly Leu (SEQ ID NO: 9) Thr Tyr Trp Gln Leu Asn Gln Asn Leu (SEQ ID NO: 10) Ala Tyr Ala Glu Ala Thr Ser Ser Leu (SEQ ID NO: 11) Asp Tyr Met Ala Ile His Arg Ser Leu (SEQ ID NO: 12) Phe Tyr Arg Ser Leu Leu Thr Ile Leu (SEQ ID NO: 13) His Tyr Gln Thr Leu Cys Thr Asn Phe (SEQ ID NO: 14) Phe Tyr Met Thr His Gly Leu Gly Thr Leu (SEQ ID NO: 1
5) Gly Glu Thr Ser Gly Ile Arg Arg Ala (SEQ ID NO: 16) Asp Leu Ser Tyr Ile Lys Ser Phe Val (SEQ ID NO: 17) Asp Tyr Ser Gln Gly Ala Phe Thr Pro Leu (SEQ ID NO: 1)
8) Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu (SEQ ID NO: 1
9) Ile Tyr Val Leu Val Met Leu Val Leu (SEQ ID NO: 20) Ile Tyr Val Leu Val Met Leu Val Leu Leu (SEQ ID NO: 2)
1) Ser Tyr Ala Ala Ala Gln Arg Lys Leu (SEQ ID NO: 22)

[0042] [Example 3] Specific CD8 ⁺ CTL epitope peptide HCMV Screening invention specific CD8 ⁺ CTL epitope candidate peptides with HCMV, for the amino acid sequence of the HCMV protein, HLA-A2
4. BioInformatics & Molecul published on the Internet, which can search epitope peptides consisting of 9 to 10 amino acids having each binding motif of A26 and B61.
HLA PeptideBinding Pr of ar Analysis Section (BIMAS)
edictions (http://bimas.dcrt.nih.gov/molbio/hla_bi
nd / index.html.), and screening a large number of antigen epitope candidate peptides consisting of 9 to 10 amino acids having the respective binding motifs of HLA-A24, A26, and B61.
100 kinds of epitope candidate peptides were synthesized.

Example 4 Identification of CD8 ⁺ CTL Epitope Peptide Specific for HCMV Preparation of Various Materials (1) Acquisition of Blood and Skin Fibroblasts Lymphocytes were isolated from peripheral blood of healthy adults already infected with HCMV. HLA type was measured using a serum method. For some people, skin biopsies were performed to obtain fibroblasts. That is, after cutting the skin biopsy tissue finely with scissors, 10% fetal calf serum, L-glutamine, benicillin,
Dulbecco's modified Ea with streptomycin
Skin fibroblasts were cultured using gle medium (GIBCO, Grand Island, NY). (2) CMV AD169 strain CMV AD169 strain is an American Type Culture Collecti
Obtained from on (ATCCVR-538).

(3) Cells for peptide presentation HLA-A24, A26, and B61 genes were introduced into 174CEM.T2 (hereinafter, referred to as T2 cells) as cells for peptide presentation, respectively.
Cells displaying each binding peptide of A24, A26, and B61 (hereinafter, referred to as T2-A24, T2-A26, and T2-B61 cells, respectively) were obtained. Each T
2-A24, T2-A26, T2-B61 cells are Iscove's modified Dulbecco's medium (GIBCO) with 10% fetal calf serum, L-glutamine, penicillin, streptomycin, and geneticin
Was used for culturing.

2. Establishment of culture of CD8 ⁺ CTL specific for HCMV (1) CD8 ⁺ CTL specific for HCMV The peripheral blood lymphocytes obtained in 1. (1) above were previously established from the same person. 1) H to skin fibroblasts
The cells were infected with CMV and mixed in a flask. If the blood donor is positive for HLA-A24, A26 or B61, some of the HLA-A24, A26 or B61 molecules on the surface of that person's dermal fibroblasts will contain 9-10 CMV proteins. An antigenic epitope consisting of the following amino acids was presented, and CD8 ⁺ CTLs that responded thereto started to proliferate. To aid the growth of CD8 ⁺ CTL, IL-2 was added to the culture. (2) Establishment of HCMV-specific CD8 ⁺ CTL clone From the established HCMV-specific CD8 ⁺ CTL strain, a CD8 ⁺ CTL clone was established using the limiting dilution method.

3. Elispot assay 96-well MultiScreen-HA plates (Mi11ipore) were coated overnight at 4 ° C. with anti-IFNγ monoclonal antibody (R & D Systems, Minneapolis, MN). After washing each well with PBS,
T2-A24, A26, and B61 cells were seeded in each well. Each epitope candidate peptide synthesized in Example 1 was placed in each well, and 30
After standing at room temperature for a minute, an appropriate number of CD8 ⁺ CTLs specific for CMV
Was added and the cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 20 hours. CD8 ⁺ CTLs that reacted with the epitope candidate peptide secreted IFN during this culture period. The next day, the plate was washed with PBS supplemented with 0.05% Tween-20, and then anti-IFNγ rabbit serum (Genzyme, Cam
bridge, MA) and peroxidase-labeled anti-rabbit IgG goat serum were reacted at room temperature for 90 minutes each. 3-am
0 containing ino-9-ethylcarbasole (Sigma) 0.015% H ₂ O _2.
Add 1M sodium acetate buffer (pH 5.0) to each well,
The reaction was carried out at room temperature for 40 minutes to visualize the IFNγ spot. Spots were counted with a stereomicroscope. An actual IFNγ spot photograph using T2-A24 as an antigen presenting cell is shown in FIG. 6A. It was also produced in response to each of the epitope candidate peptides.
Figure 6 shows the results of counting IFNγ spots under a stereomicroscope.
Shown in B. The bar graph shows the number of spots per well. In the figure, peptide numbers 60, 81 and 83 correspond to the SEQ ID NOs of the present invention.
These correspond to 30, 23, and 24, respectively.

In addition, to confirm that the CD8 ⁺ CTL epitope peptide is being processed endogenously, HCM
V, a vaccinia virus expressing the gB protein of HCMV and a vaccinia virus expressing pp65 of HCMV were HLA-A2
4-positive dermal fibroblasts were infected and used as antigen-presenting cells, and an ELISPOT assay was performed in which they were reacted with a CD8 ⁺ CTL clone specific for the peptide of SEQ ID NO: 23 (derived from pp65 of HCMV). FIG. 7 shows the result. In the figure, Mock
Shows the results of reacting HLA-A24-positive skin fibroblasts not infected with the virus with the CTL clone. The bar graph of each item is CD8 ⁺ C per well from the left.
Shown are the dilution series of TL clones, 200, 100, 50, 25. As a result, the CD8 ⁺ CTL clone specific for the peptide of SEQ ID NO: 23 reacted only with HCMV-infected cells and pp65-expressing vaccinia virus-infected cells. According to the method as described above, CD8 ⁺ CTLs specific to HCMV and the CD8 ⁺ CTL epitope peptides of the following SEQ ID NOS: 23 to 32 to which the CD8 ⁺ CTL clones reacted were obtained.

Gln Tyr Asp Pro Val Ala Ala Leu Phe (SEQ ID NO: 23) Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe (SEQ ID NO: 2)
4) Val Glu Leu Arg Gln Tyr Asp Pro Val Ala (SEQ ID NO: 2
5) Asp Val Pro Ser Gly Lys Leu Phe Met (SEQ ID NO: 26) Asp Val Ala Phe Thr Ser His Glu His Phe (SEQ ID NO: 2)
7) Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile (SEQ ID NO: 2
8) Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr (SEQ ID NO: 2
9) Asn Tyr Leu Asp Leu Ser Ala Leu Leu (SEQ ID NO: 30) Gln Tyr Arg Ile Gln Gly Lys Leu (SEQ ID NO: 31) Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr (SEQ ID NO: 3)
2)

Example 5 Vaccine Injection Peptides of SEQ ID NOs: 1 to 32 were added to DMSO at a final concentration of 20 mg / ml.
, And sterilized by filtration. Dispense and tightly stopper the resulting peptide-containing solution in sterile vials by 1 ml each,
A vaccine injection was prepared.

Example 6 Method of Quantifying HCMV-Specific CD8 ⁺ CTLs by Measuring Cytokines Lymphocytes were isolated from 5 adult peripheral blood cells, and the HCMV-derived peptide of SEQ ID NO: 23 and a negative control Was stimulated with an HLA-A24 binding peptide derived from AIDS virus (HIV). IFN-γ produced and accumulated in the cells was stained with a FITC-labeled antibody. CD69, an activation marker for T lymphocytes
Was simultaneously stained with a PE-labeled antibody. The stained cells were analyzed using a flow cytometer (Becton-Dickinson, FACSc).
an). FIG. 8 shows the result. In the figure, the percentage shown in the upper right of each graph is CD8 ⁺ CT reacted with the peptide.
The ratio of L is shown. From these results, it is clear that this method can specifically detect epitope peptide-reactive CD8 ⁺ CTL.

[Example 7] Method of quantifying HCMV-specific CD8 ⁺ CTL in peripheral blood using MHC-tetramer prepared from CD8 ⁺ CTL epitope peptide Preparation of MHC-tetramer (1) Preparation of MHC A large amount of HLA A * 2402 heavy chain and β2 microglobulin were prepared and purified using a recombinant protein expression system by Escherichia coli. The amino acid sequence recognized by biotin ligase was added to the C-terminus of the HLA A * 2402 heavy chain in advance. Purified HLA A *
2402 heavy chain and β2 microglobulin were each dissolved in 8M urea. 200 ml refolfing buffer (pH8.0; 1
00 mM Tris-HCl, 400 mM L-arginine-HCl, 2 mM EDTA,
0.5 mM oxidative glutathione, 5 mM reduced glutath
Ion) were injected with 12 mg of the peptide of SEQ ID NO: 23, 18.6 mg of HLA A * 2402 heavy chain, and 13.2 mg of β2 microglobulin using a syringe equipped with a 27 gauge needle. After stirring for 48 to 72 hours in a 10 ° C constant temperature bath to promote the formation of MHC,
Is reconstituted in 1.8 L distilled water for 24 hours at 4 ° C in a constant temperature bath.
fer to Centriprep 10 (MILLIPORE, Bedford, MA)
And concentrated to 2 ml. Superdex 200 HR (Amersham
(Pharmacia Biotech AB, Uppsala, Sweden). MHC eluted per 45 KD molecular weight was isolated by gel filtration chromatography using a column.

(2) Preparation of biotinylated MHC Using biotin ligase (AVIDITY, Denver, CO)
Biotin was bound to a specific site at the C-terminus of the LA A * 2402 heavy chain. Biotin-added MHC was purified by gel filtration chromatography using a Superdex 200 HR column.

(3) Preparation of MHC-tetramer PE-labeled streptavidin (Molecular Probe, Eugene,
OR) and purified biotinylated MHC were mixed at a molar ratio of 1: 4. Su
By gel filtration chromatography using a perdex 200 HR column, an MHC-tetramer containing SEQ ID NO: 23 flowing out per 480 KD molecular weight was isolated. Centricon 10 (MILLIP
(ORE) to about 3 mg / ml and stored at 4 ° C. Preservatives include NaAzide, EDTA, Leupeptin, Pepstati
n was added.

2. Quantification of HCMV-specific CD8 ⁺ CTL using MHC-tetramer HCMV epitope peptide-specific CD8 ⁺ T cell clone (2
× 10 ⁵ ) or peripheral blood lymphocytes (2 × 10 ⁶ ) isolated from an adult infected with HCMV were suspended in 50 μL of PBS containing 2% FCS in a 1.5 ml Eppendorf tube. MHC-tetramer, FITC-labeled anti-CD8 antibody (CALTAG, burlingame, C
A) was added in an amount of 1 μL each, and allowed to stand in a thermostat at 37 ° C. for 15 minutes to react. After the reaction, the cells were washed three times with 1 ml of PBS containing 2% FCS, and then washed with PBS containing 0.5% paraformaldehyde (1 m
l). CD8 ⁺ CTL bound to MHC-tetramer containing SEQ ID NO: 23 was stained with a labeling dye, FACS Calibu
r (Becton Dickinson) was used to count tetramer-positive cells in FITC-CD8 ⁺ T cells and calculate%.

FIG. 9 shows the result of the analysis. The HCMV-specific CD8 ⁺ CTL bound to the MHC-tetramer is stained and moves to the right. In the figure, A is an EBV-specific CD8 ⁺ CTL clone (negative control), and B is a CD8 ⁺ CTL clone specific to the peptide of SEQ ID NO: 23 (positive control). C is HCMV antibody-negative adult peripheral blood, and D is HCMV antibody-positive adult peripheral blood (the population at the upper right point in FIG. D is tetramer-positive cells). From this result, MHC- containing SEQ ID NO: 23
It is clear that the tetramer specifically stains HCMV-reactive CD8 ⁺ CTL.

[0056]

According to the present invention, C-specific EBV or HCMV
A D8 ⁺ CTL epitope peptide can be provided. In addition, the EBV or HCMV infection can be managed, treated or prevented using the epitope peptide. Furthermore, EBV
Alternatively, CD8 ⁺ CTL specific to HCMV can be quantified.

[0057]

[Sequence List] SEQUENCE LISTING <110> Aichi Prefecture <120> CD8 ⁺ cytotoxic CD8 ⁺ Lymphocyte epitope peptide <130> P01-0272 <160> 32 <170> PatentIn Ver. 2.1 <210> 1 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 1 Arg Tyr Ser Ile Phe Phe Asp Tyr Met 1 5 <210> 2 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 2 Leu Tyr Ala Leu Ala Leu Leu Leu Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 3 Ala Tyr Arg Arg Arg Trp Arg Arg Leu 1 5 <210> 4 <211> 10 <212> PRT <213> Epstien-Barr virus <400> 4 Arg Tyr Cys Cys Tyr Tyr Cys Leu Thr Leu 1 5 10 <210> 5 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 5 Thr Tyr Pro Val Leu Glu Glu Met Phe 1 5 <210> 6 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 6 Ser Tyr Lys Thr Leu Arg Glu Phe Phe 1 5 <210> 7 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 7 Asp Tyr Asn Phe Val Lys Gln Leu Phe 1 5 <210> 8 <211> 9 <212> PRT <213> Epstien-Barr virus <400 > 8 Thr Tyr Thr Ser Gly Glu Ala Cys Leu 1 5 <210> 9 <211> 9 <212> PRT <213> Epstien-B arr virus <400> 9 Cys Tyr Glu Asn Asp Asn Pro Gly Leu 1 5 <210> 10 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 10 Thr Tyr Trp Gln Leu Asn Gln Asn Leu 1 5 <210> 11 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 11 Ala Tyr Ala Glu Ala Thr Ser Ser Leu 1 5 <210> 12 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 12 Asp Tyr Met Ala Ile His Arg Ser Leu 1 5 <210> 13 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 13 Phe Tyr Arg Ser Leu Leu Thr Ile Leu 1 5 <210> 14 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 14 His Tyr Gln Thr Leu Cys Thr Asn Phe 1 5 <210> 15 <211> 10 <212> PRT <213> Epstien-Barr virus <400> 15 Phe Tyr Met Thr His Gly Leu Gly Thr Leu 1 5 10 <210> 16 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 16 Gly Glu Thr Ser Gly Ile Arg Arg Ala 1 5 <210> 17 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 17 Asp Leu Ser Tyr Ile Lys Ser Phe Val 1 5 <210> 18 <211> 10 <212> PRT <213> Epstien-Barr virus <400> 18 Asp Tyr Ser Gln Gly Ala Phe Thr Pro Leu 1 5 10 <210> 19 <211> 10 <212> PRT <213> Epstien-Barr virus <400> 19 Leu Tyr Leu Gln Gln Asn Trp Trp Thr Leu 1 5 10 <210> 20 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 20 Ile Tyr Val Leu Val Met Leu Val Leu 1 5 <210> 21 <211> 10 <212> PRT <213> Epstien-Barr virus <400> 21 Ile Tyr Val Leu Val Met Leu Val Leu Leu 1 5 10 <210 > 22 <211> 9 <212> PRT <213> Epstien-Barr virus <400> 22 Ser Tyr Ala Ala Ala Gln Arg Lys Leu 15 <210> 23 <211> 9 <212> PRT <213> Human cytomegalovirus <400> 23 Gln Tyr Asp Pro Val Ala Ala Leu Phe 1 5 <210> 24 <211> 10 <212> PRT <213> Human cytomegalovirus <400> 24 Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe 1 5 10 <210 > 25 <211> 10 <212> PRT <213> Human cytomegalovirus <400> 25 Val Glu Leu Arg Gln Tyr Asp Pro Val Ala 1 5 10 <210> 26 <211> 9 <212> PRT <213> Human cytomegalovirus <400> 26 Asp Val Pro Ser Gly Lys Leu Phe Met 1 5 <210> 27 <211> 10 <212> PRT <213> Human cytomegalovirus <400> 27 Asp Val Ala Phe Thr Ser His Glu His Phe 1 5 10 <210 > 28 <211> 10 <212> PRT <213> Human cytomegalovir us <400> 28 Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile 1 5 10 <210> 29 <211> 10 <212> PRT <213> Human cytomegalovirus <400> 29 Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr 1 5 10 <210> 30 <211> 9 <212> PRT <213> Human cytomegalovirus <400> 30 Asn Tyr Leu Asp Leu Ser Ala Leu Leu 1 5 <210> 31 <211> 8 <212> PRT <213> Human cytomegalovirus <400> 31 Gln Tyr Arg Ile Gln Gly Lys Leu 1 5 <210> 32 <211> 10 <212> PRT <213> Human cytomegalovirus <400> 32 Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr 1 5 10

[Brief description of the drawings]

FIG. 1 is a diagram showing the recognition mechanism of virus-infected cells by CD8 ⁺ CTL. In the figure, the CD8 ⁺ CTL epitope peptide is the peptide of the present invention.

FIG. 2 is a diagram showing a method for preparing an MHC-tetramer.

FIG. 3 shows the binding between MHC-tetramer and EBV or HCMV-specific CTL.

FIG. 4 is a diagram showing a method for preparing MHC-magnetic beads.

FIG. 5: EBV or HCMV-specific CTL using MHC-magnetic beads
FIG.

FIG. 6A. Specificity for HCMV by ELISPOT assay.
FIG. 2 is a view showing a photograph of an IFNγ spot in an identification example of a CD8 ⁺ CTL epitope peptide.

FIG. 6B. Specificity for HCMV by ELISPOT assay.
FIG. 9 is a view showing a spot count result in an identification example of a CD8 ⁺ CTL epitope peptide.

FIG. 7 shows that the identified epitope peptide is endogenously processed and presented on the surface of infected cells.

FIG. 8: CD8 ⁺ T reacting with peptide using IFN-γ as an index
It is a figure which shows the example of quantification of a lymphocyte.

FIG. 9 is a diagram showing an example of the identification of HCMV-specific CD8 ⁺ T lymphocytes by a tetramer prepared using the epitope peptide of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 31/18 A61P 31/18 31/22 31/22 35/00 35/00 37/04 37/04 C07K 14/05 C07K 14/05 14/405 14/405 C12Q 1/06 C12Q 1/06 G01N 33/53 G01N 33/53 KY 33/569 33/569 J F term (reference) 4B063 QA01 QQ03 QQ08 QQ79 QQ91 QR48 QR51 QS24 QS36 4C085 AA03 AA13 BA78 BA80 BA83 BB11 CC08 CC21 4H045 AA11 BA15 CA01 DA86 EA29 EA31 FA10

Claims (12)

[Claims] 1. A CD8 ⁺ cytotoxic T lymphocyte epitope peptide specific for Epstein-Barr virus or human cytomegalovirus. 2. The Epstein-Barr virus-specific CD8 ⁺ cytotoxic T lymphocyte epitope peptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 22. Peptide. 3. A CD specific for human cytomegalovirus
8 ⁺ at least one peptide of claim 1, wherein those comprising an amino acid sequence that cytotoxic T lymphocyte epitope peptide is selected from the group consisting of SEQ ID NO: 23 to SEQ ID NO: 32. 4. A vaccine for treating or preventing Epstein-Barr virus infection, comprising the peptide according to claim 2 as an active ingredient. 5. A vaccine for treating or preventing human cytomegalovirus infection, comprising the peptide according to claim 3 as an active ingredient. 6. A vaccine for treating or preventing Epstein-Barr virus infection, comprising an antigen-presenting cell pulsed with the peptide according to claim 2 as an active ingredient. 7. A vaccine for treating or preventing human cytomegalovirus infection, comprising an antigen-presenting cell pulsed with the peptide according to claim 3 as an active ingredient. 8. An Epstein-containing CD8 ⁺ cytotoxic T lymphocyte obtained by stimulating peripheral blood lymphocytes with the peptide of any one of claims 1 to 3 or an antigen-presenting cell pulsed with the peptide. A passive immunotherapeutic agent for bur virus or human cytomegalovirus. 9. A major histocompatibility complex and / or a major histocompatibility complex-tetramer prepared from the peptide according to any one of claims 1 to 3 and reacted with peripheral blood lymphocytes to obtain said major tissue. Compliant antigen complexes and / or major histocompatibility complex - tetramer CD8 ⁺ cytotoxic T lymphocytes to form a conjugate bound obtained by isolating from the conjugate CD8 ⁺ cytotoxic T lymphocytes A passive immunotherapeutic agent against Epstein-Barr virus or human cytomegalovirus, comprising: 10. A major histocompatibility complex-labeled magnetic bead obtained by reacting a major histocompatibility complex-labeled magnetic bead prepared from the peptide according to claim 1 with peripheral blood lymphocytes. To form a conjugate to which CD8 ⁺ cytotoxic T lymphocytes are bound, and to contain Epstein-containing CD8 ⁺ cytotoxic T lymphocytes obtained by isolation from the conjugate.
A passive immunotherapeutic agent for bur virus or human cytomegalovirus. 11. stimulate peripheral blood with the peptide according to claim 1, to obtain a specific CD8 ⁺ cytotoxic T lymphocytes to the virus, the CD8 ⁺ cytotoxic T lymphocytes A method for quantifying Epstein-Barr virus or human cytomegalovirus-specific CD8 ⁺ cytotoxic T lymphocytes, comprising measuring cytokines and / or chemokines produced by E. coli. 12. A major histocompatibility complex and / or a major histocompatibility complex-tetramer is prepared from the peptide according to any one of claims 1 to 3, and the major histocompatibility complex and / or the major histocompatibility complex-tetramer is prepared. CD8 ⁺ specific for Epstein-Barr virus or human cytomegalovirus in peripheral blood, which reacts the histocompatibility complex-tetramer with peripheral blood
A method for quantifying cytotoxic T lymphocytes.