JP2003064096A - Hepatitis c virus-specific cytotoxic t cell recognition epitope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hepatitis C virus (HCV)-specific cytotoxic T cell recognition epitope corresponding to a human leukocyte antigen (HLA) type. SOLUTION: The HCV-specific CTL (cytotoxic T lymphocyte) is isolated, and the constraint and the recognition epitope are specified by producing a recombinant vaccinia virus (rVAC) expressing a cDNA of each genetic region of the HCV, using a product obtained by infecting a B-cell line (B-LCL) established by infecting a patient peripheral blood mononucleosis (PBMC) with an EB (Epstein-Barr) virus, with the rVAC, as a target cell, isolating CD8<+> T-cell from the patient PBMC, using a product obtained by culturing the isolated CD8<+> T-cell with a recombinant interleukin 2, an anti-CD3 antibody and a normal-subject PBMC subject to an X-ray irradiation treatment, as an effector cell, and measuring the impaired of the effector cell to the target cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hepatitis C virus-specific cytotoxic T cell recognition epitope.

[0002]

2. Description of the Related Art There are more than 2 million hepatitis C patients in Japan, of which about 300,000 are cirrhotic patients, about 1.3 million are chronic hepatitis, and about 400,000 are normal liver functions. . Interferon administration is known as a treatment method for hepatitis C, but since its effective rate is low at about 20 to 30%, hepatitis C is still a refractory viral infection,
New treatments are desired.

Infection with a virus such as hepatitis C virus (HCV) causes elimination of the virus by innate immunity, followed by induction of a specific immune response, and elimination of the virus that cannot be eliminated by innate immunity. In specific immunity, viruses in body fluids are eliminated by neutralizing antibodies, and viruses in cells are eliminated by cytotoxic T cells (CTL). That is, CTL specifically recognizes a viral antigen (CTL epitope) consisting of 8 to 11 amino acids presented by HLA class I molecules on the surface of infected cells, and eliminates the virus by damaging infected cells. Therefore, CT specific for such viruses
Identifying the L epitope is important in creating a therapeutic vaccine against the virus. For example, preparation of a vaccine against HBV based on a hepatitis B virus (HBV) -specific CTL epitope has been reported (J. Immunol., 15
9, 1383-1392 (1997); J. Clin. Invest., 95, 341-349.
(1995)).

Regarding the identification of HCV-specific CTL epitopes, there is a report that the response of HCV type 1a to all proteins was screened and the specific CTL epitopes were identified (J. Virol., 75, 1229-1235 (2001). ).

HCV-specific CTL epitopes are HL that patients have
Since it depends on the A type, it is necessary to identify the epitope for each HLA.

[0006]

The present invention provides a novel HCV
The purpose is to provide a specific CTL epitope. Moreover, it aims at providing the HCV therapeutic vaccine based on this HCV specific CTL epitope.

[0007]

[Means for Solving the Problems]
About 80% of hepatitis C patients have recombinant vaccinia virus (rVAC) that expresses cDNA of each gene region of HCV type 1b, and established by infecting patients' peripheral blood mononuclear cells (PBMC) with EB virus. B cell line (B-LCL) infected with rVAC was used as a target cell, and CD8 ⁺ T cells were isolated from patient PBMC, and recombinant interleukin-2, anti-CD3 antibody and X
HCV-specific CTLs were obtained by measuring the effector cells' damage to target cells by using the cells cultured with PBMCs of healthy humans that had been irradiated with radiation as effector cells.
Was successfully separated, and the present invention was completed.

The present invention provides the following peptides (the peptides of the present invention). (1) A peptide consisting of 8 residues having the amino acid sequence shown in SEQ ID NO: 1. (2) A peptide consisting of 9 residues having the amino acid sequence shown in SEQ ID NO: 2. (3) A peptide consisting of 9 residues having the amino acid sequence shown in SEQ ID NO: 3. (4) A peptide consisting of 15 residues having the amino acid sequence shown in SEQ ID NO: 4. (5) A peptide consisting of 9 residues having the amino acid sequence shown in SEQ ID NO: 5. (6) A peptide consisting of 20 residues having the amino acid sequence shown in SEQ ID NO: 6. (7) A peptide consisting of 15 residues having the amino acid sequence shown in SEQ ID NO: 7. (8) A peptide consisting of 15 residues having the amino acid sequence shown in SEQ ID NO: 8.

The present invention also provides the peptide (1),
A vaccine containing as an active ingredient one or more peptides selected from the group consisting of the peptide of (2), the peptide of (3), and the peptide of (6), and the peptide of (4), the peptide of (5), The peptide of (7), and
There is provided a vaccine containing, as an active ingredient, one or more peptides selected from the group consisting of the peptide of (8).

Further, the present invention provides DNA encoding the peptide of (1) and DN encoding the peptide of (2).
A, DNA encoding the peptide of (3), and
A vaccine containing, as an active ingredient, one or more DNAs selected from the group consisting of DNAs encoding the peptides of (6),
And DNA encoding the peptide of (4),
Provided is a vaccine containing one or more DNAs selected from the group consisting of a DNA encoding the peptide of (5), a DNA encoding the peptide of (7), and a DNA encoding the peptide of (8) as an active ingredient. .

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. The peptides of the present invention are HLA-A * 0206 or HLA-B *
It is a peptide consisting of the amino acid sequence of the CTL epitope recognized by 5603-restricted CTL.

The amino acid sequences shown in SEQ ID NOs: 1, 2, 3 or 6 are those of the CTL epitope recognized by HLA-A * 0206-restricted CTL, and the amino acid sequences shown in SEQ ID NOs: 4, 5, 7 or 8. Is a CTL epitope recognized by HLA-B * 5603 restricted CTL. The peptide of the present invention can be obtained by chemical synthesis.

A peptide consisting of the amino acid sequence of the CTL epitope is generally known to induce a CTL response (J. Immunol., 159, 1383-1392 (1997); J. Clin.
Invest., 95, 341-349 (1995)), and thus the peptide of the present invention can be used as a reagent for studying the mechanism of CTL response, and C whose amino acid sequence is a CTL epitope.
It can be used to elicit a CTL response against HCV in patients with HLA type corresponding to TL restriction.

Accordingly, the present invention also provides a vaccine prepared based on the amino acid sequence of the CTL epitope.

The vaccine of the first aspect activates a CTL response of a patient having HLA-A * 0206, and the vaccine according to (1) above.
To a vaccine containing one or more peptides selected from the group consisting of (3) and (6) as an active ingredient, as well as a DNA encoding the peptide of (1) above and a DNA encoding the peptide of (2) above A vaccine comprising, as an active ingredient, at least one DNA selected from the group consisting of the DNA encoding the peptide of (3) above and the DNA encoding the peptide of (6) above.

The vaccine of the second aspect activates the CTL response of patients with HLA-B * 5603, and comprises the peptides of (4), (5), (7) and (8) above. A vaccine containing one or more peptides selected from the group as an active ingredient, a DNA encoding the peptide of (4) above, a DNA encoding the peptide of (5) above,
A vaccine comprising, as an active ingredient, one or more DNAs selected from the group consisting of the DNA encoding the peptide of (7) above and the DNA encoding the peptide of (8) above.

HCV-specific CTLs are induced by using a peptide vaccine containing the peptide of the present invention or a DNA vaccine containing a DNA encoding the peptide of the present invention
Can be eliminated.

A vaccine containing the peptide of the present invention as an active ingredient can be produced by formulating the peptide of the present invention by a method usually used for producing a peptide vaccine containing the peptide as an active ingredient. The peptide of the present invention can be formulated by mixing with an adjuvant, a pharmaceutically acceptable carrier such as physiological saline, or by modifying it so as to have high antigenicity (for example, J. Immuno.
l., 159, 1383-1392 (1997); J. Clin. Invest., 95, 3
41-349 (1995)). The dose, administration means, etc. may be the same as those usually employed for peptide vaccines, and are appropriately selected so that the peptide of the present invention is administered in an amount sufficient to enhance the immune response.

The vaccine containing the DNA encoding the peptide of the present invention such that an effective amount of the peptide of the present invention is expressed is usually used for the production of a DNA vaccine containing the DNA encoding the peptide of the present invention. It can be produced by formulating by the method used in (for example, Vaccine, 17, 3160-3170 (1999); Hep
atology, 32, 618-625 (2000)). The dose, administration means and the like may be the same as those usually adopted for DNA vaccines, and are appropriately selected so that the peptide of the present invention is expressed in an amount sufficient to enhance the immune response in the organism to which the vaccine is administered. To be selected.

The vaccine of the present invention can be used for the prevention and treatment of diseases caused by HCV infection.

[0021]

EXAMPLES Next, the present invention will be described in detail with reference to examples, but the following examples are given only as an aid to gain a concrete recognition of the present invention, and the scope of the present invention is thereby defined. It is not limited in any way.

[0022]

Example 1 HCV gene regions (core, E1, E2, NS2,
The cDNA of NS3, NS4, NS5) was incorporated into the pAK10 plasmid, and recombinant vaccinia virus (rVAC) was prepared by homologous recombination with vaccinia virus.

[0023] Approximately 11 months after recovery from acute hepatitis, mononuclear cells (PBMC) were isolated from peripheral blood collected from patients using Ficoll. CD8 ⁺ memory T cells were isolated from PBMC using magnetic beads, rIL-2, anti-CD3 antibody, and X-ray irradiation-treated healthy subjects
Cultured in 96-well plate with PBMC. rIL-2 is 1
After addition for 2 weeks, the cells were cultured for 2 weeks and the cells grown were used as effector cells. Further, a patient PBMC prepared as described above was infected with EB virus to establish a B cell line (B-LCL). B-LCL was infected with the above-mentioned rVAC for 16 to 18 hours, which was used as a target cell.

The effector cells and target cells were reacted for 6 hours, and a ⁵¹ Cr release test was conducted. The activity was determined by the following calculation formula, and the value obtained by subtracting the activity value in the target cells infected with wild-type VAC from the activity value in the target cells infected with rVAC incorporating each region was 15%.
The above was regarded as positive. The reaction was performed using 100 effector cells per well, and as a result, 5 out of 960 wells became positive.

[0025]

## EQU1 ## Specific destruction rate (%) = (measured release-spontaneous release) / (maximum release-spontaneous release) × 100

CTL activity positive cells were cloned together with rIL-2, PHA and feeder cells in a 96 well plate. As a result, 1 CTL clone that recognizes the NS3 region was identified.
Individually isolated.

The HLA restriction of the isolated CTL clone was confirmed by B-LC
It was identified using a panel of L. That is, the patient's HLA type (A * 0206, A * 2402, B * 5201, B * 5901, Cw * 0102, Cw * 120
Using B-LCL with a partial match in 2), the HLA restriction of the isolated clones was examined. As a result, it was revealed that this clone was HLA-A * 0206-restricted, because A * 0206 showed damage to the matching target cells.

Next, 63 kinds of 20-amino acid peptides each overlapping 10 amino acids were prepared based on the 631 amino acid sequence forming the NS3 region, and the epitopes recognized by the isolated clones were identified as follows. Examine the existing areas.

To B-LCL, 16 μg of the above peptide at 10 μg / ml was added.
What was reacted for time was used as a target cell. The isolated clone was allowed to react with the target cells for 4 hours, and a ⁵¹ Cr release test was conducted. The activity was determined by the same calculation formula as above, and a positive value was obtained when the value obtained by subtracting the activity value without peptide from the activity value of each peptide was 15% or more.

As a result, the CTL clone showed a damaging effect on the cells reacted with the No. 42 peptide. further,
A peptide of 8 to 9 amino acids was synthesized based on the sequence of No. 42 (the amino acid sequence is shown in SEQ ID NO: 6), and the cytotoxic activity was examined in the same manner. As a result, the sequence of 8 amino acids of FTGDFDSV (SEQ ID NO: 1) was found. Was identified as an epitope (Table 1).

[0031]

[Table 1]   Table 1 ───────────────────────────────────   Peptide name Result Amino acid sequence SEQ ID NO ───────────────────────────────────   No. 42 + T D A L M T G F T G D F D S V I D C N T 6   GF10 − G F T G D F D S V I 9   GY10 − G Y T G D F D S V I 10   GF10C + G F T G D F D S V 2   GF10N + F T G D F D S V I 3   GF8 − G F T G D F D S 11   FV8 + F T G D F D S V 1 ───────────────────────────────────

[0032]

Example 2 A CTL clone was isolated from another patient in the same manner as in Example 1 except that peripheral blood collected about 3 months after the recovery of acute hepatitis was used.

As a result, 34 wells out of 960 wells were positive in the CTL assay, and 15 CTL clones recognizing the NS5 region were isolated by cloning. The following analysis was performed on one clone out of the 15 clones isolated.

HLA restriction of the isolated CTL clone was confirmed by B-LC
It was identified using a panel of L. That is, the patient's HLA type (A * 1101, A * 2402, B * 5201, B * 5603, Cw * 0102, Cw * 120
Using B-LCL with a partial match in 2), the HLA restriction of the isolated clones was examined. As a result, it was revealed that this clone was HLA-B * 5603-restricted, because B * 5603 showed damage to the matching target cells.

Next, 108 kinds of 15-amino acid peptides each overlapping 10 amino acids were prepared based on the 591-amino acid sequence forming the NS5 region, and the isolated clone recognized the same as in Example 1. The region where the epitope is present was examined.

As a result, the CTL clones were No. 97 and No. 105.
And showed a damaging effect on cells reacted with No. 106 peptide. The sequence of peptide No. 97 is GKYLFNWAVKTK
It was LKL (SEQ ID NO: 4). Furthermore, a peptide of 8-9 amino acids was synthesized based on the sequence of the region common to the two peptides of No. 105 (amino acid sequence is shown in SEQ ID NO: 7) and No. 106 (amino acid sequence is shown in SEQ ID NO: 8). Then, when the cytotoxic activity was similarly examined, the 9-amino acid sequence of RPRWFMLCL (SEQ ID NO: 5) was identified as an epitope (Table 2).

[0037]

[Table 2]   Table 2 ───────────────────────────────────   Peptide name Result Amino acid sequence SEQ ID NO ───────────────────────────────────   No. 105 + H S L S R A R P R W F M L C L 7   No. 106 + A R P R W F M L C L L L L S V 8   AC9 − A R P R W F M L C 12   RL9 + R P R W F M L C L 5   PRL9 − P R W F M L C L L 13 ───────────────────────────────────

[0038]

INDUSTRIAL APPLICABILITY The present invention provides a newly identified HCV-specific CTL epitope. Using a vaccine created based on this epitope, HLA-A * 0206 and HL
It is possible to activate the CTL response of patients with AB * 5603, and it is expected that HCV-infected cells will be completely eliminated.

[0039]

[Sequence list] <110> Mitsubishi Kagaku Bio-Clinical Labor atories, Inc.) <120> Hepatitis C virus-specific cytotoxic T cell recognition epitope <130> P-9066 <160> 13 <210> 1 <211> 8 <212> PRT <213> hepatitis C virus <400> 1 Phe Thr Gly Asp Phe Asp Ser Val 1 5 <210> 2 <211> 9 <212> PRT <213> hepatitis C virus <400> 2 Gly Phe Thr Gly Asp Phe Asp Ser Val 1 5 <210> 3 <211> 9 <212> PRT <213> hepatitis C virus <400> 3 Phe Thr Gly Asp Phe Asp Ser Val Ile 1 5 <210> 4 <211> 15 <212> PRT <213> hepatitis C virus Gly Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu Lys Leu 1 5 10 15 <210> 5 <211> 9 <212> PRT <213> hepatitis C virus <400> 5 Arg Pro Arg Trp Phe Met Leu Cys Leu 1 5 <210> 6 <211> 20 <212> PRT <213> hepatitis C virus <400> 6 Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile 1 5 10 15 Asp Cys Asn Thr             20 <210> 7 <211> 15 <212> PRT <213> hepatitis C virus <400> 7 His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Leu Cys Leu 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> hepatitis C virus <400> 8 Ala Arg Pro Arg Trp Phe Met Leu Cys Leu Leu Leu Leu Ser Val 1 5 10 15 <210> 9 <211> 10 <212> PRT <213> hepatitis C virus <400> 9 Gly Phe Thr Gly Asp Phe Asp Ser Val Ile 1 5 10 <210> 10 <211> 10 <212> PRT <213> hepatitis C virus <400> 10 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile 1 5 10 <210> 11 <211> 8 <212> PRT <213> hepatitis C virus <400> 11 Gly Phe Thr Gly Asp Phe Asp Ser 1 5 <210> 12 <211> 9 <212> PRT <213> hepatitis C virus <400> 12 Ala Arg Pro Arg Trp Phe Met Leu Cys 1 5 <210> 13 <211> 9 <212> PRT <213> hepatitis C virus <400> 13 Pro Arg Trp Phe Met Leu Cys Leu Leu 1 5

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Michio Imawara             1-847 Amanumacho, Saitama City, Saitama Prefecture             Oka Medical Center F term (reference) 4C084 AA13 NA14 ZA752 ZB332                 4C085 AA03 BA92 CC08 DD62 EE01                 4C086 AA01 AA02 EA16 MA01 MA04                       NA14 ZA75 ZB33                 4H045 AA10 AA30 BA15 BA17 CA02                       CA42 DA86 EA29 FA74

Claims (12)

[Claims] 1. A peptide consisting of 8 residues having the amino acid sequence shown in SEQ ID NO: 1. 2. A peptide consisting of 9 residues having the amino acid sequence shown in SEQ ID NO: 2. 3. A peptide consisting of 9 residues having the amino acid sequence shown in SEQ ID NO: 3. 4. A peptide consisting of 15 residues having the amino acid sequence shown in SEQ ID NO: 4. 5. A peptide consisting of 9 residues having the amino acid sequence shown in SEQ ID NO: 5. 6. A peptide consisting of 20 residues having the amino acid sequence shown in SEQ ID NO: 6. 7. A peptide consisting of 15 residues having the amino acid sequence shown in SEQ ID NO: 7. 8. A peptide consisting of 15 residues having the amino acid sequence shown in SEQ ID NO: 8. 9. The peptide according to claim 1, the peptide according to claim 2, the peptide according to claim 3, and claim 6.
A vaccine comprising, as an active ingredient, one or more peptides selected from the group consisting of the described peptides. 10. A DNA encoding the peptide according to claim 1, a DNA encoding the peptide according to claim 2,
A vaccine comprising, as an active ingredient, one or more DNAs selected from the group consisting of the DNA encoding the peptide according to claim 3 and the DNA encoding the peptide according to claim 6. 11. A vaccine comprising one or more peptides selected from the group consisting of the peptide according to claim 4, the peptide according to claim 5, the peptide according to claim 7, and the peptide according to claim 8 as an active ingredient. . 12. A DNA encoding the peptide according to claim 4, a DNA encoding the peptide according to claim 5,
A vaccine comprising, as an active ingredient, one or more DNAs selected from the group consisting of the DNA encoding the peptide according to claim 7 and the DNA encoding the peptide according to claim 8.