KR0137153B1 - Peptide controlling immune response against hcv - Google Patents

Abstract

The present invention relates to a novel series of peptides having an immune regulatory function of the human body against hepatitis C virus. More specifically, the present invention provides a method for preparing cytotoxic T lymphocytes (CTL) against hepatitis virus, or for inducing immune tolerance against the virus. It relates to an amino acid sequence of a peptide corresponding to a specific epitope. Five novel peptides, such as C7, C8, C14, C22 and C24, of the present invention activate CTLs that recognize hepatitis C virus antigens present in the human body, and can be recognizable by the CTLs. It may be used to prevent and treat related diseases.

Description

Peptides with Human Immune Regulation Against Hepatitis C Virus

1 (A), 1 (B), and 1 (C) are graphs showing changes in MHC Class I molecules expressed on the surface of T2 cells by fluorescence intensity. 2 is a graph showing the killing ability of T2 cells by CTL.

The present invention relates to a novel group of peptides having an immune regulatory function of the human body against hepatitis C virus.

More specifically, the present invention induces cytotoxic T lymphocytes (“CTL”) against hepatitis virus among the hepatitis C virus protein antigens, or provides immunological tolerance against the virus. The present invention relates to a method for inducing cytotoxic T-lymphocytes having specificity for hepatitis C virus and by using the amino acid sequence of the peptide corresponding to a specific epitope of the antigen to be induced. When the hepatitis C virus is infected by the human body, various physiological phenomena occur in the human body to remove the virus. One of the most important phenomena is the destruction and removal of the already infected cells. Ultimately, it can recover from viral infections. The destruction of these infectious cells is caused by another type of cell with cytotoxicity, called 'cytotoxic T-lymphocytes' (CTLs). In order for this CTL to exert its cell killing capacity, it must first recognize some of the proteins that make up the infected virus. Generally, viral proteins are synthesized in infected cells and then converted into short peptide forms consisting of 8 to 15 amino acids through intracellular degradation. When some of the generated peptides appear on the cell surface in combination with major histocompatibility complex molecules (hereinafter referred to as 'MHC'), CTL recognizes and destroys infected cells. On the other hand, MHC is divided into two types, the first group (Class I) and the second group (Class II), of which the first group (Class I) is known to play a more important role in the induction of CTL. However, it is known that the site (peptide) of the protein recognized by the CTL is limited to a specific amino acid sequence among peptides resulting from the degradation of the viral protein [Fremont, D. et al., Science, 257: 919-926 (1992); Matsumura, M. et al., Sinence, 257: 929-934 (1992). Thus, knowing the amino acid sequence of such a specific site enables the chemical synthesis and production of peptides corresponding to the amino acid sequence. By artificially inducing a CTL that specifically recognizes hepatitis virus in the human body, or by regulating other immune responses such as immunosuppression, it is effective in preventing and treating diseases caused by hepatitis virus. Below, several researchers have made efforts to determine the amino acid sequence of peptides recognized by CTLs among the amino acid sequences of hepatitis virus proteins, and as a result, several specific amino acid sequences have already been reported and several more are expected in the future. However, in the prevention and treatment of hepatitis virus-related diseases, In order to use silver peptides, not only it is necessary to use as many types of peptides as possible, but also the efficacy of the peptides may vary depending on the type of peptide, and thus the amino acid sequence of the newly discovered peptides has a very important meaning. The inventors of the present invention have found a group of peptides that have not been reported so far among the peptides of hepatitis C virus recognized by CTL, and completed the present invention.

Amino acid sequences used in the present invention are abbreviated as follows according to the IUPAC nomenclature;

Amino acid abbreviation

Alanina A

Arginine R

Asparagine N

Aspartic acid D

Cysteine c

Glutamine Q

Glutamic acid E

Glycine g

Histidine H

Isoleucine I

Leucine l

Lysine K

Methionine M

Phenylalanine F

Proline p

Serine s

Threonine T

Tryptophan W

Tyrosine y

valine V

The novel peptide sequence of the present invention is as shown in Table 1 below.

TABLE 1

Amino acid sequence and position in the protein recognized by CTL among hepatitis C virus proteins

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Discovery of Peptides of Hepatitis C Virus and Determination of Amino Acid Sequences The peptides used in the present invention are based on the fact that peptides capable of binding to MHC class I molecules have a specific amino acid sequence and Given the structural characteristics of the Class I molecules, the amino acid sequences of the peptides that are likely to bind to the MHC Class I molecules were determined from the entire amino acid sequence of the hepatitis C virus antigen protein. In other words, the peptide capable of binding to HLA-A2, the MHC class I molecule of humans, is composed of leucine, valine, or iso-amino acid at the second position from the N terminus and at the C terminus. Based on the fact that it consists of leucine, etc. (Matsumura, M. et al., Sinence, 257: 929-934 (1992)), an amino acid sequence that satisfies these conditions may be used for the hepatitis C virus antigen protein. It was found from the entire amino acid sequence, and among them, the one most likely to bind appropriately to the tertiary structure of the HLA-A2 molecule was selected and chemically synthesized according to their amino acid sequence.

Example 2 Synthesis of Peptides and Determination of Binding to MHC Molecules Chemical methods of 24 peptides having different amido acid sequences, including peptides as shown in Table 1 above, from amino acid sequences of hepatitis C virus proteins Synthesis was carried out, and these were dissolved in phosphate buffered saline (hereinafter referred to as 'PBS'). To determine whether these peptides can bind to MHC molecules, especially Class I molecules that are involved in the induction of CTLs, these peptides can be started at 100 μg / ml in T2 cell lines, starting with 50, 25, 12.5, 6.25, 3.1, 1.6, 0.8, 0.4 ㎍ / ml to each of the concentration was added and reacted for 4 hours at 37 ℃. The cells were then washed with PBS to remove unreacted peptides, and the cells were treated with anti-HLA-A2.1 antibody BB7.2, a type of human MHC Class I molecule. Was added, and then reacted at 4 ° C. for 1 hour. The cells were washed again with PBS and added thereto with fluorescein isothiocyanate (FITC) (antimouse immunoglobulin antibody) for 1 hour and reacted at 4 ° C. Spectrofluorometer) was used, and the results are shown in FIG. As shown in FIG. 1, 9 types of peptides such as C1, C2, C7, C8, C14, C15, C20, C22 and C24 have good cooperation with MHC Class I molecules on T2 cell surface. appear. In addition, the types of peptides used in this experiment are as shown in Table 2 below.

TABLE 2

Amino Acid Sequences of Synthetic Peptides

Amino acid sequences are described according to the nomenclature of IUPAC.

The T2 cell line used in this example has an abnormality in the intracellular peptide's migration pathway, and thus MHC Class I molecules cannot properly bind to the peptide, resulting in destabilization of the molecular structure, resulting in normal growth temperature. Incubation at 37 ° C. maintains a low level of expression of MHC molecules. However, when a peptide capable of binding an MHC molecule in the culture of T2 cells is present in the culture medium, the peptide binds to the MHC molecule on the surface of the T2 cell, resulting in an increase in the molecular stability of the MHC molecule. Since the expression of the protein was increased, the degree of binding of the specific peptide to the MHC molecule on the T2 cell surface could be quantified by measuring the degree of binding of the BB7.2 antibody to the cell surface.

Example 3 Measurement of Recognition Between Peptide and CTL

The fact that a particular peptide binds to the MHC molecule is not necessarily recognized by CTL, so it is recognized by the CTL that recognizes hepatitis C virus that actually exists in the human body with nine peptides determined to have good binding capacity. The following experiment was conducted to see if it was possible. In other words, blood collected from general blood donors was determined to have positive hepatitis C virus antibody (ELISA value: 1.0 or more), and the cells were collected by centrifugation to remove red blood cells from the blood cells. Peptides were added to a concentration of 10 μg / ml, and then cultured in a CO 2 incubator containing 5% carbon dioxide at 37 ° C. Two days after the start of the culture, 10 units of interleukin-2 was added to the culture medium, and further cultured for 3 days, and then the blood cells of the same MHC as stimulating cells were inactivated with mitomycin C. Peptide and interleukin-2 were added. After further culturing them for 7 days, it was determined whether these blood cells recognized and destroyed T2 cells bound to the surface of a specific peptide, and the results are shown in Table 3 and FIG. 2. In FIG. 2, the percentage of TTL cells lysing the degree to which CTL recognizes each peptide according to the effector, that is, the ratio of target cells to CTL, that is, T2 cells. NC indicates negative control.

TABLE 3

Apoptosis of Peptide Specific T Cells to Target Cells Sensitized with Synthetic Peptides

The method for measuring the destruction of T2 cells by cultured blood cells was as follows: 100 μg / ml peptide was added to T2 cells and reacted at 37 ° C. for 3 hours, and then the cells were washed sufficiently to remove unreacted peptides. The T2 cells were removed, and radioactive sodium chromate (Cr51) was added and reacted for 2 hours in a CO ₂ incubator at 37 ° C.

After the reaction, the blood cells washed with PBS were mixed at a constant rate and reacted for 4 hours in a CO ₂ incubator at 37 ° C. By collecting the reacted cultures and measuring the intensity of gamma rays, the ability of blood cells to kill T2, a target cell, was measured.

As shown in Table 3 and FIG. 2, C2, C7, C8, C14, C20, C22, and C24 were found to be recognized by cultured blood cells. Among them, C2 and C20 are known, It was confirmed that the experimental method used in this example serves as a positive control showing that the appropriate. In the above, as described and demonstrated in detail, five peptides of the present invention, such as C7, C8, C14, C22, and C24, activate the CTL that recognizes the hepatitis C virus antigen present in the human body, and recognize it again by the CTL. It may be used as a peptide for the prevention and treatment of hepatitis virus-related diseases.

Claims (5)

Hepatitis C hepatitis virus peptide recognized by Cytotoxic T lymphocytes and consisting of the following amino acid sequences: S M V G N W A K V (wherein the amino acid sequence is described according to the nomenclature of IUPAC) Hepatitis C hepatitis virus peptide recognized by Cytotoxic T lymphocytes, consisting of the following amino acid sequence: C L V D Y P Y R L (wherein the amino acid sequence is described according to the nomenclature of IUPAC) Hepatitis C hepatitis virus peptide recognized by Cytotoxic T lymphocytes and consisting of the following amino acid sequences: R L V P G A A Y A V (wherein the amino acid sequence is described according to the nomenclature of IUPAC) Hepatitis C hepatitis virus peptide recognized by Cytotoxic T lymphocytes and consisting of the following amino acid sequences: I L A G Y G A G V (wherein the amino acid sequence is described according to the nomenclature of IUPAC) Hepatitis C hepatitis virus peptide recognized by Cytotoxic T lymphocytes and consisting of the following amino acid sequence: R L V F P D L G V (wherein the amino acid sequence is described according to the nomenclature of IUPAC)