KR0166425B1 - Hantan virus-derived peptide having immuno-modulation function in human - Google Patents

Abstract

The present invention relates to a novel series of peptides having an immunomodulatory function of the human body against hantan virus. More specifically, the present invention induces cytotoxic T-lymphocytes (CTL) against the N protein of Ghantan virus and Ghan, G2 protein antigens, or provides immunological tolerance against the virus. It relates to the amino acid sequence of the peptide corresponding to the epitope of the antigen to be induced. Nine novel peptides, including H1, H2, H3, H4, H7, H8, H11, H33, H37, of the present invention activate CTLs that recognize the Hantan virus antigens present in the human body, and can be recognized by the CTLs again. As a peptide, it may be used for the prevention and treatment of Hantan virus-related diseases.

Description

Peptide with human immunomodulatory function against Hantan virus

1 is a graph showing the change in MHC Class I molecules expressed on the surface of T2 cells by fluorescence intensity.

2 (a) and (b) are graphs showing the killing ability of T2 cells by CTL.

The present invention relates to a novel group of peptides having an immunomodulatory function of the human body against hantan virus. More specifically, the present invention induces cytotoxic T lymphocytes (hereinafter referred to as 'CTLs') against, or is immunized against, the Hantan virus of the N, G1 and G2 protein antigens of the Hantan virus. An amino acid sequence of a peptide corresponding to an epitope of an antigen inducing tolerance and a method for inducing cytotoxic T-lymphocytes having specificity for agar virus.

When Hantan virus is infected in the human body, various physiological phenomena occur in the human body to remove the virus. One of the most important phenomena is destroying and removing already infected cells. It can recover from infection. The destruction of these infected 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. In general, 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. (See: Fremont, D. et al., Science. 257: 919-926 (1992); Matsumura, M. et al., Science, 257: 929-934 (1992). Therefore, knowing the amino acid sequence of such a specific site, it is possible to synthesize and produce a peptide corresponding to the amino acid sequence by chemical method, by using this artificially induced or immunized CTL that specifically recognizes several viruses in the human body By regulating other immune responses, such as inhibition, they can be effective in the prevention and treatment of diseases caused by viruses.

Against this background, several researchers have identified the amino acid sequences of peptides recognized by CTL among the amino acid sequences of several antigenic proteins for human immunodeficiency virus (HIV) hepatitis B and C and influenza viruses. Efforts have been made to find and, as a result, several specific amino acid sequences have already been reported, and the inventors have also revealed new amino acid sequences of CTL inducing peptides against hepatitis B and C viruses.

However, in the case of Hantan virus, much research has been conducted only on humoral immunity that induces neutralizing antibodies by G1 and G2 glycoproteins (Michael, NP et al., J. Virol., 62: 696). (1988); Jiro, A. et al., J. Gen, Virol., 70: 615 (1989); Xiao, X, et al., Am. J. Trop. Med. Hyg., 47: 397 (1992) )), Cellular immunity against N protein and G1 and G2 glycoproteins, or CTL induction, has not been studied at the peptide level. Accordingly, the present inventors have tried to find the amino acid sequence of the peptide recognized by CTL among the amino acid sequences of the N, G1 and G2 proteins of the Hantan virus in order to prevent and treat Hantan virus disease by the immunomodulatory function by the CTL inducing peptide. Finally, for the first time, a group of peptides having a specific amino acid sequence recognized by CTL was found.

After all, the main object of the present invention is to provide the amino acid sequence of a peptide recognized by CTL in the N, G1 and G2 proteins of the Hantan virus.

It is another object of the present invention to provide a method of inducing cytotoxic T-lymphocytes having specificity for agar virus using an electric peptide.

The novel peptide sequences found in the present invention are shown in Table 1 below.

In addition, the present invention includes not only peptides including the foregoing nine amino acids, but also all peptides and functional equivalents thereof further including at least one to five amino acids before and after the foregoing peptides. In the present invention, the term "functional equivalent" refers to all peptides in which one or two amino acid sequences of the peptide are substituted. For example, substitutions may include Gly, Ala; Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And combinations such as Phe, Tyr.

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 according to the gist of the present invention.

Example 1

Discovery of Astrocytes Virus Peptides and Determination of Amino Acid Sequences

In view of the structural characteristics of the peptide MHC group 1 (Class I) molecules used in the present invention, peptides capable of binding to MHC Class I molecules from the whole amino acid sequence of the antigenic proteins of the Hantan virus, N, G1 and G2 Amino acid sequence was determined. That is, the teptide capable of binding to HLA-A2, the first group of human MHC (Class I) molecules, 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 aliphatic nonpolar amino acids such as isoleucine (Matsumura, M. et al., Science, 257: 929-934 (1992)), these conditions are met. The amino acid sequences to be found were found from the whole amino acid sequences of the Hanta virus antigen proteins, and among them, those most likely to be appropriately bound to the tertiary structure of the HLA-A2 molecule were selected and chemically synthesized according to their amino acid sequences.

Example 2

Synthesis of Peptides and Measurement of Binding to MHC Molecules

37 peptides having different amino acid sequences, including peptides as shown in Table 1 above, from the amino acid sequence of the Hantan virus protein were synthesized by chemical methods, and these were referred to as phosphate buffered saline (PBS). Dissolve). To determine whether these peptides could bind to MHC molecules, especially the first group (Class I) molecules involved in the induction of CTL, these teptides were started at 100 μg / ml in T2 cell lines starting from 64, 32, 16, 8, 4, 2, 1 ㎍ / ㎖ was added to each concentration 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. After the addition, it was reacted again at 4 ° C. for 1 hour. The cells were washed again with PBS, added with anti-mouse immunoglobulin antibody attached to fluorescein isothiocyanate (FITC), and reacted at 4 ° C. for 1 hour. The fluorescence intensity of the cells was measured using a spectrofluorometer, and the results are shown in FIG. As shown in FIG. 1, 20 types of H1, H2, H3, H4, H5, H7, H8, H10, H11, H17, H20, H21, H25, H26, H28, H30, H31, H33, H36 and H37 Peptides showed good binding to MHC class 1 molecules on the surface of T2 cells. In addition, the types of peptides used in this experiment are as shown in Table 2 below. Up to H1-H8 are peptides derived from N protein, H-9-H26 is from G1 protein, and up to H27-37 are peptides from G2 protein.

T2 cell line used in this example is abnormal in the migration pathway of intracellular peptides, MHC class 1 molecules can not be properly combined with the peptides resulting in destabilization of the molecular structure and consequently the normal growth temperature 37 When cultured at ℃, the expression level of MHC molecules is maintained low. However, when a peptide capable of binding MHC molecules in the culture of T2 cells is present in the culture medium, the peptide binds to the MHC molecules on the surface of the T2 cells. As a result, the molecular stability of the MHC molecules increases, resulting in MHC. Since the expression of the molecule is increased, the degree of binding of the specific peptide to the MHC molecule of the T2 cell surface can be quantified by measuring the degree of binding of the BB7.2 antibody to the cell surface.

Example 3

Measurement of Awareness Between Peptide and CTL

The fact that a specific peptide binds to MHC molecules is not immediately recognized by CTL, so it is determined whether CTL recognizes a peat virus that actually exists in the human body with 20 peptides determined to have good binding ability. In order to find out, the following experiment was performed.

In other words, blood collected from general blood donors was confirmed to have a positive anti-BTS virus (ELISA value: 1.0 or higher), and the cells were collected by centrifugation to remove red blood cells from the blood cells, and the peptide was added to the remaining white blood cells. The solution was added to a concentration of 10 µg / ml, and then cultured in a CO incubator containing 5% carbon dioxide at 37 ° C. After 2 days of incubation, 10 units of interleukin-2 was added to the culture medium, and further cultured for 3 days, and then inactivated blood cell cells of the same MHC as mitomycin C as stimulating cells. Peptide and interleukin-2 were added. After further culturing them for 7 days, it was measured 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.

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. Sodium Chromatide (Cr) ) Was added and reacted for 2 hours in a CO incubator at 37 ℃. After completion of the reaction, the solution was sufficiently washed with RPMI culture to remove unreacted sodium chromate, and the blood cells washed with PBS were mixed at a constant rate and reacted for 4 hours in a CO incubator at 37 ° C. By collecting the reaction medium and measuring the intensity of gamma rays, the ability of blood cells to kill T2, a target cell, could be measured.

As shown in Table 3, H1, H2, H3, H4, H7, H8, H11, H33, and H37 were found to be recognized by the cultured blood cells.

As described and demonstrated in detail above, nine peptide groups, such as H1, H2, H3, H4, H7, H8, H11, H33, H37, of the present invention activate CTLs that recognizes a hantan virus antigen present in the human body, As a peptide that can be recognizable by the CTL, it may be used for the prevention and treatment of a disease related to hantan virus.

Claims (10)

Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTL) and exhibit cell killing ability by virus: S M L S Y G N V L (Amino acid sequence is described according to the nomenclature of IUPAC.) Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTL) and exhibit cell killing ability by virus: Y L T S F V V P (Amino acid sequence is described according to the nomenclature of IUPAC). Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTLs) and exhibit cell killing ability by virus (I L L K A L Y M L (wherein the amino acid sequence is described according to the nomenclature of IUPAC). Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTLs) and exhibit cell killing ability by virus: G L Y P A Q I K A (Amino acid sequence is described according to the nomenclature of IUPAC). Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTL) and exhibit cell killing ability by virus: K L L P D T A A V (Amino acid sequence is described according to IUPAC nomenclature). Peptides comprising the following peat virus peptides and are recognized by cytotoxic T lymphocytes (CTLs) and exhibit cell killing ability by viruses: T L A Q S L I D V (Amino acid sequence is described according to the nomenclature of IUPAC). Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTL) and exhibit cell killing ability by virus: (V, A S L V X P V (Amino acid sequence is described according to the nomenclature of IUPAC). Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTLs) and exhibit cell killing ability by virus: G I F S G N W I V (Amino acid sequence is described according to the nomenclature of IUPAC). Peptides comprising the following Hantan virus peptides and are recognized by cytotoxic T lymphocytes (CTL) and exhibit cell killing ability by virus: (VL L S I L C P V Y L (Amino acid sequence is described according to the nomenclature of IUPAC). A method of inducing cytotoxic T lymphocytes (CTLs) having specificity for a hantan virus from mammals other than humans using at least one peptide of claim 1.