RU2189989C2 - PEPTIDE ANTIGEN OBTAINED FROM HEPATITIS b PROTEIN A (VARIANTS), PH-SENSITIVE LIPOSOME FOR INDUCTION OF CELLULAR IMMUNITY AGAINST HEPATITIS B VIRUS - Google Patents

Abstract

FIELD: medicine, pharmacy, biotechnology. SUBSTANCE: invention relates to liposomes comprising novel peptide antigens that participate in regulation of human immunity against hepatitis B virus, namely, to groups of peptides corresponding to antigen epitopes obtained from hepatitis B virus (HBV) protein X. Peptide antigens induce cytotoxic T-lymphocytes against hepatitis B virus or immunological tolerance with respect to virus. pH- Sensitive liposomes comprise peptide antigens for induction of cellular immunity to provide their production by cytotoxic T-lymphocytes showing specificity to virus. Indicated liposomes can be used for the development of potential medicinal agents used for prophylaxis and treatment of hepatitis B virus-associated diseases since peptide antigens obtained from the protein X (for example, X3, X4, X5, X6 and X7) activate cytotoxic T- lymphocytes that can recognize antigens of hepatitis B virus in human body and they can be recognized by cytotoxic T-lymphocytes also. EFFECT: antigen and liposome indicated above, valuable medicinal properties. 12 cl, 5 tbl, 4 dwg, 6 ex

Description

 The invention relates to liposomes containing new peptide antigens that are involved in the regulation of human immunity against hepatitis B virus ("HBV"), and more particularly to groups of peptides corresponding to epitopes of antigens derived from HBV protein X that induce cytotoxic T lymphocytes ( "CTL") against viruses, or immunological tolerance to viruses, and pH-sensitive liposomes containing the indicated groups of peptides, to induce cellular immunity in such a way that CTL specific Sgiach against this virus.

Description of the prior art
When the human body is infected with HBV, various physiological reactions occur in it to remove the virus. Among these reactions, one of the most important is the destruction and removal of infected cells, which, in the end, leads to a recovery from a viral infection. The destruction of infected cells is carried out by cells of another type, which have cytotoxicity, and which are called CTL. In order to exhibit cytotoxicity, CTLs must first recognize certain peptides derived from invading viral proteins. Usually, viral proteins are synthesized inside infected cells, and then they turn into short peptides containing from 8 to 15 amino acids as a result of intracellular degradation. If some of the peptides thus formed appear on the cell surface in combined form with a molecule of the intracellular major histocompatibility complex ("MHC"), CTL recognizes them in order to destroy the infected cells. On the other hand, MHCs are subdivided into class I and class II, and it was known that class MHCs play a more important role in CTL induction compared to class II CTL. However, it was reported that the sites of proteins (peptides) recognized by CTL are limited only to those that contain specific amino acid sequences among all peptides that are formed upon the destruction of viral proteins (see: Fremont, D. et al., Science, 257: 919- 926 (1992); Matsumura, M. et al., Science, 257: 929-934 (1992)).

 Accordingly, elucidation of the amino acid sequences of these specific sites will allow the chemical method to synthesize and obtain peptides corresponding to these amino acid sequences, and such use of these peptides will artificially induce CTLs that specifically recognize hepatitis virus in the human body, or regulate other immune reactions, such as immunosuppression, which will lead to effective prevention and treatment of diseases caused by the hepatitis virus.

 Knowing this, many researchers have attempted to find the amino acid sequences of peptides recognized by CTL among peptides resulting from the breakdown of hepatitis virus proteins. As a result, several specific amino acid sequences were found, and it was expected that several more sequences would be found in the future. In addition, in order to use such peptides for the prevention and treatment of hepatitis-related diseases, it is necessary that as many peptides as possible be used simultaneously, and their effectiveness could vary depending on the type of peptides. Therefore, the disclosure of the amino acid sequences of such peptides is very important.

 Peptides for the prevention and treatment of hepatitis B that are reliably recognized by CTL come mainly from protein S (surface antigen) and protein C (nuclear) HBV. However, peptides derived from protein X, one of the HBV antigenic proteins, have not yet been reported, despite the fact that a number of studies using transgenic mice have clearly shown that protein X is directly related to HBV-associated liver cancer (see: Hoyhne et al, EMBO J., 9: 1137-1145 (1990); Kim et al., Nature, 351: 317-320 (1991)).

 On the other hand, if phospholipids are dispersed in water, the polar head groups are oriented toward water, and the hydrophobic tails are assembled due to hydrophobic interactions, spontaneously forming spherical closed bilayer vesicles, the so-called "liposomes". Such liposomes are widely used as carriers in drug delivery systems, as well as in modeling studies of biological membranes (see: Lee, Jw and Kim, H., Arch. Biochem. Biophys., 297: 354 (1992); Hahn, KH and Kim , H., J. Biochem., 110: 635 (1991); Yun, CH and Kim, H., J. Biochem., 105: 406 (1989); Kim, J. and Kim, H., Biochemistry, 25 : 7867 (1986); Kim, J. and Kim, H., 15 Korean Biochem. J., 18: 403 (1985)).

 It has recently been reported that liposomes can be used in the field of immunology, which accelerates the study of liposomes as carriers of vaccines or adjuvants (see: Rooijen, NV, Vaccine, 11: 1170 (1993). Typically, if the liposomes are administered intravenously, they are easily captured by macrophages (" MO "), which are present in large quantities in the blood, which plays an important role in the processing of liposomal antigen, as well as in the immunological processing of antigen.

 In addition, methods have recently been developed to induce a CTL reaction specific for certain proteins or peptides using pH-sensitive liposomes (see: Readdy, R. et al., J. Immunol. Methods, 141: 157 (1991); Zhou, F. et al., J. Immnunol. Methods, 145: 143 (1991); Nair, S. et al., J. Exp. Med., 175: 609 (1992); Harding, CV et al., J. Immunol. 147: 2860 (1991); Zhou, F. and Huang, L., Vaccine, 11: 1139 (1993)), and pH-sensitive liposomes have been widely used as carriers and adjuvant systems of a protein or peptide antigen to produce a subunit vaccines. In this regard, the authors of the present invention have disclosed a method of obtaining a pH-sensitive liposome, which allows for the selective transport of anti-blastoma drugs (see: Choi, M.J. et al., J. Biochem., 112: 694 (1992)).

SUMMARY OF THE INVENTION
The present invention provides a series of peptides specifically recognized by CTL among peptides derived from HBV protein X. Also proposed are pH-sensitive liposomes containing peptide antigens derived from HBV protein X. The inventors of the present invention have found that these liposomes can be used to create the intended therapeutic agents for the prevention and treatment of HBV-related diseases by inducing cellular immunity so that CTLs specific for HBV peptide antigens can be generated.

 Therefore, the main objective of the invention is the creation of peptide antigens derived from HBV protein X, which can be recognized by CTL and exhibit cytotoxicity against the virus.

 Another objective of the invention is the creation of pH-sensitive liposomes, including these peptide antigens that can induce cellular immunity.

Brief Description of the Drawings
The above and other objects and features of the present invention will become more apparent from the following description, given together with the accompanying drawings, where:
FIG. 1 is a graph showing a change in the number of MHC class I molecules expressed on the surface of a T2 cell, which is presented as fluorescence intensity. Figure 2 is a graph showing CTL cytotoxicity against T2 cells.

 Fig. 3A is a graph showing tumor formation in nude mice infected with human hepatocellular carcinoma.

 FIG. 3B is a graph showing tumor regression in nude mice after treatment with CTL stimulated with peptide antigens derived from HBV protein X.

DETAILED DESCRIPTION OF THE INVENTION
Based on the discovery that peptides that bind to MHC class I molecules have specific amino acid sequences, peptides with potential to bind to MHC class I molecules were sequenced in the complete amino acid sequences of HBV protein X. That is, since peptides that can bind to HLA-A2, the human MHC class I molecule, contain leucine, valine or isoleucine at the C-ends and in the second position from the N-ends (see: Matsumura, M. et al. , Science, 257: 929-934 (1992)), amino acid sequences satisfying these conditions were screened from the complete amino acid sequences of HBV protein X, and peptides that have the potential to bind to an HLA-A2 molecule, i.e. peptide antigens that can induce a CTL response by binding to the human MHC class I molecule were selected from peptides resulting from the degradation of HBV antigen protein X, based on the three-dimensional structure of the molecule (see table 1 below). Then, the peptides were chemically synthesized in accordance with the amino acid sequences thus determined. Amino acid sequences of peptides derived from HBV X protein, i.e. X3, X4, X5, X6 and X7 are presented further in table 1.

 The present invention encompasses not only the peptides described above consisting of 9 amino acids, but also peptides to which are added 1 to 5 amino acids from the N- or C-termini of these peptides, as well as their functional equivalents. In the present invention, the term "functional equivalents" is used to mean all peptides substituted with 1 or 2 amino acids among the amino acid sequences of these peptides. For example, such substitutions include combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Turkish.

 To obtain pH-sensitive liposomes, including peptide antigens derived from HBV protein X, phosphatidylethanolamine-β-oleoyl-γ-palmitoyl (POPE) and cholesterol hemisuccinate (CHOH) are mixed in a molar ratio of from 6: 4 to 8: 2, more preferably 7 : 3, or POPE, phosphatidylethanolamine (PE) and CHOH are mixed in a molar ratio of from 3: 3: 4 to 4: 4: 2, more preferably 3.5: 3.5: 3, to obtain a thin phospholipid membrane. Then, the liposomes thus obtained are added to a buffer solution containing the indicated peptide antigens obtained from HBV protein X. In this regard, liposomes comprising peptide antigens derived from HBV protein X are prepared by mixing 1 or more types of peptide antigens with a phospholipid in a molar ratio of from 1: 5 to 1:25, more preferably 1:20. In addition, pH-sensitive liposomes prepared in accordance with the present invention may further include up to 1 mol% of monophosphoryl lipid A as a phospholipid.

 The liposomes of the present invention encapsulate peptide antigens that induce a CTL response in humans, and pH-sensitive liposomes that are used as peptide carriers can also induce a CTL reaction specific for the virus from which the encapsulated peptides are derived. Naturally, it has been clearly shown that pH-sensitive liposomes including these peptides can induce cellular immunity in humans.

When describing the amino acid sequences of the peptides of the present invention, the following single-letter characters are used in accordance with the IUPAC-IUB standards:
Amino Acid - Symbol
Alanine - A
Arginine - R
Asparagine - N
Aspartic Acid - D
Cysteine - C
Glutamine - Q
Glutamic acid - E
Glycine - G
Histidine - N
Isoleucine - I
Leucine - L
Lysine - K
Methionine - M
Phenylalanine - F
Proline - P
Serene - S
Threonine - T
Tryptophan - W
Tyrosine - Y
Valin - V
Further, the present invention is illustrated by examples, which should not be construed as limiting the scope of the invention.

Example 1. Determination of peptides with binding potential with MHC molecules of protein X
Since peptides that can bind to HLA-A2, the human MPC class I molecule, contain leucine, valine or isoleucine from the C ends, and in the second position from the N ends (see: Matsumura, M. et al., Science , 257: 929-934 (1992)), amino acid sequences satisfying these conditions are first screened from the full amino acid sequences of HBV protein X, and ultimately peptides that have the potential for binding to HLA-A2 molecules are selected, given the three-dimensional structure of the molecule (see table 1). Chemical peptides are then synthesized by methods known to those skilled in the art based on the amino acid sequences thus determined.

Example 2. The synthesis of peptides and the determination of their binding to MHC molecules
Seven different peptides, including the peptides indicated previously in Table 1, which were obtained from HBV protein X, were chemically synthesized and dissolved in phosphate buffered saline (hereinafter referred to as “PBS”). To study the possibility of binding of these peptides to MHC class I molecules associated with induced CTL, each of the peptides is added to T2 cell lines at a concentration of 100, 64, 32, 16, 8, 2, and 1 μg / ml, respectively, and allowed to interact at 37 ^o C for 4 hours. The cells are then washed with PBS to remove unreacted peptides, BB7.2 anti-HLA-2A.1 antibody, MHC class I molecules are added and allowed to react at 4 ^{° C.} for 1 hour. The cells were washed again with PBS, fluoresceinisothiocyanate (FITC) conjugated to a mouse immunoglobulin antibody was added, and the reaction was allowed to proceed at 4 ^{° C.} for 1 hour. Then determine the fluorescence of the cells using a fluorocytometer (FACScan, BECTON DICKINSON), and the results are presented in figure 1 below.

 As can be seen in FIG. 1, 5 peptides were discovered, such as X3, X4, X5, X6 and X7, which successfully bind to MHC molecules of class I on the surface of T2 cells. The peptides used in these experiments are characterized in table 2.

The T2 cell line used in this example has a violation in the scheme of intracellular transport of peptides, and does not allow proper binding of the MHC class I molecule to the peptide with the formation of an unstable molecular structure, which leads to low expression of MHC molecules during cultivation at 37 ^{° C.} normal temperature for growth. However, if T2 cells are cultured in a medium containing peptides that can bind to MHC molecules, these peptides bind to MHC molecules on the surface of T2 cells, increasing the stability of MHC molecules, which leads to increased expression of MHC molecules. Knowing this, the binding of specific peptides to MHC molecules on the surface of T2 cells can be analyzed by determining the degree of binding of BB7.2 antibodies to the cell surface.

Example 3: Determination of CTL Peptide Recognition
The binding of a particular peptide to an MHC molecule cannot prove the recognition of the CTL peptide. Accordingly, the following experiment was conducted to investigate whether the peptides used to determine binding to MHC molecules in Example 2 are recognized by CTLs that can recognize HBV in humans.

That is, blood that is positive for anti-HBV antibodies (ELISA value: 1.0 or more) is taken from conventional donors and centrifuged to collect blood cells. These peptides are added at a concentration of 10 μg / ml to the white blood cells that remained after the removal of red blood cells (erythrocytes) from blood cells, and incubated at 37 ^{° C.} in a CO ₂ incubator in which a concentration of 5% CO _{2 is} maintained. After 2 days of cultivation, 10 units of interleukin-2 are added to the culture medium and the cells continue to cultivate for another 3 days. Then, blood cells with the same MHCs are inactivated with mitomycin C and added to the culture medium as stimulating cells. Both peptides and interleukin-2 are added to the medium, and the cells continue to cultivate for 7 days. It is then determined whether blood cells are recognized and whether T2 cells containing a particular peptide on their surface are destroyed, the results obtained are presented in table 3 and figure 2, respectively.

The destruction of T2 cells by cultured blood cells is determined as follows: 100 μg / ml of the peptide is added to T2 cells and allowed to react at 37 ^{° C.} for 3 hours. The cells are then washed thoroughly to remove unreacted peptides, radioactive sodium chromate (Cr ⁵¹ ) is added and allowed to react at 37 ^{° C.} for 2 hours in a CO ₂ incubator. Then they are thoroughly washed with RPMI medium to remove unreacted chromate, mixed with blood cells washed with PBS in a certain ratio and allowed to react at 37 ^° C for 4 hours in a CO ₂ incubator. The resulting medium is collected and the intensity of gamma rays is determined to determine the cytotoxicity of blood cells in relation to T2 cells, i.e. target cells. As can be seen from table 3, it was found that X3, X4, X6, X7 and P5 are recognized by cultured blood cells, with P5 being a known peptide that plays the role of a positive control (see: Nayersina, R. Et al., J. Immunol., 150: 4659-4671 (1993)), which indicates the suitability of the experimental techniques used in this example.

Example 4: Obtaining pH-sensitive liposomes containing peptide antigens
A phospholipid containing POPE / CHOH (7: 3 molar ratio) or POPE / PE / CHOH (3.5: 3.5: 3 molar ratio) is added to a glass vessel and dissolved in an organic solvent. The vessel is then filled with nitrogen gas to form a thin lipid film in the vessel. Then, a buffer (pH 8.0) is added to the lipid film containing the peptides to be encapsulated for sufficient moisture, and mixed vigorously to obtain multilayer vesicles ("MLV"), from which small single-layer vesicles ("SUV") are prepared in the result of sonication. SUVs thus obtained are incubated at room temperature for 2 to 12 hours, and the rapid freezing of SUVs with liquid nitrogen and the slow thawing at room temperature are repeated 3-4 times to obtain liposomes including peptides. Thus, liposomes are ultimately obtained by mixing peptides and phospholipids in a molar ratio of 1:20. In this regard, the peptides used are peptide antigens that are derived from HBV protein X and which can induce a CTL reaction by binding to MHC class I molecules (see table 1).

The physical stability and pH sensitivity of the liposomes thus obtained are examined as follows, which showed a weak destruction of the liposomes when treated with ethanol and a very small decrease in the content of liposomes compared to conventional pH-sensitive liposomes:
(1) Determination of physical stability
The physical stability of the liposomes thus obtained is determined by measuring the change in the turbidity of the solution with the addition of ethanol. Since ethanol treatment destroys liposomes, which leads to a decrease in turbidity, the stability of liposomes is determined by measuring the change in absorption at 400 nm. As a result, it was found that liposomes containing the peptides of the present invention have higher physical stability than conventional pH-sensitive liposomes.

(2) Determination of pH sensitivity
12.5 mM disodium salt of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), 45 mM paraxylene bis (pyridinium) bromide (DPX), 68 mM NaCl and 10 mM HEPES buffered solution (pH 8.0) encapsulate in liposomes containing peptides, and FNTA and DPX that were not encapsulated are removed using a Sephadex G-50 column (1 x 20 cm) to collect only the liposome solution. The concentration of phospholipids in liposomes is determined by the method of Vaskovsky (see: Vaskovsky, VE, Kostetsky, EY and Vasendin, IM, J. Chromatogr., 114: 129 (1975)).

In this experiment, to determine the pH sensitivity, the fluorescence intensity of the liposome solution passed through the column is taken as "0% scattering", and the fluorescence intensity of the liposome stream destroyed by the Triton X-100 is taken as "100% scattering". Concentrations of liposomes (30 μM each) are added to 2 ml of buffer solution with the corresponding pH values (7.5, 6.9, 5.9, 5.4, 4.9 and 4.5), and left to stand at 37 ^o С within 30 minutes. Then determine the pH sensitivity by measuring fluorescence. In this case, fluorescence is measured using exciting radiation with a wavelength of 360 nm and measuring emission at a wavelength of 545 nm using a spectrofluorimeter (FP-777, JASCJ, Japan).

 As a result, it turned out that liposomes that contain the peptides of the present invention exhibit a lower decrease in liposome content (about 20%) compared to conventional pH-sensitive liposomes, which are used as a control (about 80%).

Example 5: Induction of cellular immunity by pH-sensitive liposomes containing peptides
To determine whether pH-sensitive liposomes, as a carrier of peptides, can induce a CTL reaction specific for the virus from which these peptides are derived, pH-sensitive liposomes in which synthetic peptides are encapsulated are prepared and cytotoxicity studies are carried out in a serial manner. Since the cellular immunity of peptides that can bind HLA-A2, human MHC class I molecules must be investigated using the human body, an indirect experiment is carried out using peptides derived from HBV and HCV proteins that can bind murine MHC class I molecules ( see table 4). To do this, obtain pH-sensitive liposomes, including peptides, using a phospholipid composition containing POPE / CHOH (7: 3 molar ratio). Then immunize mice with liposomes and determine the cytotoxicity of T-lymphocytes after recognition of target cells labeled with Cr ⁵¹ .

In the case of HIV, Balb / C mice are immunized with pH-sensitive liposomes including R15K or T26K, and the spleen is excised 4 weeks after booster immunization and homogenized in 2 ml of RPMI cell culture medium using a homogenizer. 2 ml of the homogenate thus obtained are added to 2 ml of ficoll and centrifuged at 1500 rpm for 30 minutes. T cells located between ficoll and the cell culture medium are isolated, added to 15 ml of RPMI-10 cell culture medium, centrifuged at 1000 rpm and washed three times with RPMI-10 medium. T lymphocytes are diluted to a cell concentration of 5 x 10 ⁶ / ml in a 24-well plate and the immunized peptide antigen is added to a final concentration of 2 μM. Then the cells were cultured at 37 ^{° C.} for 3 days in a CO ₂ incubator containing 5% CO ₂ and used as effector cells. In the case of HCV, five-week-old mice of strain C57BL / 6 are immunized with liposomes containing the D8L peptide antigen and T cells are isolated in the same manner as described above and used as effector cells. On the other hand, p815 cells (ATCC TIB64) in which MHC class I are the same as in Balb / C mice are used as target cells in the case of HIV, and RMA cells (see: Hosken, NA and Bevan, MJ , J.Exp. Med., 175: 719 (1992)) or EL4 cells (ATCC TIB39), in which MHC class I are the same as in C57BL / 6 mice, are used as their target cells in the case of HCV .

100 μM of the immunized peptide was added to the respective target cells, allowed to react at 37 ^{° C.} for 16 hours and washed twice with RPMI cell culture medium. Then, radioactive sodium chromate (Cr ⁵¹ ) was added to the cells and allowed to react at 37 ^° C for 2 hours in a CO ₂ incubator. After the reaction, the cells are thoroughly washed with RPMI cell culture medium to remove unreacted sodium chromate, mixed with effector cells washed with phosphate-buffered saline in various ratios, shown in Table 5, and allowed to react at 37 ^{° C.} for 4 hours in a CO ₂ incubator . The resulting medium is collected and the intensity of gamma rays is measured to determine the cytotoxicity of the effector cells against target cells (see table 5).

 As can be seen from table 5, it turned out that effector cells isolated from the spleens of mice immunized with R15K, T26K and D8L peptide antigens can recognize and destroy target cells. Moreover, it was found that if mice were immunized with peptide antigens that induce the CTL reaction using pH-sensitive liposomes, cellular immunity resulting in the production of virus-specific CTLs can be induced.

 In indirect experiments on the induction of cellular immunity, which were carried out using pH-sensitive liposomes containing peptides derived from HIV and HCV proteins, it was clearly shown that: liposomes containing peptide antigens derived from HBV protein X can induce cellular immunity, which, in turn, induces a CTL response, since peptide antigens derived from HBV protein X can induce a CTL response in humans (see Example 3), and pH-sensitive liposomes containing these peptides can also induce l response CTL reaction specific for the virus from which these peptides are derived.

представляют вводимые дозы 2•10⁷ и 1•10⁶ соответственно, а Т.Р. представляют моменты обработки CTL.Example 6: Destruction of human hepatocellular carcinoma by HBx-specific T cells
The destruction of CTL tumors that were stimulated with peptide antigens derived from HBV protein X (i.e., HBx peptide) is investigated as follows. Human hepatocarcinoma cells are subcutaneously injected into mice of nude strain Ba1b / C in a different number of cells from 1 • 10 ⁷ to 2 • 10 ⁷ , and a tumor test is performed on nude mice. After 3 days, tumors can already be determined, and after 5 days, CTLs stimulated with HBc peptide are adoptively transferred to the tail vein of mice that have formed tumors in the amount of 1.5 x 10 ⁸ cells (see FIG. 3A). As a result, it was shown that the size of the tumor begins to decrease by 2 days after treatment, and the tumor completely disappears after treatment (see figv). In FIGS. 3A and 3B, the icons (- • -) and

represent the administered doses of 2 • 10 ⁷ and 1 • 10 ^6, respectively, and T.R. represent moments of CTL processing.

 As was clearly illustrated above, the present invention provides pH-sensitive liposomes that include groups of peptides corresponding to epitopes of antigens derived from HBV protein X that induce CTL against the virus or immunological tolerance to the virus, so that cellular immunity against HBV. Since peptide antigens derived from protein Xa, such as X3, X4, X5, X6 and X7, activate CTLs that can recognize HBV antigens present in the human body, and CTLs that are indicated are pH-sensitive liposomes including peptide antigens derived from HBV protein X can be used to create potential therapeutic agents for the prevention and treatment of HBV-associated diseases.

 A list of sequences is provided at the end of the description.

Claims (12)

 1. A peptide antigen derived from hepatitis B virus protein X, which is recognized by cytotoxic T lymphocytes, for cytotoxicity against hepatitis B virus, its amino acid sequence being represented as SEQ ID No: 1: HLSLRGLFV.  2. A peptide antigen derived from hepatitis B virus protein X, which is recognized by cytotoxic T lymphocytes, for the manifestation of cytotoxicity against hepatitis B virus, and its amino acid sequence is presented as SEQ ID No: 2: VLHKRTLGL.  3. A peptide antigen derived from hepatitis B virus protein X, which is recognized by cytotoxic T lymphocytes, for cytotoxicity against hepatitis B virus, and its amino acid sequence is presented as SEQ ID No: 3: AMSTTDLEA.  4. A peptide antigen derived from hepatitis B virus protein X, which is recognized by cytotoxic T-lymphocytes, for cytotoxicity against hepatitis B virus, its amino acid sequence being represented as SEQ ID No: 4: CLFKDWEEL.  5. A peptide antigen derived from hepatitis B virus protein X, which is recognized by cytotoxic T lymphocytes, for cytotoxicity against hepatitis B virus, and its amino acid sequence is represented as SEQ ID No: 5: EIRLKVFVL.  6. pH-sensitive liposome, including peptide antigens, designed to induce cellular immunity against hepatitis B virus, which is obtained by mixing in a molar ratio of 5: 1 to 25: 1 phospholipid and one or more peptide antigens according to claims. 1-5.  7. The pH-sensitive liposome of claim 6, wherein the peptide antigen is selected from the group consisting of HLSLRGLFV, VLHKRTLGL, AMSTTDLEA, CLFKDWEEL and EIRLKVFVL.  8. The pH-sensitive liposome of claim 6, wherein the phospholipid contains 50% or more phosphatidylethanolamine-β-oleoyl-γ-palmitoyl.  9. The pH-sensitive liposome of claim 6, wherein the phospholipid is prepared by mixing phosphatidylethanolamine-β-oleoyl-γ-palmitoyl and cholesterol hemisuccinate in a molar ratio of from 6: 4 to 8: 2.  10. pH-sensitive liposome according to claim 9, where the phospholipid is obtained by additional inclusion up to 1 mol. % monophosphoryl lipid A.  11. The pH-sensitive liposome of claim 6, wherein the phospholipid is prepared by mixing phosphatidylethanolamine-β-oleoyl-γ-palmitoyl, phosphatidylethanolamine and cholesterol hemisuccinate in a molar ratio of from 3: 3: 4 to 4: 4: 2.  12. pH-sensitive liposome according to claim 11, where the phospholipid is obtained by additional inclusion of up to 1 mol. % monophosphoryl lipid A.

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