JP2014005205A - Ebola virus liposome vaccine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide and a vaccine which can effectively induce a cytotoxic T lymphocyte to an Ebola virus antigen and can be expected to have a therapeutic effect or a preventive effect on Ebola virus infection and diseases caused by the infection.SOLUTION: The vaccine contains: a peptide which can induce a cytotoxic T lymphocyte restricted to HLA-A*0201, having an amino acid sequence represented by a specific sequence and an amino acid length of 9 to 11; or a liposome to which the peptide is bonded, containing a phospholipid having an acryl group with one unsaturated bond and a carbon number of 14 to 24 or a hydrocarbon group with one unsaturated bond and a carbon number of 14 to 24 and a stabilizer for the liposome, wherein the peptide is bonded to the surface of the liposome.

Description

  The present invention relates to peptide-bonded liposomes, peptides, and their uses useful as Ebola virus vaccines.

  Ebola virus is a minus-strand RNA virus of the Filoviridae family. Ebola virus causes severe Ebola hemorrhagic fever in primates, including humans. The fatality rate is extremely high, sometimes exceeding 90%. Therefore, strict containment of BSL4 is necessary when conducting research. Although bats have been reported to be natural hosts for Ebola virus, there are many unclear points. Ebola hemorrhagic fever was a pandemic in Sudan and Zaire in 1976, followed by sporadic epidemics in Central and West Africa. Originally considered a regional disease in Africa, the epidemic in the monkey quarantine room in the United States in 1989 led to a new recognition of the threat of Ebola virus in developed countries. In recent years, the import of pet monkeys, the spread of Ebola virus by travelers, or the increased risk of bioterrorism using Ebola virus has been pointed out, and the development of an effective prevention or treatment method for Ebola hemorrhagic fever is desired. However, there are still no effective prevention or treatments and vaccines.

  Various research results on immune responses to Ebola virus have been reported. It has been reported that CTLs and antibodies against Ebola virus antigens exist in humans who have been infected with Ebola virus but have not died (Non-patent Document 1). In infection tests using mice, it has been reported that neutralizing antibodies and CTLs against Ebola virus antigens were effective (Non-Patent Documents 2 and 3). In addition, in infection tests using monkeys, it has been reported that DNA vaccines and recombinant virus vaccines were effective in suppressing infection by inducing cellular immunity and humoral immunity (Non-Patent Documents 4 to 7). . On the other hand, there is a report that neutralizing antibodies against Ebola virus antigens were ineffective (Non-patent Document 8). In humans, antibodies against Ebola virus (humoral immunity) are not very effective, but CTL (cellular immunity) ) Is presumed to be effective.

  On the other hand, as a method for enhancing the immune response of a living body, a method using a liposome preparation is known.

  Patent Document 1 can efficiently and specifically enhance CTL for killing pathogen-infected cells or cancer cells using liposomes to which antigens are bound, and is useful for prevention and treatment of infectious diseases and cancers. A method for preparing a T cell activator is disclosed.

  Patent Document 2 discloses that an antigenic epitope particularly effective for the preparation of cytotoxic T lymphocytes was found from the highly conserved internal protein sequence of avian influenza virus, and the peptide containing the epitope was bound to the surface. It has been described that the prepared liposomes can induce extremely specific antigen-specific cytotoxic T lymphocytes.

JP 2008-37831 A International Publication No. 2010/061924

Nat. Immunol., 8: 1159, 2007 Science, 287: 1664, 2000 J Virol, 75: 2660, 2001 Nature, 408: 605, 2000 Nature, 424: 681, 2003 Nat Med, 11: 786, 2005 J Virol, 81: 6379, 2007 PLos Pathogens, 3: e9, 2007

  The problem to be solved by the present invention is that peptides capable of efficiently inducing cytotoxic T lymphocytes against Ebola virus antigens and expected to have therapeutic or preventive effects on Ebola virus infection and diseases resulting from the infection To provide a vaccine.

  In view of the above-mentioned problems, the present inventors have conducted intensive studies and searched for antigen epitopes that are particularly effective for inducing cytotoxic T lymphocytes in Ebola virus antigens, and as a result, found several excellent antigen epitopes. It was. In addition, when vaccinated with liposomes to which peptides containing this epitope are bound, antigen-specific cytotoxic T lymphocytes can be induced extremely strongly, and Ebola virus infection and diseases associated with the infection can be treated or prevented. As a result, the present invention has been completed.

That is, the present invention provides the following.
[1] It can induce cytotoxic T lymphocytes restricted to HLA-A * 0201, comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 14 and having a length of 9 to 11 amino acids peptide.
[2] A liposome to which the peptide of [1] is bound,
The liposome contains a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, and a liposome stabilizer. And the peptide is bound to the surface of the liposome;
Peptide-bound liposomes.
[3] The peptide-bonded liposome according to [2], wherein the phospholipid is a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond.
[4] The peptide-bonded liposome according to [2], wherein the acyl group is an oleoyl group.
[5] The phospholipid is at least one selected from diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacylphosphatidylethanolamine. The peptide-bonded liposome of [2].
[6] The peptide-bonded liposome according to [2], wherein the liposome stabilizer is cholesterol.
[7] The peptide has an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond contained in the phospholipid membrane constituting the liposome. The peptide-bonded liposome of [2], which is bound to a phospholipid.
[8] The peptide-bonded liposome of [2], wherein the liposome has the following composition:
(A) a phospholipid having 1 to 99.8 mol% of an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(B) Liposome stabilizer 0.2-75 mol%.
[9] The peptide-bonded liposome of [2], wherein the liposome has the following composition:
(I) An acidic phospholipid having 1 to 85 mol% of an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(II) 0.01 to 80 mol% of a neutral phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(III) Phospholipids having a peptide-bound acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond 0.2 to 80 mol %;
(IV) Liposome stabilizer 0.2-75 mol%.
[10] A cytotoxic T lymphocyte activator comprising the peptide of [1] or the peptide-bonded liposome of [2].
[11] The cytotoxic T lymphocyte activator according to [10], further comprising an adjuvant.
[12] An Ebola virus vaccine comprising the peptide of [1] or the peptide-bound liposome of [2].
[13] The Ebola virus vaccine of [12], further comprising an adjuvant.

  By using the peptide or peptide-bound liposome of the present invention, cytotoxic T lymphocytes against the Ebola virus antigen can be efficiently induced, and excellent therapeutic or preventive effects on Ebola virus infection and diseases caused by the infection are expected. it can.

Induction of CD8 ⁺ / IFN-γ ⁺ T cells by various peptides derived from Ebola virus antigen protein. The vertical axis represents IFN-γ expression, and the horizontal axis represents CD8 expression. The numbers in the graph indicate the percentage of gated CD8 ⁺ / IFN-γ ⁺ T cells. Induction of CD8 ⁺ / IFN-γ ⁺ T cells by various peptide-bound liposomes. (× 2) indicates immunization twice. All others were immunized once. The vertical axis represents IFN-γ expression, and the horizontal axis represents CD8 expression. The numbers in the graph indicate the percentage of gated CD8 ⁺ / IFN-γ ⁺ T cells. Induction of CTL activity in vivo by various peptide-bound liposomes. The number of immunizations is one. The vertical axis represents the number of cells, and the horizontal axis represents CFSE expression. The numbers in the graph indicate the percentage of cells in the M2 gate that were killed.

1. Peptide The present invention is a peptide capable of inducing cytotoxic T lymphocytes restricted by HLA-A * 0201,
(1) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 14, or (2) an amino acid sequence in which one or two amino acids are substituted in the amino acid sequence represented by any of SEQ ID NOs: 1 to 14 And a peptide (peptide of the present invention) having a length of 9 to 11 amino acids.

The present invention has been completed based on the discovery of the following excellent epitope sequences (epitope sequences of the present invention) in the Ebola virus antigen protein:
Amino acid sequence represented by any one of SEQ ID NOs: 1 to 14 Amino acid sequence in which one or two amino acids are substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 14 (wherein the amino acids Peptides consisting of sequences can induce cytotoxic T lymphocytes restricted to HLA-A * 0201).

The amino acid sequences represented by SEQ ID NOs: 1 to 14 are Nucleoprotein (NP) (SEQ ID NOs: 1-2), which is a protein of the Ebola virus Zaire strain,
virion structural protein 40 (VP40) (SEQ ID NO: 3),
Glycoprotein (GP) (SEQ ID NOs: 4-7),
virion structural protein 30 (VP30) (SEQ ID NO: 8) or RNA-dependent RNA polymerase (L) (SEQ ID NOs: 9 to 14)
It corresponds to a continuous partial sequence contained in the amino acid sequence.

The amino acid sequence represented by SEQ ID NO: 1 corresponds to a partial sequence of 9 amino acids long starting from the 56th isoleucine contained in the NP amino acid sequence of Ebola virus Zaire strain (NP-56).
The amino acid sequence represented by SEQ ID NO: 2 corresponds to a partial sequence of 9 amino acids continuous starting from the 401st arginine contained in the NP amino acid sequence of the Ebola virus Zaire strain (NP-401).
The amino acid sequence represented by SEQ ID NO: 3 corresponds to a partial sequence of 9 amino acids continuous starting from the 73rd phenylalanine contained in the VP40 amino acid sequence of the Ebola virus Zaire strain (VP40-73).
The amino acid sequence represented by SEQ ID NO: 4 corresponds to a continuous partial sequence of 9 amino acids long starting from the 25th isoleucine contained in the amino acid sequence of GP of Ebola virus Zaire strain (GP-25).
The amino acid sequence represented by SEQ ID NO: 5 corresponds to a continuous partial sequence of 9 amino acids long starting from the 160th phenylalanine contained in the GP amino acid sequence of Ebola virus Zaire strain (GP-160).
The amino acid sequence represented by SEQ ID NO: 6 corresponds to a partial sequence of 9 amino acids continuous starting from the 252nd phenylalanine contained in the amino acid sequence of GP of Ebola virus Zaire strain (GP-252).
The amino acid sequence represented by SEQ ID NO: 7 corresponds to a partial sequence of 9 amino acids long starting from the 546th glycine contained in the GP amino acid sequence of Ebola virus Zaire strain (GP-546).
The amino acid sequence represented by SEQ ID NO: 8 corresponds to a continuous 9-amino acid long partial sequence starting from the 94th serine contained in the VP30 amino acid sequence of the Ebola virus Zaire strain (VP30-94).
The amino acid sequence represented by SEQ ID NO: 9 corresponds to a continuous partial sequence of 9 amino acids long starting from the 209th alanine contained in the L amino acid sequence of the Ebola virus Zaire strain (L-209).
The amino acid sequence represented by SEQ ID NO: 10 corresponds to a continuous 9-amino acid long partial sequence starting from the 293rd lysine contained in the L amino acid sequence of the Ebola virus Zaire strain (L-293).
The amino acid sequence represented by SEQ ID NO: 11 corresponds to a partial sequence of 9 amino acids long starting from the 771st arginine contained in the L amino acid sequence of the Ebola virus Zaire strain (L-771).
The amino acid sequence represented by SEQ ID NO: 12 corresponds to a continuous partial sequence of 9 amino acids long starting from the 932rd glycine contained in the L amino acid sequence of the Ebola virus Zaire strain (L-932).
The amino acid sequence represented by SEQ ID NO: 13 corresponds to a continuous 9-amino acid long partial sequence starting from the 1099th alanine contained in the L amino acid sequence of the Ebola virus Zaire strain (L-1099).
The amino acid sequence represented by SEQ ID NO: 14 corresponds to a partial sequence of 9 amino acids long starting from the 1955th valine contained in the L amino acid sequence of the Ebola virus Zaire strain (L-1955).

  The mode of substitution in (2) is not particularly limited as long as the peptide consisting of the amino acid sequence can induce cytotoxic T lymphocytes restricted by HLA-A * 0201, but an Ebola virus strain different from the Zaire strain ( Type / subtype) (eg, Sudan strain, Reston strain, Côte d'Ivoire strain, Bundibugi strain) reflecting known amino acid mutations in the corresponding epitope sequences in the same antigen is a preferred embodiment.

A peptide comprising the epitope sequence of the present invention (epitope peptide of the present invention) has the following excellent properties:
(A) The epitope peptide of the present invention is excellent in binding to HLA-A * 0201, which is the world's most popular human major histocompatibility antigen (HLA) type. Presented stably.
(B) Since the epitope peptide of the present invention has the above-mentioned characteristics (A), it can strongly induce cytotoxic T lymphocytes restricted by HLA-A * 0201. “Antigen induces cytotoxic T lymphocytes restricted by HLA” means that a mammal (eg, human, transgenic mouse, etc.) expressing a specific HLA (eg, HLA-A * 0201) is expressed as an antigen. In the mammal, the number and / or activity of cytotoxic T lymphocytes that are restricted by the HLA and specifically recognize the antigen (for example, IFN-γ production or cytotoxicity). Activity). As described above, since HLA-A * 0201 is the most popular HLA in the world, the epitope peptide of the present invention can be used in many humans (especially HLA-A * 0201) in the world regardless of race. Can induce cytotoxic T lymphocytes and activate cellular immunity.

  The peptide of the present invention can be the above-described epitope peptide of the present invention, or can be cleaved by the action of a proteasome or the like in a cell (preferably a human dendritic cell) to give the epitope peptide of the present invention. Therefore, the peptide of the present invention has substantially the same excellent characteristics as the epitope peptide of the present invention, and the cytotoxic T lymphocyte activator and Ebola virus vaccine of the present invention as described below are produced. When used in the above, it can be expected to exhibit excellent effects in killing cells infected with Ebola virus and protecting against Ebola virus infection.

  The length of the peptide of the present invention is not particularly limited, but is usually 9 to 11 amino acids, preferably 9 to 10 amino acids, and more preferably 9 amino acids. When the length of the peptide is 10 amino acids or more, the peptide has an additional sequence on the N-terminal side and / or C-terminal side of the epitope sequence of the present invention. The length of the additional sequence and the amino acid sequence are not particularly limited as long as the above properties of the peptide are not impaired. For example, the additional sequence may be an amino acid sequence actually present adjacent to the epitope sequence in the protein amino acid sequence of the Ebola virus Zaire strain from which each epitope sequence is derived. For example, when the peptide of the present invention includes the amino acid sequence represented by SEQ ID NO: 1, the additional sequence that can be included in the peptide is from SEQ ID NO: 1 included in the amino acid sequence of Nucleoprotein (NP) of the Ebola virus Zaire strain. It may be an amino acid sequence that actually exists adjacent to the partial amino acid sequence. In addition, when the peptide is presented on the major histocompatibility complex (MHC) in the cell, residues that can overcome the selection of the presented antigen due to individual differences (polymorphisms) of MHC are also added. Preferred as an array.

  The peptide of the present invention can be prepared by a known peptide synthesis technique such as liquid phase synthesis or solid phase peptide synthesis. Alternatively, the peptide is produced by culturing a transformant (such as E. coli) into which an expression vector capable of expressing the peptide is introduced, and isolating the peptide from the culture by a well-known purification technique such as an affinity column. can do. An expression vector capable of expressing the peptide can be constructed by linking a polynucleotide encoding the peptide downstream of a promoter in an appropriate expression vector using well-known genetic engineering techniques.

2. Peptide-bound liposome The present invention is a liposome to which the peptide of the present invention is bound,
The liposome contains a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, and a liposome stabilizer. And the peptide of the present invention is bound to the surface of the liposome;
Peptide-bound liposomes (peptide-bound liposomes of the present invention) are provided.

One of the problems of the present invention is that cytotoxic T lymphocytes (CD8) for killing Ebola virus-infected cells (preferably, cells presenting the peptides of the present invention on HLA-A * 0201 by Ebola virus infection). ⁺ T cells, CTL) efficiently and specifically. From this viewpoint, the peptide of the present invention used for the peptide-bonded liposome of the present invention is preferably an amino acid represented by SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 10, 11, 13 or 14. It contains an array.

  The phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention is such that the phospholipid that is an amphiphilic surfactant forms an interface with the polar group facing the aqueous phase, and the hydrophobic group is on the opposite side of the interface It has a structure suitable for. Here, the liposome refers to a phospholipid bilayer membrane having a closed space.

  The peptide contained in the peptide-bonded liposome of the present invention can be bound to the surface of the liposome via a functional group that it has. Examples of the functional group in the peptide used for binding to the liposome surface include an amino group, a thiol group, a carboxyl group, a hydroxyl group, a disulfide group, or a hydrophobic group composed of a hydrocarbon group (alkyl group or the like) having a methylene chain. It is done. Among these, amino group, thiol group, carboxyl group, hydroxyl group and disulfide group are covalently bonded, amino group and carboxyl group are bonded by ionic bond, hydrophobic group is hydrophobic group by hydrophobic bond, and the peptide is attached to the surface of liposome. Can be combined. The peptide is preferably bound to the surface of the liposome via an amino group, carboxyl group or thiol group.

  Since the peptide is stably bound to the liposome via the functional group of the peptide contained in the peptide-bonded liposome of the present invention, the phospholipid membrane constituting the liposome has an amino group, a succinimide group, a maleimide group, a thiol group, It is desirable to have a functional group such as a hydrophobic group composed of a carboxyl group, a hydroxyl group, a disulfide group, or a hydrocarbon group having a methylene chain (such as an alkyl group). The functional group possessed by the phospholipid membrane constituting the liposome is preferably an amino group, a succinimide group or a maleimide group. The combination of the functional group of the peptide involved in the binding of the peptide to the liposome and the functional group of the phospholipid membrane constituting the liposome can be freely selected within a range that does not affect the effects of the present invention. Preferred combinations include an amino group and an aldehyde group, an amino group and an amino group, an amino group and a succinimide group, a thiol group and a maleimide group, respectively. The ionic bond and the hydrophobic bond are preferable from the viewpoint of easy preparation of the peptide bond to the liposome, and the covalent bond is preferably a peptide bond point or peptide bond on the liposome surface. It is preferable from the viewpoint of storage stability when the liposome is put to practical use. One feature of the peptide-bound liposome of the present invention is that a peptide having a cytotoxic T lymphocyte activation effect is bound to the surface of the liposome, which is a constituent component thereof. Therefore, it is preferable from the viewpoint of further enhancing the effect of the present invention that the peptide is stably bound to the surface of the liposome even after being administered into the living body, for example, by an injection action in a practical stage. From such a viewpoint, the bond between the peptide and the liposome is preferably a covalent bond.

  The phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention comprises an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond. It contains a phospholipid having, and a liposome stabilizer.

  In the phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond, the carbon number of the acyl group is preferably 16 to 22, more preferably 18 to 22, and most preferably 18 It is. Specific examples of the acyl group include a palmitooleoyl group, an oleoyl group, and an elcoyl group, and an oleoyl group is most preferred.

  In the phospholipid having a C 14-24 hydrocarbon group having one unsaturated bond, the carbon number of the hydrocarbon group is preferably 16-22, more preferably 18-22, most preferably 18 is preferable. Specific examples of the hydrocarbon group include a tetradecenyl group, a hexadecenyl group, an octadecenyl group, a C20 monoene group, a C22 monoene group, and a C24 monoene group.

  The unsaturated acyl group or unsaturated hydrocarbon group bonded to the 1-position and 2-position of the glycerin residue of the phospholipid may be the same or different. From the viewpoint of industrial productivity, the 1-position and 2-position groups are preferably the same.

  As the phospholipid, a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond is preferably used.

One of the problems of the present invention is that cytotoxic T lymphocytes (CD8) for killing Ebola virus-infected cells (preferably, cells presenting the peptides of the present invention on HLA-A * 0201 by Ebola virus infection). ⁺ T cells, CTL) efficiently and specifically. From the viewpoint of enhancing CTL activity to a practically sufficient level, the phospholipid preferably has an acyl group having 14 to 24 carbon atoms having one unsaturated bond. When the carbon number of the acyl group is less than 13, the stability of the liposome may be deteriorated, and the CTL activity enhancing effect may be insufficient. Moreover, when the carbon number of the acyl group exceeds 24, the stability of the liposome may deteriorate.

  Examples of phospholipids having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond include acidic phospholipids, neutral phospholipids and peptides. Examples include reactive phospholipids having a functional group capable of binding. These can be selected as appropriate according to various requirements.

  As the acidic phospholipid, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol and the like can be used. Diacylphosphatidyl having an acyl group having 14 to 24 carbon atoms having one unsaturated bond from the viewpoint of enhancing the CTL activity to a practically sufficient level, industrial availability, quality for use as a pharmaceutical, and the like. Serine, diacylphosphatidylglycerol, diacylphosphatidic acid, and diacylphosphatidylinositol are preferably used. The acidic phospholipid imparts an anionic ionizing group to the surface of the liposome, and thus imparts a negative zeta potential to the surface of the liposome. For this reason, liposomes have a charge repulsive force and can exist as a stable preparation in an aqueous solvent. Thus, acidic phospholipids are important in ensuring the stability of the liposome when the peptide-bound liposome of the present invention is in an aqueous solvent.

  As the neutral phospholipid, for example, phosphatidylcholine and the like can be used. The neutral phospholipid that can be used in the present invention can be used by appropriately selecting the type and amount thereof within the scope of achieving the enhancement of CTL activity addressed by the present invention. The neutral phospholipid has a higher function of stabilizing the liposome and can improve the stability of the membrane, compared to the phospholipid to which the acidic phospholipid and the peptide of the present invention are bound. From this viewpoint, the phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention preferably contains a neutral phospholipid. The amount of neutral phospholipid used can be determined after ensuring the contents of acidic phospholipids used to achieve the CTL activity enhancing effect, reactive phospholipids for peptide bonds, and liposome stabilizers.

  In the peptide-bonded liposome of the present invention, the peptide of the present invention contains an acyl group having 14 to 24 carbon atoms having one unsaturated bond or 14 carbon atoms having one unsaturated bond contained in the phospholipid membrane constituting the liposome. It binds to the surface of liposomes by binding to phospholipids having ˜24 hydrocarbon groups.

  As the phospholipid for binding the peptide, a reactive phospholipid having a functional group to which the peptide of the present invention can bind is used. Reactive phospholipids having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, depending on various requirements, The ratio is appropriately selected. Similar to the phospholipid, also in the reactive phospholipid, it is not preferable that the unsaturated acyl group or unsaturated hydrocarbon group contained in the phospholipid has more than 24 or less than 14.

  Examples of the reactive phospholipid include phosphatidylethanolamine or a terminally modified product thereof. Further, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and terminally modified products thereof can also be used as reactive phospholipids. From the viewpoint of industrial availability, simplicity of the coupling step with the peptide of the present invention, yield, etc., phosphatidylethanolamine or a terminally modified product thereof is preferably used. Phosphatidylethanolamine has an amino group capable of binding the peptide of the present invention at its terminal. Furthermore, in terms of enhancing the CTL activity to a practically sufficient level, stability in liposomes, industrial supply, quality for use as a pharmaceutical, etc., the number of carbon atoms having one unsaturated bond is 14 to 14 Most preferably used is diacylphosphatidylethanolamine having 24 acyl groups or a terminally modified product thereof.

  Diacylphosphatidylethanolamine can be obtained, for example, by base exchange reaction of choline and ethanolamine using phospholipase D using diacylphosphatidylcholine as a raw material. Specifically, a crude reaction product can be obtained by mixing a chloroform solution in which diacylphosphatidylcholine is dissolved with water in which phospholipase D and ethanolamine are dissolved in an appropriate ratio. The crude reaction product can be purified on a silica gel column using a chloroform / methanol / water solvent to obtain the desired diacylphosphatidylethanolamine. A person skilled in the art can carry out by appropriately selecting column purification conditions such as a solvent composition ratio.

  Examples of the terminally modified product include a diacylphosphatidylethanolamine terminally modified product in which one end of a divalent reactive compound is bonded to the amino group of diacylphosphatidylethanolamine. As the divalent reactive compound, a compound having at least one aldehyde group or succinimide group capable of reacting with the amino group of diacylphosphatidylethanolamine can be used. Examples of the divalent reactive compound having an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde, terephthalaldehyde and the like. Preferably, glutaraldehyde is used. As a divalent reactive compound having a succinimide group, dithiobis (succinimidyl propionate), ethylene glycol-bis (succinimidyl succinate), disuccinimidyl succinate, disuccinimidyl suberate, or Examples include disuccinimidyl glutarate.

  Further, as a divalent reactive compound having a succinimide group at one end and a maleimide group at the other end, N-succinimidyl-4- (p-maleimidophenyl) butyrate, sulfosuccinimidyl-4- (p-maleimide) Phenyl) butyrate, N-succinimidyl-4- (p-maleimidophenyl) acetate, N-succinimidyl-4- (p-maleimidophenyl) propionate, succinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, Examples include sulfosuccinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, N- (γ-maleimidobutyryloxy) succinimide, N- (ε-maleimidocaproyloxy) succinimide, and the like. When such a divalent reactive compound is used, a diacylphosphatidylethanolamine terminal modified product having a maleimide group as a functional group can be obtained. A functional group at one end of the divalent reactive compound as described above can be bonded to the amino group of diacylphosphatidylethanolamine to obtain a diacylphosphatidylethanolamine terminal-modified product.

  Examples of a method for binding a peptide to the surface of the liposome include a method of preparing a liposome containing the above-mentioned reactive phospholipid and then adding the peptide to bind the peptide to the reactive phospholipid of the liposome. it can. In addition, by previously binding the peptide to the reactive phospholipid, and then mixing the obtained reactive phospholipid bound to the peptide with a phospholipid other than the reactive phospholipid and a liposome stabilizer. In addition, it is possible to obtain a liposome having a peptide bound to its surface. Methods for conjugating peptides to reactive phospholipids are well known in the art.

  The phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention comprises an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond. It contains at least one, for example two or more, preferably three or more phospholipids.

  For example, the phospholipid membrane constituting the liposome portion of the peptide-bonded liposome of the present invention includes diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacyl. At least one selected from phosphatidylethanolamine, for example, 2 or more, preferably 3 or more, 14 to 24 carbon atoms having one unsaturated bond, or 14 to 24 carbon atoms having one unsaturated bond A phospholipid having a hydrocarbon group.

The phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention is
An acidic phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
Neutral phospholipids having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, and 14 carbon atoms having one unsaturated bond It is preferable to contain at least one reactive phospholipid having a C24-24 hydrocarbon group having 1 to 24 acyl groups or one unsaturated bond.

  In the present invention, sterols and tocopherols can be used as the liposome stabilizer. The sterols may be those generally known as sterols, and examples thereof include cholesterol, sitosterol, campesterol, stigmasterol, and brassicasterol, and particularly preferably from the viewpoint of availability, Cholesterol is used. As said tocopherol, what is generally known as tocopherol should just be mentioned, For example, commercially available alpha-tocopherol is mentioned preferably from points, such as availability.

  Furthermore, as long as the effects of the present invention are not impaired, the phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention may contain a known component capable of constituting the liposome.

Examples of the composition of the phospholipid membrane constituting the liposome portion of the peptide-bonded liposome of the present invention include the following:
(A) a phospholipid having 1 to 99.8 mol% of an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(B) Liposome stabilizer 0.2-75 mol%
In addition, content of each component is displayed as mol% with respect to all the structural components of the phospholipid membrane which comprises the liposome part of a peptide bond liposome.
The content of the component (A) is preferably 10 to 90 mol%, more preferably 30 to 80 mol%, still more preferably 50 to 70 mol%, from the viewpoint of liposome stability.
The content of the component (B) is preferably 5 to 70 mol%, more preferably 10 to 60 mol%, and still more preferably 20 to 50 mol%, from the viewpoint of liposome stability. When the content of the stabilizer exceeds 75 mol%, the stability of the liposome is impaired, which is not preferable.

The component (A) includes the following:
(A) a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, to which no peptide is bound, and (b) ) A phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, to which a peptide is bound.
Content of the said component (a) is 0.01-85 mol% normally, Preferably it is 0.1-80 mol%, More preferably, it is 0.1-60 mol%, More preferably, it is 0.1-50 mol% It is.
The content of the component (b) is usually 0.2 to 80 mol%, preferably 0.3 to 60 mol%, more preferably 0.4 to 50 mol%, still more preferably 0.5 to 25 mol%. It is. When the content is less than 0.2 mol%, the amount of the peptide of the present invention decreases, and it becomes difficult to activate cytotoxic T lymphocytes at a practically sufficient level, and exceeds 80 mol%. And the stability of the liposomes is reduced.

  The above component (a) phospholipids usually include the above-mentioned acidic phospholipids and neutral phospholipids. Moreover, the above-mentioned reactive phospholipid is contained in the phospholipid of said component (b).

  The content of acidic phospholipid is usually 1 to 85 mol%, preferably 2 to 80 mol%, more preferably 4 to 60 mol%, and further preferably 5 to 40 mol%. When the content is less than 1 mol%, the zeta potential is reduced, the stability of the liposome is lowered, and it becomes difficult to activate cytotoxic T lymphocytes to a practically sufficient level. On the other hand, if the content exceeds 85 mol%, as a result, the content of the phospholipid bound to the peptide of the liposome decreases, and it is difficult to activate cytotoxic T lymphocytes to a practically sufficient level. Become.

  The content of neutral phospholipid is usually 0.01 to 80 mol%, preferably 0.1 to 70 mol%, more preferably 0.1 to 60 mol%, still more preferably 0.1 to 50 mol%. is there. When the content exceeds 80 mol%, the content of acidic phospholipids, peptide-bound phospholipids and liposome stabilizers contained in the liposomes decreases, and cytotoxic T lymphocytes are reduced to a practically sufficient level. It becomes difficult to activate.

  The peptide-bound phospholipid is obtained by binding the peptide to the reactive phospholipid described above, and the ratio of the reactive phospholipid binding to the peptide is used for binding as long as the effect of the present invention is not hindered. The type of functional group, coupling treatment conditions, and the like can be selected as appropriate.

  For example, when a terminal modified form of diacylphosphatidylethanolamine obtained by binding one end of disuccinimidylsuccinate, a divalent reactive compound, to the terminal amino group of diacylphosphatidylethanolamine is used as a reactive phospholipid. Depending on the selection of the binding treatment conditions, 10-99% of the reactive phospholipid can be bound to the peptide. In this case, the reactive phospholipid not bound to the peptide becomes acidic phospholipid and is contained in the liposome.

Preferred embodiments of the phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention can include the following compositions:
(I) 1 to 85 mol% of acidic phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(II) 0.01 to 80 mol% of a neutral phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(III) Phospholipids having a peptide-bound acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond 0.2 to 80 mol %;
(IV) Liposome stabilizer 0.2-75 mol%.
(Total 100 mol%)

As a more preferred embodiment of the phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention, the following composition can be mentioned:
Component (I) 2-80 mol%
Component (II) 0.1 to 70 mol%
Component (III) 0.3-60 mol%
10-70 mol% of the above component (IV)
(Total 100 mol%)

As a more preferred embodiment of the phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention, the following composition can be mentioned:
Component (I) 4-60 mol%
Component (II) 0.1-60 mol%
Component (III) 0.4-50 mol%
Component (IV) 20-60 mol%
(Total 100 mol%)

Particularly preferred embodiments of the phospholipid membrane constituting the liposome portion of the peptide-bonded liposome of the present invention can include the following compositions:
Component (I) 5-40 mol%
Component (II) 0.1-50 mol%
Component (III) 0.5-25 mol%
Component (IV) 25-55 mol%
(Total 100 mol%)

  The peptide-bonded liposome of the present invention is characterized in that the unsaturated acyl group or unsaturated hydrocarbon group contained in the phospholipid in the phospholipid membrane constituting the liposome part has 14 to 24 carbon atoms. A phospholipid containing an unsaturated acyl group or unsaturated hydrocarbon group having less than 14 or more than 24 carbon atoms may be included as long as the effects of the invention are not hindered. The number of carbon atoms is 14 to 24 with respect to the total number of all unsaturated acyl groups or unsaturated hydrocarbon groups contained in the phospholipid in the phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention. The ratio of the number of saturated acyl groups or unsaturated hydrocarbon groups is, for example, 50% or more, preferably 60% or more, more preferably 75% or more, still more preferably 90% or more, and most preferably 97% or more (for example, substantially 100%).

  The phospholipid membrane constituting the liposome portion of the peptide-bonded liposome of the present invention is a non-phospholipid lipid having an acyl group or a hydrocarbon group having 14 to 24 carbon atoms unless the effects of the present invention are hindered. May be included. The content of the lipid is usually 40 mol% or less, preferably 20 mol% or less, more preferably 10 mol% or less, still more preferably 5 mol% or less (for example, substantially 0 mol%).

  The liposome portion of the peptide-bonded liposome of the present invention is appropriately formulated and processed using phospholipids, reactive phospholipids, liposome stabilizers, peptides, and the like, which are added to a suitable solvent, etc. It can be obtained by the method.

  Extrusion method, vortex mixer method, ultrasonic method, surfactant removal method, reverse phase evaporation method, ethanol injection method, prevesicle method, French press method, W / O / W emulsion method, annealing method, freeze-thaw And other manufacturing methods. The form of the liposome is not particularly limited, and liposomes having various sizes and forms such as multilamellar liposomes, small unilamellar liposomes and large unilamellar liposomes are produced by appropriately selecting the above-described liposome production method. be able to.

  The particle size of the liposome is not particularly limited, but from the viewpoint of storage stability, the particle size is 20 to 600 nm, preferably 30 to 500 nm, and preferably 40 to 400 nm, and more preferably. Is 50-300 nm, most preferably 70-230 nm.

  In the present invention, in order to improve the physicochemical stability of the liposome, a saccharide or a polyhydric alcohol is added to the inner aqueous phase and / or the outer aqueous phase of the liposome after the liposome preparation process or after the preparation. Also good. In particular, when long-term storage or storage during formulation is required, saccharides or polyhydric alcohols are added and dissolved as a liposome protective agent, and water is removed by freeze-drying to freeze-dry the phospholipid composition. It is preferable to use a product.

  Examples of the saccharide include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; disaccharides such as saccharose, lactose, cellobiose, trehalose and maltose; trisaccharides such as raffinose and melezitose; oligosaccharides such as cyclodextrin Sugar; polysaccharides such as dextrin; sugar alcohols such as xylitol, sorbitol, mannitol, maltitol and the like. Among these saccharides, monosaccharides or disaccharides are preferable, and glucose or saccharose is more preferable in terms of availability.

  Examples of the polyhydric alcohols include glycerin-based compounds such as glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, octaglycerin, nonaglycerin, decaglycerin, polyglycerin; sorbitol, mannitol Sugar alcohol compounds such as ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol, nonaethylene glycol and the like. Among these, glycerin, diglycerin, triglycerin, sorbitol, mannitol, and polyethylene glycol having a molecular weight of 400 to 10,000 are preferably mentioned from the viewpoint of availability.

  The concentration of saccharides or polyhydric alcohols contained in the inner aqueous phase and / or outer aqueous phase of the liposome is, for example, 1 to 20% by weight, preferably 2 to 10% by weight, based on the weight of the liposome liquid. Can be mentioned.

  When producing the peptide-bonded liposome of the present invention, the peptide-bonded liposome of the present invention can be easily obtained by preparing the liposome before binding the peptide and then binding the peptide.

  For example, a suspension of liposomes containing phospholipids, liposome stabilizers, and reactive phospholipids for binding peptides to the membrane surface is prepared, and sucrose which is one of the above sugars in its outer aqueous phase 2 to 10% by weight is added and dissolved. The sugar-added preparation is transferred to a 10 ml glass vial, placed in a shelf-type freeze dryer, cooled to −40 ° C. or the like to freeze the sample, and a freeze-dried product is obtained by a conventional method.

  The lyophilized product of the liposome obtained here can be stored for a long time since the water has been removed, and the final peptide of the present invention can be obtained by adding a specific peptide when necessary and carrying out the subsequent steps. Bound liposomes can be obtained simply and quickly. In the case where the interaction between the peptide and the liposome is strong and unstable, it is very convenient to store the liposome at the lyophilized product and bind the peptide when necessary.

The phospholipid membrane constituting the liposome part of the peptide-bonded liposome of the present invention can have a phospholipid to which a peptide is bound. Examples of a method for obtaining liposomes containing phospholipids to which peptides are bound include the following methods (A) and (B).
(A) A liposome containing a phospholipid, a reactive phospholipid, and a liposome stabilizer, a peptide and a divalent reactive compound are added thereto, and the functional group of the reactive phospholipid contained in the liposome And a functional group of the peptide linked via a divalent reactive compound. As the divalent reactive compound that can be used here, those used in the preparation of a terminal modified form of a reactive phospholipid can be similarly used. Specific examples of the divalent reactive compound having an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde, and terephthalaldehyde. Preferably, glutaraldehyde is used. Furthermore, dithiobis (succinimidyl propionate), ethylene glycol-bis (succinimidyl succinate), disuccinimidyl succinate, disuccinimidyl suberate as divalent reactive compounds having a succinimide group Or disuccinimidyl glutarate. Further, as a divalent reactive compound having a succinimide group at one end and a maleimide group at the other end, N-succinimidyl-4- (p-maleimidophenyl) butyrate, sulfosuccinimidyl-4- (p-maleimide) Phenyl) butyrate, N-succinimidyl-4- (p-maleimidophenyl) acetate, N-succinimidyl-4- (p-maleimidophenyl) propionate, succinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, It is possible to use sulfosuccinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, N- (γ-maleimidobutyryloxy) succinimide, N- (ε-maleimidocaproyloxy) succinimide, etc. it can. When such a divalent reactive compound is used, a terminal modified product of a reactive phospholipid having a maleimide group as a functional group (for example, phosphatidylethanolamine) can be obtained.
(B) A liposome containing a phospholipid, a reactive phospholipid, and a liposome stabilizer is prepared, a peptide is added thereto, and the functional group of the reactive phospholipid and the functional group of the peptide are contained in the liposome. A method of connecting and joining.

  Examples of the type of bond in (A) and (B) include an ionic bond, a hydrophobic bond, and a covalent bond, and a covalent bond is preferable. Specific examples of the covalent bond include a Schiff base bond, an amide bond, a thioether bond, and an ester bond.

  In any of the above two methods, the peptide can be bound to the reactive phospholipid contained in the phospholipid membrane constituting the liposome, and the phospholipid bound to the peptide is formed in the liposome.

  In the method (A) described above, a specific example of the method of binding the liposome as a raw material and the peptide via a divalent reactive compound is, for example, a method using a Schiff base bond. As a method for linking a liposome and a peptide via a Schiff base bond, a liposome having an amino group on its surface is prepared, the peptide is added to the suspension of the liposome, and then a divalent reactive compound is formed as a divalent reactive compound. An example is a method in which an aldehyde is added to bind the amino group on the liposome surface and the amino group in the peptide via a Schiff base.

As a specific example of this coupling procedure, for example, the following method may be mentioned.
(A-1) In order to obtain a liposome having an amino group on its surface, an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond A reactive phospholipid (eg, phosphatidylethanolamine) is mixed into a liposome raw material lipid (phospholipid, liposome stabilizer, etc.) to prepare a liposome having a predetermined amount of amino groups on the surface of the liposome.
(A-2) A peptide is added to the liposome suspension.
(A-3) Next, glutaraldehyde is added as a divalent reactive compound and reacted for a predetermined time to form a Schiff base bond between the liposome and the peptide.
(A-4) Thereafter, in order to deactivate the reactivity of excess glutaraldehyde, glycine as an amino group-containing water-soluble compound is added to the liposome suspension and reacted.
(A-5) The peptide of the present invention is obtained by removing unbound peptide, reaction product of glutaraldehyde and glycine, and excess glycine by a method such as gel filtration, dialysis, ultrafiltration, and centrifugation. A bound liposome suspension is obtained.

  As a specific example of the method (B), a reactive phospholipid having a functional group capable of forming an amide bond, a thioether bond, a Schiff base bond, an ester bond, etc. is applied to the phospholipid membrane constituting the liposome. The method to introduce is mentioned. Specific examples of such functional groups include succinimide group, maleimide group, amino group, imino group, carboxyl group, hydroxyl group, thiol group and the like.

  As an example of the reactive phospholipid introduced into the phospholipid membrane constituting the liposome, the above-mentioned 14 to 24 carbon acyl group having one unsaturated bond or 14 to 24 carbon atoms having one unsaturated bond is used. A terminal modified product of the amino group of a reactive phospholipid having a hydrocarbon group (eg, phosphatidylethanolamine) can be used.

As a specific example of this coupling procedure, the case where diacylphosphatidylethanolamine is used will be described below as an example.
(B-1) A diacylphosphatidylethanolamine having a C 14-24 acyl group having one unsaturated bond and a disuccinimidyl succinate are reacted only at one end by a known method, and a succinimide group as a functional group To a disuccinimidyl succinate linked diacyl phosphatidylethanolamine.
(B-2) The disuccinimidyl succinate-linked diacyl phosphatidylethanolamine and other liposome constituents (phospholipid, liposome stabilizer, etc.) are mixed by a known method, and a succinimide group as a functional group on the surface To make liposomes.
(B-3) A peptide is added to the liposome suspension, and the amino group in the peptide is reacted with the succinimide group on the liposome surface.
(B-4) Unreacted peptides, reaction byproducts and the like are removed by a method such as gel filtration, dialysis, ultrafiltration, and centrifugation to obtain a suspension of the peptide-bound liposome of the present invention.

  When a liposome and a peptide are bound, it is practically preferable to target an amino group or a thiol group that is often contained as a functional group. When an amino group is intended, a Schiff base bond can be formed by reacting with an succinimide group. When a thiol group is intended, a thioether bond can be formed by reacting with a maleimide group.

3. Use of Peptide of the Present Invention and Peptide-Binding Liposomes The present invention provides a cytotoxic T lymphocyte activator and an Ebola virus vaccine comprising the peptide of the present invention or the peptide-bonded liposome of the present invention. If the peptide of the present invention or the peptide-bonded liposome is used, it becomes possible to strongly induce cytotoxic T lymphocytes (CTLs) that recognize the peptide of the present invention or the epitope peptide in an HLA-A * 0201-restricted manner. . Cytotoxic T lymphocytes induced by the peptide of the present invention and peptide-bound liposomes kill cells that present the peptide of the present invention or epitope peptide on HLA-A * 0201, as a result of Ebola virus infection, etc. Cells can be removed. Therefore, the peptides and peptide-bound liposomes of the present invention are useful as cytotoxic T lymphocyte activators and Ebola virus vaccines for the treatment and prevention of Ebola virus infection and diseases caused by the infection (Ebola hemorrhagic fever, etc.).

The type of strain of Ebola virus targeted by the Ebola virus vaccine of the present invention is not particularly limited as long as a preventive or therapeutic effect is exhibited against the Ebola virus infection. Examples of Ebola virus strains include Zaire strain, Sudan strain, Reston strain, Côte d'Ivoire strain, and Bundibuyo strain. Since the epitope sequences represented by SEQ ID NOs: 1 to 14 are included in the amino acid sequence of the corresponding protein (NP, VP40, GP, L or VP30) of the Zaire strain, the Ebola virus vaccine of the present invention is Is expected to be particularly effective. Further, the Ebola virus vaccine of the present invention is expected to exert an excellent effect on Ebola virus strains other than the Zaire strain in which the epitope sequence of the present invention is conserved as well as the Zaire strain. Ebola virus strains in which the epitope sequences of the present invention are conserved are listed below.
SEQ ID NO: 1: Zaire strain, Sudan strain, Côte d'Ivoire strain, Bundi Bugyo strain SEQ ID NO: 2: Zaire strain, Sudanese strain, Reston strain, Côte d'Ivoire strain, Bundi Bugyo strain 4: Zaire strain SEQ ID NO: 5: Zaire strain, Sudan strain, Reston strain, Côte d'Ivoire strain, Bundi Bugyo strain SEQ ID NO: 6: Zaire strain SEQ ID NO: 8: Zaire strain SEQ ID NO: 8: Zaire strain, Sudan strain, Côte d'Ivoire strain, Bundibuguyo Strain SEQ ID NO: 9: Zaire Strains SEQ ID NO: 10: Zaire Strains SEQ ID NO: 11: Zaire Strains, Sudan Strains, Reston Strains, Cote d'Ivoire Strains, Bundi Bugyo Strains SEQ ID NO: 12: Zaire Strains, Cote d'Ivoire Strains SEQ ID NO: 13: Zaire Strains, Coats Givoar strain SEQ ID NO: 4: Zaire strain

  Even if it is a strain other than the strains listed above, those skilled in the art know whether or not the target epitope sequence is conserved in the Ebola virus strain to be evaluated, such as RT-PCR and direct sequencing. It can be easily determined by this nucleotide sequence analysis method.

  When the peptide of the present invention or peptide-bound liposome is used as a cytotoxic T lymphocyte activator or Ebola virus vaccine, it can be formulated according to conventional means. The peptides and peptide-bonded liposomes of the present invention have low toxicity and can be used as a liquid or as a pharmaceutical composition in an appropriate dosage form as a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat, Dogs, monkeys, etc.), birds (chicken, geese, ducks, ostriches, quail, etc.) and the like can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.). The subject of administration of the peptide of the present invention or peptide-bound liposome is usually a mammal (eg, human) that can be infected with the target Ebola virus, preferably a human or transgenic mammal that expresses HLA-A * 0201. (Example: Transgenic mice such as Hsc mice (Pascolo S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Perarnau B. The Journal of Experimental Medicine 1997; 185 (12): 2043-2051)) Preferably, it is a human that expresses HLA-A * 0201. The peptides and peptide-bound liposomes of the present invention are usually administered parenterally.

  The cytotoxic T lymphocyte activator and Ebola virus vaccine of the present invention may be administered as an active ingredient peptide or peptide-bound liposome itself, or may be administered as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the peptide or peptide-bound liposome and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving or suspending the peptide or peptide-bound liposome in a sterile aqueous solvent usually used for injection. Examples of the aqueous solvent for injection include distilled water; physiological saline; phosphate buffer, carbonate buffer, Tris buffer, acetate buffer, and other buffer solutions. As for pH of such an aqueous solvent, 5-10 is mentioned, Preferably it is 6-8. The prepared injection solution is preferably filled in a suitable ampoule.

  Further, the peptide preparation of the present invention or the powder preparation of the peptide-binding liposome of the present invention can be obtained by subjecting the solution of the peptide of the present invention or the suspension of the peptide-bonded liposome of the present invention to a treatment such as vacuum drying or freeze drying. It can also be prepared. The peptide of the present invention or the peptide-bonded liposome of the present invention is stored in a powder state, and can be used by dispersing the powder with an aqueous solvent for injection at the time of use.

  The cytotoxic T lymphocyte activator and Ebola virus vaccine of the present invention may further contain an adjuvant in order to enhance the effect. Examples of the adjuvant include aluminum hydroxide gel, complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, poly (I, C), CpG-DNA and the like, and CpG-DNA is particularly preferable. CpG-DNA is a DNA containing a bacterial unmethylated CpG motif and is known to act as a ligand for a specific receptor (Toll-like receptor 9) (for details, see Biochim. Biophys. Acta 1489, 107-). 116 (1999) and Curr. Opin. Microbiol. 6, 472-477 (2003)). CpG-DNA can enhance the induction of cytotoxic T lymphocytes by the peptides or peptide-bound liposomes of the present invention by activating dendritic cells (DC).

  The content of the active ingredient (the peptide of the present invention or the peptide-bound liposome) in the pharmaceutical composition is usually about 0.1 to 100% by weight, preferably about 1 to 99% by weight, more preferably the whole pharmaceutical composition About 10 to 90% by weight.

  When the cytotoxic T lymphocyte activator or Ebola virus vaccine of the present invention contains an adjuvant, the content of the adjuvant (for example, CpG-DNA) is appropriately determined within a range that can enhance the induction of cytotoxic T lymphocytes. Although it can be set, it is usually about 0.01 to 10% by weight, preferably about 0.1 to 5% by weight of the whole pharmaceutical composition.

  The dose of the peptide of the present invention or the peptide-bonded liposome of the present invention varies depending on the subject to be administered, the administration method, the administration form, etc., and for example, activates cytotoxic T lymphocytes in vivo by subcutaneous administration or nasal administration. In the range of 1 μg to 1000 μg, preferably in the range of 20 μg to 100 μg, as a peptide of the present invention per adult (body weight 60 kg), usually 2 times over 4 weeks to 18 months. 3 times. When Ebola virus infection is prevented by subcutaneous administration, the peptide of the present invention is in the range of 1 μg to 1000 μg, preferably in the range of 20 μg to 100 μg, usually twice for 4 weeks to 18 months. 3 times. Further, when treating diseases caused by Ebola virus infection (eg, Ebola hemorrhagic fever) by subcutaneous administration, the peptide of the present invention is in the range of 1 μg to 1000 μg, preferably in the range of 20 μg to 100 μg, usually for 4 weeks. 2 to 3 doses over the next 18 months.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

[Reference Example 1]
Preparation of liposome 1) Preparation of lipid mixed powder Synthesis of reactive phospholipid (succinimidyl group-dioleoylphosphatidylethanolamine) consisting of terminally modified phosphatidylethanolamine Dissolve 2 g of dioleoylphosphatidylethanolamine and 180 μl of triethylamine in 50 ml of chloroform. And added to a 300 ml four-necked flask. While stirring the flask at room temperature with a magnetic stirrer, a solution prepared by dissolving 3 g of disuccinimidyl suberate, a divalent reactive compound prepared separately, in 80 ml of chloroform was dropped over 4 hours according to a conventional method. One end of disuccinimidyl suberate was reacted with the amino group of dioleoylphosphatidylethanolamine. This crude reaction solution was transferred to an eggplant type flask, and the solvent was distilled off by an evaporator. Next, a small amount of chloroform capable of dissolving the crude reaction product was added to the flask to obtain a high concentration crude reaction solution, and silica gel equilibrated with chloroform / methanol / water (65/25/1, volume ratio) was used. Perform column chromatography according to a conventional method, collect only the fraction in which one end of disuccinimidyl suberate is bound to the amino group of the desired dioleoylphosphatidylethanolamine, and evaporate the solvent to obtain the desired reactivity. A succinimide group-dioleoylphosphatidylethanolamine which is a phospholipid was obtained.
2) Preparation of lipid mixed powder 1.3354 g (1.6987 mmol) of dioleoylphosphatidylcholine, 0.2886 g (0.2831 mmol) of succinimide group-dioleoylphosphatidylethanolamine prepared in the previous section, 0.7663 g (1.9818 mmol) of cholesterol and dioleoylphosphatidyl 0.4513 g (0.5662 mmol) of glycerol Na salt was placed in an eggplant-shaped flask, and 50 ml of a mixed solvent of chloroform / methanol / water (65/25/4, volume ratio) was added and dissolved at 40 ° C. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Further, 30 ml of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen and dried in a freeze dryer for 24 hours to obtain a lipid mixed powder.
3) Preparation of liposomes Next, 60 ml of separately prepared buffer solution (1.0 mM Na ₂ HPO ₄ / KH ₂ PO ₄ , 0.25 M saccharose, pH 7.4, hereinafter abbreviated as buffer solution) was added to the eggplant containing the above-mentioned lipid mixed powder. Lipids were hydrated with stirring in a mold flask at 40 ° C. to obtain liposomes. Next, the particle size of the liposome was adjusted using an extruder. First, an 8 μm polycarbonate filter was passed, followed by a filter in the order of 5 μm, 3 μm, 1 μm, 0.65 μm, 0.4 μm and 0.2 μm. An average particle size of 206 nm (measured by a dynamic light scattering method) of the liposome particles was obtained.

[Example 1]
Search for CTL epitopes 94 types of epitope candidates for HLA-A * 0201-restricted CTL were selected from the amino acid sequence of Ebola virus (Zaire strain) polyprotein based on the motif sequence and the like. About the peptide (9 amino acid length) which consists of these epitopes, the binding property with respect to HLA-A * 0201 was evaluated by the following method.

  RMA-S-HHD cells were incubated overnight at 26 ° C. Thereafter, RMA-S-HHD cells were incubated at 37 ° C. for 3 hours with various concentrations of the peptides to be evaluated. The cells were collected, treated with a conformation-sensitive anti-HLA-A * 0201 monoclonal antibody (BB7.2) (primary antibody), and subsequently stained with a FITC-labeled secondary antibody, followed by flow cytometry. The expression of A * 0201 was analyzed. Since RMA-S-HHD cells are deficient in TAP, at 26 ° C., they stably express empty HLA-A * 0201 to which no self peptide is bound. On the other hand, at 37 ° C., empty HLA-A * 0201 to which no peptide is bound is unstable, and BB7.2 cannot recognize this, so it is incubated at 37 ° C. in the presence of the peptide to be evaluated. By analyzing the expression of HLA-A * 0201 after BB7.2, the binding property of the peptide to be evaluated to HLA-A * 0201 can be evaluated.

BL ₅₀ (μM) was calculated for each peptide. BL ₅₀ means the concentration of each peptide that gives half-maximal MFI of RMA-S-HHD cells incubated at 26 ° C. for 14-16 hours. The results are shown in Table 1.

[Example 2]
About 94 types of epitope peptides as in Example 1 to Ebola virus-specific ^{CD8 +} / ^IFN-γ + T cell inducibility was evaluated Ebola virus-specific ^{CD8 +} / ^IFN-γ + T cell inducibility.
HHD mice were immunized with 2 × 10 ⁷ allogeneic syngeneic spleen cells pulsed with the peptide to be evaluated (10 μM). In HHD mice, mouse-specific MHC class I gene H-2D and mouse β2-microglobulin gene are knocked out, and human HLA-A * 0201 and human β2-microglobulin genes are introduced and expressed (Pascolo). S, Bervas N, Ure JM, Smith AG, Lemonnier FA, Perarnau B. The Journal of Experimental Medicine 1997; 185 (12): 2043-2051). One week later, spleen cells from the immunized mice were incubated with 10 μM of the corresponding peptide to be evaluated in the presence of Brefeldin A for 5 hours in a CO ₂ incubator.
Cells were harvested and anti-mouse CD16 / CD32 monoclonal antibody was added to block Fc receptors on the cells and stained with FITC-labeled anti-mouse CD8α monoclonal antibody. Cells were fixed, permeabilized, further stained with PE-labeled anti-mouse IFN-γ monoclonal antibody and analyzed by flow cytometry. CTL inducibility was assessed by increasing the proportion of CD8 ⁺ / IFN-γ ⁺ T cells.

  Of the 94 epitope peptides evaluated, 24 epitope peptides shown in FIG. 1 induced CTL specific for each epitope.

[Example 3]
Evaluation of Ebola virus-specific CD8 ⁺ / IFN-γ ⁺ T cell inducing ability of peptide-bound liposome 1) Preparation of peptide-bound liposome preparation Using 24 types of epitope peptides that showed CTL-inducing activity in Example 2, peptide binding Liposomes were prepared by the following method.
1.5 ml of the liposome of Reference Example 1 (preparation of liposome) was collected in a test tube, 3 ml of each peptide pool solution prepared separately was added, and the mixture was gently stirred at 5 ° C. for 48 hours for reaction. This reaction solution was subjected to gel filtration according to a conventional method using Sepharose CL-4B equilibrated with a buffer solution. Since the liposome fraction is cloudy, the target fraction can be easily confirmed, but it may be confirmed with a UV detector or the like.
Phosphorus concentration in the obtained liposome suspension was measured (Phospholipid Test Wako), and the concentration was diluted with a buffer so that the phospholipid-derived phosphorus concentration was 2 mM. Got.

2) Evaluation of Ebola virus-specific CD8 ⁺ / IFN-γ ⁺ T cell induction ability HHD mice were immunized with the peptide-bonded liposomes to be evaluated together with CpG. One week later, spleen cells from the immunized mice were incubated with 10 μM of the corresponding peptide to be evaluated in the presence of Brefeldin A for 5 hours in a CO ₂ incubator.
Cells were harvested and anti-mouse CD16 / CD32 monoclonal antibody was added to block Fc receptors on the cells and stained with FITC-labeled anti-mouse CD8α monoclonal antibody. Cells were fixed, permeabilized, further stained with PE-labeled anti-mouse IFN-γ monoclonal antibody and analyzed by flow cytometry. CTL inducibility was assessed by increasing the proportion of CD8 ⁺ / IFN-γ ⁺ T cells.

Of the 24 types of epitope peptides evaluated, peptide-bound liposomes prepared using the following 14 types of epitope peptides significantly induced CTL.
NP-56: a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.
NP-401: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2.
VP40-73: a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3.
GP-25: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 4.
GP-160: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 5.
GP-252: a peptide consisting of the amino acid sequence represented by SEQ ID NO: 6.
GP-546: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 7.
VP30-94: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 8.
L-209: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 9.
L-293: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 10.
L-771: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 11.
L-932: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 12.
L-1099: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 13.
L-1955: A peptide consisting of the amino acid sequence represented by SEQ ID NO: 14.

  The results for these 14 types of epitope peptides are shown in FIG. Particularly, NP56, L293, and L209 had strong CTL inducing ability. VP40-73, GP160 and GP252 strongly induced CTL when immunized twice.

[Example 4]
Evaluation of ability to induce CTL activity in vivo
HHD mice were immunized once with each peptide to be evaluated (0.5 μg / μl was 100 μl / mouse) and CpG5002 (see Vaccine 25, 4914-4921 (2007)) (5 μg / mouse) bound to liposomes. Seven days after immunization, splenocytes were prepared from another naive HHD mouse. 2 × 10 ⁸ spleen cells were suspended in 2 ml of RPMI 1640, and the suspension was divided into two tubes of 1 ml each. Spleen cells were pulsed with the peptide by adding the peptide (10 μM) used for immunization to one tube and incubating at 37 ° C. for 1 hour. Splenocytes were centrifuged and the supernatant was removed. Splenocytes were washed once with 10 ml T10, centrifuged and the supernatant removed. Splenocytes were redispersed by adding 20 ml PBS / 0.1% BSA to the splenocyte pellet and vortexing rapidly. 10 μl of 5 mM CFSE is added to the splenocyte dispersion (final concentration: 2.5 μM), vortexed quickly and incubated at 37 ° C. for 10 minutes to label the peptide-pulsed splenocytes with a dense fluorescent dye (CFSE) did. The other tube was labeled with a thin fluorescent dye (CFSE) (final concentration: 0.25 μM) without adding peptide. Both splenocytes were centrifuged and the supernatant was removed. 10 ml of R10 was added to the washed splenocytes, centrifuged, and the supernatant was removed. Both splenocytes were resuspended in HBSS (or serum-free RPMI) and adjusted to a concentration of 5 × 10 ⁷ cells / ml. Spleen cells were transferred (1 × 10 ⁷ cells / mouse) by mixing the same volume of these two groups of cell suspensions and injecting them intravenously into immunized mice. The next day (4 to 12 hours after transfer), mice were euthanized and the spleen was collected, and the reduction rate of target cells labeled with a dense fluorescent dye (CFSE) 2.5 μM was analyzed by a flow cytometer. .

  As shown in FIG. 3, both peptide-bound liposomes induced CTL activity specific for each epitope in vivo. In particular, the induction ability of NP56, NP401, VP40-73, GP160, GP252, L1955, L293, L209, L1099, L771 and VP30-94 was strong.

  If the peptide or peptide-bonded liposome of the present invention is used, cytotoxic T lymphocytes against Ebola virus can be efficiently induced, and an excellent therapeutic or preventive effect against Ebola virus infection can be expected.

Claims (13)

  A peptide capable of inducing cytotoxic T lymphocytes restricted by HLA-A * 0201, comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 14 and having a length of 9 to 11 amino acids. A liposome to which the peptide of claim 1 is bound,
The liposome contains a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, and a liposome stabilizer. And the peptide is bound to the surface of the liposome;
Peptide-bound liposomes.   The peptide-bonded liposome according to claim 2, wherein the phospholipid is a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond.   The peptide-bonded liposome according to claim 2, wherein the acyl group is an oleoyl group.   The phospholipid is at least one selected from diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacylphosphatidylethanolamine. The peptide-bound liposome described.   The peptide-bonded liposome according to claim 2, wherein the liposome stabilizer is cholesterol.   The peptide is a phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond, contained in the phospholipid membrane constituting the liposome. The peptide-bonded liposome according to claim 2, which is bound. The peptide-bonded liposome according to claim 2, wherein the liposome has the following composition:
(A) a phospholipid having 1 to 99.8 mol% of an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(B) Liposome stabilizer 0.2-75 mol%. The peptide-bonded liposome according to claim 2, wherein the liposome has the following composition:
(I) An acidic phospholipid having 1 to 85 mol% of an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(II) 0.01 to 80 mol% of a neutral phospholipid having an acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond;
(III) Phospholipids having a peptide-bound acyl group having 14 to 24 carbon atoms having one unsaturated bond or a hydrocarbon group having 14 to 24 carbon atoms having one unsaturated bond 0.2 to 80 mol %;
(IV) Liposome stabilizer 0.2-75 mol%.   A cytotoxic T lymphocyte activator comprising the peptide according to claim 1 or the peptide-bonded liposome according to claim 2.   The cytotoxic T lymphocyte activator according to claim 10, further comprising an adjuvant.   An Ebola virus vaccine comprising the peptide according to claim 1 or the peptide-bonded liposome according to claim 2.   The Ebola virus vaccine according to claim 12, further comprising an adjuvant.