JPH10212300A - Peptide vaccine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide capable of functioning a peptide vaccine for influenza virus or human immunodeficiency virus, excellent in immunogenicity, applicable to infectious diseases and tumor caused by a variety of pathogenic organisms, having a specific amino acid sequence. SOLUTION: This new peptide comprises a new peptide containing an amino acid sequence of the formula F-X-A-X (F is any one of an amino acid sequence shown by amino acid number 1-5 of formula I, an amino acid sequence shown by amino acid number 2-5 and an amino acid number 3-5; X is an amino acid sequence of arbitrary 7 residues; A is any one of an amino acid sequence shown by amino acid number 1-6 of formula II, an amino acid sequence shown by amino acid number 1-4 and an amino acid sequence of amino acid number 1-3), is excellent in immunogenicity and is useful as a peptide vaccine applicable to a variety of pathogenic organisms. The peptide is obtained by synthesizing an amino acid sequence in which a part of an amino acid of an antigen protein of influenza is bonded to an amino acid sequence F by a peptide synthesizing machine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a peptide vaccine having excellent immunogenicity and applicable to a wide range of pathogens.

[0002]

BACKGROUND OF THE INVENTION An immune response is initiated by the activation of helper T cells. Activation of helper T cells requires T cell receptor, major histocompatibility complex (Major Histocompa
tibility Complex (hereinafter referred to as "MHC") and a peptide fragment of an antigen are required to bind to form a trimolecular complex. In particular, peptide fragments of antigens and MHC
Binding to the molecule is important.

Ogasawara et al., In a peptide antigen (p43-58) consisting of an amino acid sequence corresponding to positions 43-58 from the N-terminus in the amino acid sequence of pigeon cytochrome c molecule, found that the amino acid sequence of pigeon cytochrome c molecule contained
The amino acids at positions 46 and 54 from the end are mouse MHC
Ogasaw has been shown to bind to class II Ab molecules [Ogasaw
ara, K., et al. (1989) J. Immunol. 142, 1448-1456
reference]. Further, an 18-residue peptide having an amino acid sequence in which a 7-residue peptide derived from influenza virus hemagglutinin was inserted between the 43-47th and 53-58th peptides was influenza in a mouse having an Ab molecule. It was reported to produce neutralizing antibodies to the virus.

That is, the 43-47th and 53-58
It was shown that an artificial peptide having a 7-residue peptide from any antigen protein of any pathogen inserted between them could be a peptide vaccine against the pathogen [Ogasawara, K., et al. 1992) Proc. Natl. Acad.
Sci. USA 89, 8995-8999 and Naruse, H., et al. (19
94) Proc Natl. Acad. Sci. USA 91, 9588-9592].

[0005]

The first object of the present invention is to have a novel structure, to be more immunogenic than conventional peptide vaccines, and to be applicable to infections or tumors caused by a wide range of pathogens. An object of the present invention is to provide a peptide having an amino acid sequence that can function as a peptide vaccine.

A second object of the present invention is to provide a vaccine comprising the above peptide as an active ingredient.

[0007]

Means for Solving the Problems The present invention provides (1) the following general formula (I): FX-A-X (I) (wherein F is amino acid number 1 of SEQ ID NO: 1 in the sequence listing) X represents an amino acid sequence represented by any one of amino acid sequences represented by amino acid numbers 2 to 5 or amino acid numbers 3 to 5; X represents an amino acid sequence of any 7 residues; A represents any one of the amino acid sequences represented by amino acid numbers 1 to 6, the amino acid numbers 1 to 4, and the amino acid sequences represented by amino acid numbers 1 to 3 in SEQ ID NO: 2 in the sequence listing) (2) F is the amino acid sequence represented by amino acid numbers 1 to 5 of SEQ ID NO: 1 in the sequence listing, and A is the amino acid sequence of SEQ ID NO: 2 in the sequence listing. (3) F is an amino acid sequence represented by amino acid numbers 2 to 5 of SEQ ID NO: 1 in the sequence listing, wherein A is an amino acid sequence represented by amino acid sequences represented by 1 to 6. The peptide according to (1), wherein F is the amino acid sequence shown in amino acid Nos. 1 to 4 of SEQ ID No. 2 in the sequence listing, and (4) F is amino acid number 3 in SEQ ID No. 1 in the sequence listing.
The peptide according to (1), wherein A is an amino acid sequence represented by amino acid numbers 1 to 3 of SEQ ID NO: 2 in the sequence listing,
(5) The method according to any one of (1) to (4), wherein X is an amino acid sequence of seven consecutive residues in an amino acid sequence of a human pathogen or an antigen protein derived from a human tumor cell. (6) The peptide according to any one of (1) to (5), wherein X is an amino acid sequence of seven consecutive residues in the amino acid sequence of an antigenic protein of influenza virus. (7) The peptide according to any one of (1) to (6), wherein X is an amino acid sequence of seven consecutive residues in the amino acid sequence of hemagglutinin of influenza virus, 8) SEQ ID NO: 3 in Sequence Listing
A peptide comprising the amino acid sequence shown in
(9) The method according to any one of (1) to (5), wherein X is an amino acid sequence of seven consecutive residues in the amino acid sequence of the antigen protein of human immunodeficiency virus (HIV). (10) X is an amino acid sequence of seven consecutive residues in the amino acid sequence of the gp120 protein of human immunodeficiency virus (HIV), wherein (1) to (5) or (9) And (11) SEQ ID NO: 4 in the sequence listing.
A peptide comprising the amino acid sequence shown in
(12) a peptide comprising the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing, (13) a peptide comprising the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, (14) (1) to ( (13) a medicine containing the peptide according to any one of (13) as an active ingredient;
5) an immunotherapeutic agent comprising the peptide according to any one of (1) to (13) as an active ingredient, (16)
An influenza vaccine agent comprising the peptide according to any one of (1) to (8) as an active ingredient;
7) an HIV vaccine agent comprising the peptide according to any one of (1) to (5) or (9) to (13) as an active ingredient, (18) any one of (1) to (5) A tumor vaccine containing the peptide of any one of (1) to (4) as an active ingredient.

Hereinafter, the present invention will be described in detail.

[0009] The peptide of the present invention comprises, at the N-terminal side, Tyr-Glu-Gly-Phe-Ser (amino acids 1 to 5 of SEQ ID NO: 1 in the sequence listing); Glu-Gly-Phe-Ser (sequence in the sequence listing) An amino acid sequence represented by any one of Gly-Phe-Ser (amino acid numbers 3 to 5 of SEQ ID NO: 1 in the sequence listing) (hereinafter referred to as "F")
), Followed by an amino acid sequence of seven consecutive residues in the amino acid sequence derived from any human pathogen or antigenic protein of a human tumor cell (hereinafter referred to as "X"), and Lys-Ala-Lys-Gly-Ile- Thr (amino acids 1 to 6 of SEQ ID NO: 2 in the sequence listing); Lys-Ala-Lys-Gly (amino acids 1 to 4 of SEQ ID NO: 2 in the sequence listing); Lys-Ala-Lys (SEQ ID NO: 2 of the sequence listing) The amino acid sequence represented by any one of amino acids 1 to 3 (hereinafter “A”)
And an amino acid sequence X of 7 residues derived from the antigen protein of the arbitrary pathogen or tumor cell on the C-terminal side.
Having the structure represented by the general formula (I): FX-A-X (I). Note that F is 4 amino acids from the N-terminal in the amino acid sequence of pigeon cytochrome c.
A corresponds to the partial amino acid sequence at positions 3-47, 44-47 or 45-47, and A is the amino acid sequence of pigeon cytochrome c at positions 53-58, 5
This is an amino acid sequence corresponding to the partial amino acid sequence at positions 3-56 or 53-55. The present inventors have proposed FX-A-X in which X is further added to the C-terminal side of a peptide having a structure represented by FX-A, which has been conventionally known.
The present inventors have completed the present invention by synthesizing a peptide having a structure represented by the following formula, and finding that the peptide is a superior peptide vaccine for humans than conventional ones.

In the present invention, the amino acid sequence of X is a continuous amino acid sequence which is normally exposed on the surface of pathogens such as pathogenic microorganisms, viruses, etc. to humans or human tumor cells without being modified with sugar chains or the like. There is no particular limitation, but in the case of a human pathogen antigen protein, an amino acid sequence conserved between mutant strains of the same species of pathogen is more preferable.
However, even if the above conditions are met, the amino acid sequence that is also present in proteins or peptides produced by normal human cells is not suitable as X, but immunotolerant to autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. For the purpose of treating by inducing, the amino acid sequence in a typical autoantigen protein of these diseases can be selected as X.

Examples of preferred peptides in the present invention are as follows: [1] Tyr-Glu-Gly-Phe-Ser- Trp-Thr-Gly-Val-Thr-Gln-
Asn -Lys-Ala-Lys-Gly-Ile-Thr- Trp-Thr-Gly-Val-Thr-Gl
n-Asn (Peptide 1: SEQ ID NO: 3 in Sequence Listing); [2] Tyr-Glu-Gly-Phe-Ser- Ile-Gly-Pro-Gly-Arg-Ala-
Phe -Lys-Ala-Lys-Gly-Ile-Thr- Ile-Gly-Pro-Gly-Arg-Al
a-Phe (peptide 2: SEQ ID NO: 4 in the sequence listing); [3] Glu-Gly-Phe-Ser- Ile-Gly-Pro-Gly-Arg-Ala-Phe-
Lys-Ala-Lys-Gly- Ile- Gly-Pro-Gly-Arg-Ala-Phe (Peptide 5: SEQ ID NO: 8 in Sequence Listing); [4] Gly-Phe-Ser- Ile-Gly-Pro-Gly- Arg-Ala-Phe -Lys-
Ala-Lys- Ile-Gly-Pro- Gly-Arg-Ala-Phe (peptide 6: SEQ ID NO: 9 in Sequence Listing).

In the above [1], the general formula (I)
The amino acid sequence (underlined) corresponding to X of the hemagglutinin N of influenza virus A / Aichi / 2/68 (H3N2) strain (hereinafter referred to as “Aichi strain”)
This is an amino acid sequence corresponding to positions 127 to 133 from the terminal. In the above [2] to [4], the amino acid sequence corresponding to X in the general formula (I) (underlined) is the 318-324 position from the N-terminus of the human immunodeficiency virus HIV-1 IIIb type gp120 protein. The corresponding amino acid sequence.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide according to the present invention comprises:
It can be easily prepared by a peptide synthesis method commonly used in the art, which is known as “solid phase method” or “liquid phase method”. For example, “The New Chemistry Experiment Course” edited by The Biochemical Society of Japan, Vol. 1, “Protein VI”, Nos. 3-44
Pp. 1992, published by Tokyo Kagaku Dojin, etc., describes details of peptide synthesis. The peptide was synthesized using Fmoc (9-fluorenyl methyloxycar) using a peptide synthesizer (for example, model PSSM-8: manufactured by Shimadzu Corporation).
bonyl) It can be synthesized by the solid phase synthesis method according to the protocol of the same apparatus. That is, Fmoc-L- in which an amino acid corresponding to the C-terminal of each peptide to be synthesized is introduced.
The amino acid Resin resin is set in the reaction vessel of the above peptide synthesizer, and Fmoc is removed using a deprotection solution. Further, an amino acid solution corresponding to the second amino acid from the C-terminus is reacted with an activator solution, and after the reaction, the Fmoc group is again deprotected and the same operation is repeated to synthesize the target peptide. .

The peptide of the present invention is not limited to those prepared by chemical synthesis, but may be those prepared by recombinant DNA technology. For example, the above-mentioned [1] to [4]
A DNA encoding the amino acid sequence described in any one of the above is prepared, inserted into a vector capable of autonomous propagation, and transformed into a host such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast or the like as appropriate. The peptide of the present invention may be collected from a cultured body.

As a method for preparing DNA having a desired nucleotide sequence, for example, chemically synthesizing sense and antisense nucleotides which are partial sequence nucleotides of the desired DNA and having both ends overlapping, Chain reaction [Saiki, RK et
al (1988) Science 239, 487-491], and a method in which those partial sequences are linked to each other by using a DNA polymerase reaction.

By incorporating the DNA encoding the amino acid sequence of the peptide of the present invention obtained as described above into a suitable vector, prokaryotic or eukaryotic host cells can be transformed. Furthermore, by introducing an appropriate promoter and a sequence related to transformation into these vectors, the DNA can be expressed in each host cell.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
And the like. In order to express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences.
Further, it is desirable that the vector has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cells.

For example, Escherichia coli includes E. coli. coli K1
Two strains are commonly used, and the vector is generally pBR
Although 322 and pUC-type plasmids are often used, the invention is not limited thereto, and any of various known strains and vectors can be used.

Examples of the promoter include a tryptophan (trp) promoter, a lactose (lac) promoter, a tryptophan lactose (tac) promoter, a lipoprotein (lpp) promoter, a bacteriophage-derived lambda (λ) P in Escherichia coli.
L promoter, polypeptide chain elongation factor Tu (tuf
B) A promoter and the like can be used, and any promoter can be used for production of the peptide of the present invention.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 [Ohmura,
K., et al. (1984) J. Biochem. 95, 87-93] and the like, but are not limited thereto. As a promoter for Bacillus subtilis, a regulatory sequence of the α-amylase gene of Bacillus subtilis is often used, and if necessary, a DNA sequence encoding a signal peptide sequence of α-amylase may be ligated to allow extracellular secretory expression. Is also possible.

For example, when Escherichia coli is used as a host cell, an expression vector having a pBR322 origin of replication, capable of autonomous growth in Escherichia coli, and having a transcription promoter and a translation initiation signal is used. be able to. The expression vector was prepared by the calcium-chloride method [Mandel, M. and Higa, A. (1970) J. Mol.
Biol. 53, 154], Hanahan's method [Hanahan, D .;
and Meselson, M. (1980) Gene 10, 63] and electric pulse drilling [Neumann, E., et al. (1982) EMBO J.
1, 841-845], and the like, whereby cells transfected with the desired vector can be obtained.

Eukaryotic host cells include cells of vertebrates, insects, yeasts and the like. Examples of vertebrate cells include COS cells, which are monkey cells [Gluzman, Y. (1981)
Cell23, 175-182] and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO) [Urlaub, G. and Chasin, LA (1980) Proc. Natl.
Acad. Sci. USA 77, 4216-4220], human Namalva cells, hamster BHK cells and the like are often used, but are not limited thereto.

As a vertebrate cell expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc., which is usually located upstream of the gene to be expressed, can be used. May have a replication origin. An example of such an expression vector is pSV2dhfr having the SV40 early promoter [Subramani, S., et al. (1981) Mo.
l. Cell. Biol. 1, 854-864].
It is not limited to this.

As eukaryotic microorganisms, yeasts are generally used, and among them, yeasts belonging to the genus Saccharomyces,
For example, Saccharomyces cerevisiae
revisiae) is preferred. Examples of expression vectors for eukaryotes such as yeast include a promoter for an alcohol dehydrogenase gene [Bennetzen, JL and Hall, BD (198
2) J. Biol. Chem. 257, 3018-3025] and the promoter of the acid phosphatase gene [Miyanohara, A., et al.
Natl. Acad. Sci. USA 80, 1-5] can be preferably used.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription assembly signal and an RNA splice. One having a site can be used. The expression vector is a DEAE-dextran method [Luth
man, H. and Magnusson, G. (1983) Nucleic Acids Re
s. 11, 1295-1308], calcium phosphate-DNA
Coprecipitation method [Graham, FL and van der Ed, AJ (197
3) Virology 52, 456-457] and electric pulse drilling [Neumann, E., et al. (1982) EMBO J. 1, 841-845.
See] and the like,
Thus, a desired transformed cell can be obtained. When a CHO cell is used as a host cell, n serves as a G418 resistance marker together with an expression vector.
a vector capable of expressing the eo gene, such as pRSVne
o [Sambrook, J., et al. (1989) "Molecular Clonin
g: A Laboratory Manual "Cold Spring Harbor Laborat
ory, NY] or pSV2neo [Southern, PJ and
Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341
And the like, and G418-resistant colonies are selected to obtain transformed cells stably producing the peptide of the present invention.

The desired transformant obtained above can be cultured according to a conventional method, and the polypeptide of the present invention is produced intracellularly or extracellularly by the culture. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of Escherichia coli, a tryptone-yeast medium (1.6% bactotryptone, yeast extract 1) is used. 0.0%, NaCl
0.5% (pH 7.0)) or a peptone medium (manufactured by Difco) can be used. As the COS cells, those obtained by adding serum components such as fetal bovine serum (FBS) to a medium such as RPMI-1640 medium or Dulbecco's modified Eagle's minimum essential medium (DMEM) as necessary can be used.

As described above, the peptide of the present invention produced inside or outside the cells of the transformant can be separated and separated by various known separation procedures utilizing the physical and chemical properties of the protein. It can be purified. Specific examples of such a method include treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC). ), Various chromatography methods, dialysis methods, combinations thereof, and the like.

When a foreign gene is introduced into Escherichia coli or the like and expressed in a large amount, the produced peptide may form a water-insoluble aggregate called an inclusion body. In such a case, the peptide can be solubilized by denaturing the peptide with a strong denaturing agent such as guanidine isothiocyanate.

The peptide of the present invention exerts the desired therapeutic and / or prophylactic effects even when administered in a relatively crude form, but is usually purified prior to use. Purification includes, for example, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography,
Conventional methods in the art for purifying peptides or proteins, such as affinity chromatography, gel electrophoresis, and isoelectric focusing, are used, and these methods may be appropriately combined as needed. Then, depending on the final use form, the purified peptide may be concentrated and lyophilized to a liquid or solid state.

The binding of the peptide of the present invention to a human MHC molecule is determined by, for example, H, one of the human MHC molecules.
Isolating the LA-DR9 protein from a cultured cell line expressing the protein;
A peptide known to bind to the protein [Fujisao, S., et al. (1996) Hum. Immunol. 45, 131
Reference]] can be confirmed.

The effect of the peptide of the present invention on the prevention of pathogen infection or onset can be confirmed, for example, by the following experiment.

By crossing a transgenic mouse into which the human MHC gene has been introduced and a mouse having the mouse MHC gene knocked out, the MHC of the mouse can be obtained.
A mouse that does not express the gene but expresses the human MHC gene is created. That is, the mouse reacts only with an antigen that binds to a human MHC molecule to produce an antibody. The mice are immunized with the peptide of the present invention, and the obtained serum is used to determine whether an antibody against the pathogen to be tested is produced. For example, the serum is mixed with the pathogen to be tested and the infectivity of the pathogen is examined. Confirm by doing. Alternatively, after administering the test pathogen after immunizing the mouse with the peptide of the present invention, removing the tissue in which the pathogen mainly grows, whether or not the pathogen is growing in the tissue, For example, it is confirmed by measuring an enzymatic activity specific to the pathogen.

Further, as a method for confirming that immunity is established by administration of the peptide of the present invention, for example, the peptide of the present invention is subcutaneously injected into a mouse which does not express the MHC gene of the mouse but expresses the human MHC gene. One or two weeks later, a method of examining whether or not T cells collected from lymph nodes of the mouse proliferate in the presence of the peptide (antigen-specific T cell proliferation test) can be exemplified.

The vaccine comprising the peptide of the present invention will be described in detail. The vaccine comprising the peptide of the present invention generally contains one or more of the peptides of the present invention in an amount of 0.01 to 100% (w / w). , Preferably 0.
It comprises from 0.05 to 50% (w / w), more preferably from 0.5 to 5.0% (w / w). Vaccines comprising the peptide of the present invention include, in addition to the form of the peptide alone, other physiologically acceptable carriers, such as serum albumin, gelatin, carriers such as mannitol, excipients,
Includes forms as compositions with immune boosters, stabilizers, and optionally one or more other drugs. Further, a vaccine agent comprising the peptide of the present invention,
The present invention also encompasses a drug in a dosage unit form, which means that the peptide of the present invention can be produced, for example, by administering the peptide of the present invention to a daily dose or an integer multiple thereof (up to 4 times) or a submultiple thereof (1/1).
(Up to 40) means a drug in physically separate, unitary dosage form suitable for administration. Drugs in such dosage unit form include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, cataplasms, suppositories, eye drops ,
Nasal drops, sprays, injections and the like can be mentioned.

The method of using the vaccine agent comprising the peptide of the present invention will be described. The vaccine agent comprising the peptide of the present invention is generally used in mammals including humans for the purpose of preventing infection, preventing onset of infection after infection, or treating tumors. leather,
Oral, nasal, ophthalmic or injection administration. The dosage in humans varies depending on the purpose and symptoms of administration, but is usually 0.01 to 1.0 g, preferably 0.01 to 1.0 g per adult, while observing the symptoms and progress of the subject after administration.
It is usually administered once or several times with a standard of about 0.1 to 0.1 g.
When administered for the purpose of inducing immune tolerance, it is repeatedly administered once a week to once a month for about 1 to 6 months, usually in escalating doses.

[0036]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

Embodiment 1 The peptide was chemically synthesized by a method in which amino acids were bonded one by one from the carboxyl terminal side to the amino acid derivative immobilized on the synthetic resin of peptide 1 (solid phase synthesis method). The amino acid used in each cycle used was a special amino acid derivative in which the α-amino group and the reactive group at the residue were blocked with a protecting group. In this example, an amino acid in which each α-amino group was blocked by Fmoc (9-fluorenylmethyloxycarbonyl) was used (Fmoc method). In addition, peptide synthesis was performed by sequentially repeating a reaction of deprotecting the Fmoc of the α-amino group of the amino acid bound to the resin, and then binding an amino acid derivative having an activated carboxyl group.

Each peptide was prepared using a peptide synthesizer (PSSM).
-8: manufactured by Shimadzu Corporation) by the Fmoc solid-phase synthesis method described above.

That is, Fmoc-Asn into which an amino acid (Asn) corresponding to the C-terminal residue of the peptide to be synthesized is introduced.
30 mg of (Trt) -resin resin (0.44 mmol / g; manufactured by Shimadzu Corporation) was set in the reaction vessel of the peptide synthesizer, and dimethylformamide (hereinafter, “DMF”) was added.
Was washed once. Next, a deprotection solution (30% (v / v) piperidine / DMF) was added for 3 minutes for 3 minutes.
Reaction twice per minute, Fm of amino acid bound to resin
After removing the oc group, the resultant was washed five times with DMF. 150 μmol of Fmoc-Glu (OtBu) corresponding to the second amino acid from the C-terminal side
-OH / PyBOP and activator solution (0.5 M 1-hydroxybenzotriazole (HOBt) / DMF and 1
(MN-methylmorpholine / DMF) and the amino acid bound to the resin excluding the Fmoc group were reacted for 30 minutes at room temperature. Fmoc generated here
After washing the -Glu (OtBu) -Asn (Trt) -resin resin four times with DMF, the Fmoc group is deprotected again, and
After washing twice, it was reacted with an activated Fmoc-Thr (tBu) -OH / PyBOP solution.

By repeating the same operation, the desired protected peptide resin (Fmoc-Tyr (tBu) -Glu (OtBu) -Gly-
Phe-Ser (tBu) -Trp-Thr (tBu) -Gly-Val-Thr (tBu) -Gln (Tr
t) -Asn (Trt) -Lys (Boc) -Ala-Lys (Boc) -Gly-Ile-Thr (tBu)
-Trp-Thr (tBu) -Gly-Val-Thr (tBu) -Gln (Trt) -Asn (Trt)-
Resin resin).

The amino acids used in the following peptide synthesis are as follows (() indicates a protecting group for protecting a reactive group at a residue portion; Fm: Shimatsu Seisakusho Co., Ltd.): Fm
oc-Ala-OH / PyBOP, Fmoc-Arg (Pmc) -OH / PyBOP, Fmoc-Asn
(Trt) -OH / PyBOP, Fmoc-Gln (Trt) -OH / PyBOP, Fmoc-Glu (O
tBu) -OH / PyBOP, Fmoc-Gly-OH / PyBOP, Fmoc-Ile-OH / Py
BOP, Fmoc-Lys (Boc) -OH / PyBOP, Fmoc-Phe-OH / PyBOP,
Fmoc-Pro-OH / PyBOP, Fmoc-Ser (tBu) -OH / PyBOP, Fmoc-T
hr (tBu) -OH / PyBOP, Fmoc-Trp-OH / PyBOP, Fmoc-Tyr (tB
u) -OH / PyBOP, Fmoc-Val-OH / PyBOP.

The meanings of the abbreviations used in the above description are as follows: Trt = trityl; Pmc =
2,2,5,7,8-pentamethylchroman-6-sulfonyl; Boc = t-butoxycarbonyl; tBu = tert-
Butyl; PyBOP = benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate; OtBu = tert-butoxy.

First, the protected peptide resin synthesized as described above (Fmoc-Tyr (tBu) -Glu (OtBu) -Gly-Phe-Ser (tB
u) -Trp-Thr (tBu) -Gly-Val-Thr (tBu) -Gln (Trt) -Asn (Trt)
-Lys (Boc) -Ala-Lys (Boc) -Gly-Ile-Thr (tBu) -Trp-Thr (tB
u) -Gly-Val-Thr (tBu) -Gln (Trt) -Asn (Trt) -resin resin)
Was reacted twice for 5 minutes and 3 minutes to deprotect the N-terminal Fmoc group.

Next, the resultant was washed with DMF five times, washed twice with methanol and once with t-butyl methyl ether, and dried by blowing nitrogen gas for 10 minutes.

The resin was taken out, and the crevage solution (940)
Mix 0 ml of trifluoroacetic acid, 300 ml of thioanisole and 300 ml of ethanedithiol,
Of 2-methylindole) in 0.5 ml
In addition, the peptide is cleaved from the resin and the amino acid side-chain protecting group is removed by reacting at room temperature for 2 hours.
A peptide solution was obtained. To this, 10 ml of cold ether was added to precipitate the peptide. Centrifuge this (200
(0 × g, 10 minutes) The precipitate was collected, and the operation of adding and dispersing cold ether again and then collecting was repeated four times to wash the peptide. The obtained peptide was lyophilized to obtain the desired peptide.

Embodiment 2 FIG . Synthesis of Peptide 2 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Tyr (tBu) -Glu (OtBu) -Gly-Phe-Ser (tBu) -Ile-Gly-Pro-G
ly-Arg (Pmc) -Ala-Phe-Lys (Boc) -Ala-Lys (Boc) -Gly-Ile-
Thr (tBu) -Ile-Gly-Pro-Gly-Arg (Pmc) -Ala-Phe-Resin resin) was synthesized, and deprotection and other operations were performed to obtain the target peptide.

Reference Example Synthesis of Peptides 3 and 4 By performing the same operation as in Example 1, the following peptides were obtained: Tyr-Glu-Gly-Phe-Ser-Trp-Thr-Gly-Val-Thr-Gln-Asn-Ly
s-Ala-Lys-Gly-Ile-Thr (peptide 3: SEQ ID NO: 5 in Sequence Listing); Tyr-Glu-Gly-Phe-Ser-Ile-Gly-Pro-Gly-Arg-Ala-Phe-Ly
s-Ala-Lys-Gly-Ile-Thr (peptide 4: SEQ ID NO: 6 in the sequence listing) was synthesized. Peptides 3 and 4 are known from literature [Naruse, H., et al. (1994) Proc.
Acad. Sci. USA 91, 9588-9592], an amino acid corresponding to X in peptides 1 and 3, and peptides 2 and 4, respectively. The arrays are common. In the test for confirming the effect of peptide 1 or 2 described below, peptide 3 or 4 was also used for comparison.

Test Example 1 HIV neutralization test 1) Preparation of antiserum Transgenic mouse into which HLA-DQ6 gene was introduced [Nishimura, Y., et al. (1990) J. Immunol. 145,
353-360] and MHC class II gene knockout mice [Gosgrove, D., et al. (1991) Cell 66,
1051-1066], the mouse M
A mouse expressing the HLA-DQ6 gene without expressing the HC class II gene (hereinafter referred to as "DQ6 mouse") was created. This DQ6 mouse is a human-derived HLA
-Produce antibodies only against antigens that bind to DQ6.

The DQ6 mouse was treated with peptide 2 (50 nm).
ol / mouse) was intraperitoneally administered together with Freund's complete adjuvant (manufactured by Difco).
(50 nmol / mouse) was intraperitoneally administered together with Freund's incomplete adjuvant (manufactured by Difco). Three weeks later, peptide 2 (50 nmol / mouse) was again intraperitoneally administered together with Freund's incomplete adjuvant (manufactured by Difco), and one week later, serum was separated from the collected blood and kept at 56 ° C. for 30 minutes. Those were used as anti-peptide 2 antiserum in the following tests. An anti-peptide 4 antiserum was also prepared in the same manner.

2) Neutralization test MOLT4 cell line (ATCC No. CRL158)
2) was infected with HIV-1IIIB strain (National Institutes of Health, AIDS Research and Reference Reagent Program Catalog No. 398) [HIV: A Practical Approach (eds. Karn, J., et al)
21-43, IRL Press (1995)]. The cells were RPMI164 containing 10% fetal calf serum (hereinafter "FCS").
A suspension of 1 × 10 ⁵ cells / ml in 0 medium was dispensed into a 96-well culture plate at 90 μl / well, and the uninfected MOLT4 cell line (1 × 10 ⁶ cells) was added to each well.
(Cells / ml) 90 μl and anti-peptide 2 antiserum, anti-peptide 4 antiserum prepared as described above, or 20 μl of serum of an untreated mouse were added, and the cells were cultured at 37 ° C. under 5% carbon dioxide for 24 hours. 4 μl of the culture supernatant in each well was collected in a test tube, and 10 μl of Nonidet P-40 (hereinafter referred to as “NP-40”) was added to destroy the virus. Add a mixed solution for reverse transcriptase reaction (50
mM Tris-HCl (pH 8.0), 7.5 mM potassium chloride, 2 mM dithiothreitol, 4.95 mM
5. magnesium chloride, 5 μg / ml poly (A),
25 μg / ml oligo d (T), 0.2 μCi / ml
[ ³² P] dTTP 0.2 μl) was added thereto,
It was kept at 37 ° C. for 3 hours. After completion of the reaction, each reaction solution was added dropwise to DEAE filter paper (manufactured by Whatman), and then the filter paper was washed with 2 × SSC (300 mM sodium chloride, 30 mM
Washed 5 times with (trisodium citrate) and dried. This filter paper is used for bio-image analyzer Bas10.
00 (manufactured by Fuji Photo Film Co., Ltd.) and left at room temperature for 2 hours. The image plate was set on the Bas1000 body, and the energy value (photostimulated luminescence) of each sample was measured to compare the reverse transcriptase activities of the cell groups under each condition.
As a result, the group to which the anti-peptide 2 antiserum was added had lower reverse transcriptase activity than the group to which the anti-peptide 4 antiserum was added (FIG. 1). That is, it was shown that the peptide 2 of the present invention, which is of the RXX type, has a stronger activity of suppressing HIV infection than the conventionally known peptide 4 of the RXA type.

Test Example 2 Influenza infection prevention test 100 µg of peptide 1 was dissolved in 1 ml of chloroform, and 50 µl of a mixture of phosphatidylserine and phosphatidylcholine mixed at a ratio of 3: 7 (v / v) was added. After adding twice the amount of chloroform to this, chloroform was volatilized while adding nitrogen gas using an evaporator, and then dried using a vacuum pump. A solution obtained by adding 30 μl of Dulbecco's PBS (−) (hereinafter referred to as “liposome”) to the DQ6 mouse was administered through the nasal cavity of the DQ6 mouse, and the same peptide administration was repeated twice every two weeks thereafter. The control group received liposomes without peptide. Four days later, 30 μl of a 3000-fold diluted stock solution of an influenza virus Aichi strain having a hemagglutinating ability was administered through the nasal cavity to a 2048-fold dilution. Four days later, the lungs were removed from the mice of each group, and
% Bovine serum albumin (hereinafter referred to as “BSA”) and homogenized in a glass homogenizer containing Dulbecco PBS (−). A serial dilution of this lung cell lysate was used to grow MDCK cells (ATCC No. CCL-
34), 100 μl / well was added thereto, and the mixture was cultured at 37 ° C. under 5% carbon dioxide gas for 1 hour. Then, the medium was removed, and 1% BSA was added.
And washed with PBS (-) containing Then, the agar medium (2
50 ml of Bacto agar (manufactured by Difco), 50 ml of Dulbecco's modified Eagle's medium (DMEM) at a concentration of 2 cultures and 20 μl of 1% trypsin were mixed, and the mixture was kept at 42 ° C. so that the agar would not solidify). The cells were cultured at 5 ° C. under 5% carbon dioxide for 2 days. A staining solution (0.5% crystal violet, 50% ethanol, 5% formaldehyde, 4%
(5% physiological saline) was added in an amount of 60 μl each, and after several minutes, washed with running water. The cells in each well were observed under a microscope and the number of unstained white plaques per well was counted.

As a result, the number of plaques in the MDCK cells to which the cell lysate from the mouse lung to which the peptide 1 was administered was added was smaller than the number of plaques in the MDCK cells to which the cell lysate from the lungs of the control group was added (FIG. 9). 2). Therefore, it was found that the DQ6 mice to which peptide 1 was administered were not infected with the Aichi strain virus, or even if they were infected, the proliferation was suppressed.

Test Example 3 HLA molecule expressing the binding test HLA-DR9 for, (which is established by infecting Epstein-Barr virus in human peripheral blood B cells) L-KT12 cells [HLA 1991 (eds. Tsuj
i, K., et al.) 1166-1169, Oxford University Press
Inc., New York, 1992] with 10% FCS
The cells were cultured in a PMI1640 medium at 37 ° C. under 5% carbon dioxide. About 1 × 10 ⁹ cells were collected and centrifuged, and the precipitated cells were added to 1% NP-40, 5 mM sodium orthovanadate, 25 mM iodoacetamide and
mM phenylmethylsulfonyl fluoride (hereinafter “PMS
F ") and disrupted the cells by adding 4 ml of Dulbecco's PBS (-), and used as the HLA-DR9 stock solution in the operation described below.

A mouse hybridoma HU-4 producing an anti-HLA-DR monoclonal antibody [Kasahara, M., et al.
al. (1984) Immunology 53, 79] to ICR nu.
/ Nu mice were injected intraperitoneally and ascites was collected 14 days later. IgG Orientation Kit (Pierce)
Was used as described in the protocol of the kit to prepare an affinity column to which an antibody for 5 ml of the above ascites was bound.

The above-mentioned HLA-
After passage through the DR9 stock solution, 0.5% NP-40 and 0.
The column was washed with PBS containing 1% sodium dodecyl sulfate, and then washed with PBS containing 1% n-octyl-β-D-glucopyranoside. Then 1% n
-Octyl-β-D-glucopyranoside and 50 mM
By flowing a 0.15 M sodium chloride solution (pH 10.5) containing diethylamine through the column, HLA-
DR9 molecules were eluted. Immediately eluate 0.15M Tris-HCl containing 0.15M sodium chloride (pH
After 6.8) was added to neutralize the mixture, the mixture was centrifuged at 5000 × g in a centrifugal tube type ultrafiltration apparatus (Centricon-10, discharge limit: 10 kDa, manufactured by Amicon) to obtain 40%.
Concentrate to 0 μl and add 1 mM PMSF, 0.05
% NP-40, 5% dimethyl sulfoxide and 0.15 M sodium chloride in 50 mM phosphate buffer (pH 7.0) and centrifugation were repeated twice to exchange the solvent, and the protein concentration was 1 μg / ml.
The purified sample was used as a purified HLA-DR9 molecule sample.

On the other hand, it is known that it strongly binds to the HLA-DR9 molecule [Fujisao, S., et al. (1996) Huma
n Immunol. 45, 131], peptides described below: Ala-Asn-Asp-Gly-Ala-Thr-Gly-Trp-Val-Ala-Ser-Met-Se
r-Ser-Ala-Tyr (hereinafter referred to as “standard peptide”: SEQ ID NO: 7 in the sequence listing) was synthesized in the same manner as described in Example 1, and was biotinylated by the method described below. 10 mg of peptide still attached to the resin resin, 6 mg of biotin, DMF
120 μl was added, and further 5 μl of N, N-diisopropylethylamine was added. This was reacted overnight at room temperature while stirring with a rotary shaker. Next, 1 ml of DMF was added to wash the resin resin, and 11,000 rpm
The operation of removing the supernatant by centrifugation was repeated 5 times. Further, after the same operation was performed twice by replacing the washing solution with methanol, and after drying, operations such as resin resin cutting and deprotection were performed in the same manner as described in Example 1 to obtain a biotinylated standard peptide. Obtained. A standard peptide not biotinylated was also prepared.

96-well plate (3590 manufactured by Coaster)
50 μl / well of 10 μg / ml protein A (Pierce) was dispensed to the plate) and allowed to stand at room temperature for 24 hours to adsorb protein A to the bottom of the well. Duplicate each well with Dulbecco's PBS (-) containing 0.05% Tween 20 (hereinafter referred to as "PBS-T").
After washing twice, the mouse ascites fluid containing the anti-HLA-DR monoclonal antibody was diluted 10-fold with Dulbecco's PBS (-), dispensed at 50 µl / well, and allowed to stand at 4 ° C for 24 hours. An anti-HLA-DR monoclonal antibody was bound to protein A adsorbed on the plate. Each well was washed four times with PBS-T. Thereafter, 1.5 μg / well of purified HLA-DR9 molecule sample, sample (peptide 1, peptide 2, peptide 3, peptide 4 or non-biotinylated standard peptide diluted to various concentrations) and 3 μM biotinylated standard peptide Were mixed and reacted at room temperature for 72 hours. Thereafter, each well was washed four times with PBS-T, and then avidin-labeled alkaline phosphatase (manufactured by Gibco) was added to 1%
A 400-fold dilution with PBS (-) containing BSA
After dispensing 0 μl / well and allowing to stand at room temperature for 2 hours, each well was washed four times with PBS-T. And a phosphatase substrate (phosphatase substrate system for EIA;
Kirkeguard & Perry Laboratories)
Was dispensed at 50 μl / well and reacted for 60 minutes, and then the absorbance at 405 nm of each well was measured.

A standard curve was prepared by plotting the relationship between the peptide concentration and the absorbance when a non-biotinylated standard peptide was used as a sample on a graph, and based on the standard curve, the relative binding affinity of the other sample ( relative binding affin
[rba] = Cv / Cs (where Cv and Cs are the concentrations of the sample peptide and the non-biotinylated standard peptide having the same absorbance, respectively) Represent).

As a result, the peptides of the present invention (peptides 1 and 2), which are of the R-X-A-X type, are each higher in HLA than the conventionally known R-X-A-type peptides (peptides 3 and 4). -It turned out to bind strongly to DR9. In particular, peptide 3 did not bind to HLA-DR9, whereas peptide 1 bound to HLA-DR9 (FIG. 3).

Embodiment 3 FIG . Synthesis of Peptide 5 Protected peptide resin (Fmoc
-Glu (OtBu) -Gly-Phe-Ser (tBu) -Ile-Gly-Pro-Gly-Arg (Pm
c) -Ala-Phe-Lys (Boc) -Ala-Lys (Boc) -Gly-Ile-Gly-Pro-G
ly-Arg (Pmc) -Ala-Phe-Resin resin) was synthesized, and deprotection and other operations were performed to obtain the desired peptide (SEQ ID NO: 8 in the sequence listing).

Embodiment 4 FIG . Synthesis of Peptide 6 By the same operation as in Example 1, the protected peptide resin (Fmoc
-Gly-Phe-Ser (tBu) -Ile-Gly-Pro-Gly-Arg (Pmc) -Ala-Phe
-Lys (Boc) -Ala-Lys (Boc) -Ile-Gly-Pro-Gly-Arg (Pmc) -Al
a-Phe-Resin resin) was synthesized, and deprotection and other operations were performed to obtain the target peptide (SEQ ID NO: 9 in the sequence listing).

Test Example 4 Antigen-specific T cell proliferation test Peptide 2, 5 or 6 prepared in Examples 2 to 4
(20 nmol / mouse) was injected subcutaneously in the tail of DQ6 mouse tail with Freund's complete adjuvant. 1
0 days later, regional lymph nodes (inguinal lymph nodes and para-aortic lymph nodes) were excised from the mice, and nylon wool method [Ju
lius, MH et al. (1973) Eur. Immunol. 3, 645], T cells were separated and collected, and suspended in RPMI1640 medium. This T cell was placed in a 96-well flat bottom plate at 4 × 1.
0 put ⁵ cells / well.

On the other hand, the spleen was excised from a non-immunized DQ6 mouse, and spleen cells were prepared. The spleen cells were irradiated with 30 Gy of radiation, and this was used as an antigen-presenting cell in a 96-well plate containing the T cells in a concentration of 1 × 10 6. ⁵ cells / well. In addition, peptides 2, 5 or 6 are added at various concentrations,
% Carbon dioxide, from the cultured 3 days under conditions of 37 ° C., ³
H-thymidine was added at 0.5 μCi / well for another 1 hour.
The cells were cultured for 6 hours. After completion of the culture, the cells in each well were collected using a cell harvester, and then the amount of ³ H-thymidine incorporated into the cells in each well was measured using a liquid scintillation counter.

As a result, in all T cells derived from mice to which peptides 2, 5 and 6 were administered, the uptake of ³ H-thymidine was increased in a 96-well plate, depending on the peptide concentration. There was found. Therefore, it was shown that immunity was established by these peptides in DQ6 mice that produce antibodies only against antigens that bind to human-derived HLA-DQ6.

Formulation Examples Peptides obtained by the methods described in Examples 1 to 6 at a final concentration of 0.01, 0.1 or 1 mg / ml in a physiological saline containing 1% (w / v) human serum albumin as an injection stabilizer.
After dissolving in a sterilized vial, 2 ml is dispensed into a sterilized vial, freeze-dried, and sealed.

Prior to administration, 1 ml of distilled water for injection or the like is first added to a vial before administration, and the contents are uniformly dissolved before use. The product having excellent stability and containing the peptide according to the present invention as an active ingredient is useful as a dry preparation for treating or preventing infectious diseases or tumors.

[0067]

According to the present invention, a peptide having an amino acid sequence which has a novel structure, is more immunogenic than conventional peptide vaccines, and can function as a peptide vaccine applicable to a wide range of pathogens or tumors is provided. Could be provided. The peptide of the present invention can also be applied to pathogens, tumors, and pathogens for which vaccine effects could not be expected due to mutations in the amino acid sequence of antigens, for which conventional vaccine development was difficult.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of an HIV neutralization test.

FIG. 2 shows the results of an influenza infection prevention test.

FIG. 3 shows the results of a binding test for an HLA molecule.

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 2 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 3 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Glu Gly Phe Ser Trp Thr Gly Val
Thr Gln Asn Lys Ala Lys Gly 15
10 15 Ile Thr Trp Thr Gly Val Thr Gln Asn 20 25
SEQ ID NO: 4 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Glu Gly Phe Ser Ile Gly Pro Gly
Arg Ala Phe Lys Ala Lys Gly 15
10 15 Ile Thr Ile Gly Pro Gly Arg Ala Phe 20 25
SEQ ID NO: 5 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Glu Gly Phe Ser Trp Thr Gly Val Thr Gln Asn Lys Ala Lys Gly 1 5 10 15 Ile Thr SEQ ID NO: : 6 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Tyr Glu Gly Phe Ser Ile Gly Pro Gly Arg Ala Phe Lys Ala Lys Gly 1 5 10 15 Ile Thr SEQ ID NO: 7 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Asn Asp Gly Ala Thr Gly Trp Val Ala Ser Met Ser Ser Ala Tyr 1 5 10 15 SEQ ID NO: 8 Sequence length : 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Gly Phe Ser Ile Gly Pro Gly Arg Ala Phe Lys Ala Lys Gly Ile 1 5 10 15 Gly Pro Gly Arg Ala Phe 20 SEQ ID NO: 9 Sequence Length: 20 Sequence type: Amino acid Topology : Linear sequence type: peptide sequence Gly Phe Ser Ile Gly Pro Gly Arg Ala Phe Lys Ala Lys Ile Gly Pro 1 5 10 15 Gly Arg Ala Phe 20

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 14/11 C07K 14/11 14/155 14/155 14/80 14/80

Claims (18)

[Claims] 1. The following general formula (I): FX-A-X (I) (wherein F is an amino acid sequence represented by amino acid numbers 1 to 5 of SEQ ID NO: 1 in the sequence listing, amino acid number 2) X represents an amino acid sequence of any seven residues; A represents an amino acid sequence of SEQ ID NO: 2 in the sequence listing; 1 to 6, the amino acid sequence represented by amino acid numbers 1 to 4 or the amino acid sequence represented by amino acid numbers 1 to 3). 2. F is an amino acid sequence represented by amino acid Nos. 1 to 5 of SEQ ID No. 1 in the sequence listing, and A is an amino acid sequence represented by amino acid Nos. 1 to 6 of SEQ ID No. 2 in the sequence listing. The peptide according to claim 1, characterized in that: 3. The method according to claim 1, wherein F is an amino acid sequence represented by amino acid numbers 2 to 5 of SEQ ID NO: 1 in the sequence listing, and A is an amino acid sequence represented by amino acid numbers 1 to 4 of SEQ ID NO: 2 in the sequence listing. The peptide according to claim 1, characterized in that: 4. The method according to claim 1, wherein F is an amino acid sequence represented by amino acid numbers 3 to 5 of SEQ ID NO: 1 in the sequence listing, and A is an amino acid sequence represented by amino acid numbers 1 to 3 of SEQ ID NO: 2 in the sequence listing. The peptide according to claim 1, characterized in that: 5. The method according to claim 1, wherein X is an amino acid sequence of 7 consecutive amino acids in the amino acid sequence of an antigenic protein derived from a human pathogen or a human tumor cell. The peptide according to claim 1. 6. The peptide according to any one of claims 1 to 5, wherein X is an amino acid sequence of seven consecutive residues in the amino acid sequence of the antigenic protein of influenza virus. 7. The peptide according to any one of claims 1 to 6, wherein X is an amino acid sequence of seven consecutive residues in the amino acid sequence of hemagglutinin of influenza virus. 8. A peptide comprising the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. 9. The method according to claim 1, wherein X is an amino acid sequence of seven consecutive residues in the amino acid sequence of the antigen protein of human immunodeficiency virus (HIV). Peptide. 10. X is a human immunodeficiency virus (HIV).
7 in the amino acid sequence of the gp120 protein of
2. The amino acid sequence of residues.
10. The peptide according to any one of to 5 or 9. 11. A peptide comprising the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. 12. A peptide comprising an amino acid sequence represented by SEQ ID NO: 8 in the sequence listing. 13. A peptide comprising an amino acid sequence represented by SEQ ID NO: 9 in the sequence listing. 14. A medicament comprising the peptide according to any one of claims 1 to 13 as an active ingredient. An immunotherapeutic agent comprising the peptide according to any one of claims 1 to 13 as an active ingredient. 16. An influenza vaccine agent comprising the peptide according to any one of claims 1 to 8 as an active ingredient. 17. An HIV vaccine comprising the peptide according to any one of claims 1 to 5 or 9 to 13 as an active ingredient. 18. A tumor vaccine containing the peptide according to any one of claims 1 to 5 as an active ingredient.