Multiple antigenic peptides against flaviviruses and immunogenic compositions containing the same

Abstract

To provide a vaccine against viruses belonging to the genus of Flavivirus.SOLUTION: The present invention discloses (a) peptides including a specific amino acid sequence, or (b) partial peptides of polyproteins of a virus belonging to the genus of Flavivirus including an amino acid sequence corresponding to the specific amino acid sequence, and multiple antigen peptides including these peptides (a) or (b).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to multiple antigenic peptides against flaviviruses and immunogenic compositions comprising same, in particular immunogenic compositions or vaccines against flaviviruses. [Background technology]

[0002] It has been known for some time that multiple antigen peptides (MAPs) can induce an increase in antibodies in vivo without the intervention of T cells (Non-Patent Document 1). It has been revealed that MAPs can induce autoantibodies (anti-IgE antibodies), which are difficult to induce in vivo (Patent Document 1). It has also been disclosed that this method can be used to create a peptide portion of the Ebola virus to create a pan-virus MAP, and that the desired antibody can be induced in the serum by immunizing mice with the MAP (Patent Document 2). Similarly, it has been disclosed that a pan-virus MAP can be created using a partial peptide of influenza virus hemagglutinin, and that the desired antibody can be induced in the serum by immunizing mice with the MAP (Patent Document 3). Neutralizing antibodies against Flaviviruses have been developed (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US2017-0158738A [Patent Document 2] US2019-0276495A [Patent Document 3] US2019-0337989A [Non-patent literature]

[0004] [Non-Patent Document 1] Saravanan, P., et. al., Acta Virol., (48) 39-45, 2004 [Non-Patent Document 2] Yi-Chin Fan, et al. PLoS Negl.Trop. Dis., 9(10):e0004167, 2015 Summary of the Invention

[0005] The present invention provides multiple antigenic peptides against flaviviruses and immunogenic compositions comprising the same, in particular immunogenic compositions or vaccines against flaviviruses.

[0006] The present inventors have found that a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a multiple antigen peptide having a partial peptide of a polyprotein of a Flavivirus virus consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, promotes the production of IgM antibodies. The present inventors have also found that the multiple antigen peptide of the present invention directly stimulates antibody production from marginal zone B cells and B1B cells, and induces T cell-independent antibody production.

[0007] The present invention provides the following inventions. [1] A peptide having a length of 11 to 30 amino acids that is a part of the amino acid sequence of a polyprotein of a Flavivirus virus, the peptide comprising the amino acid sequence set forth in SEQ ID NO: 10, or a partial peptide of a polyprotein consisting of the amino acid sequence of the polyprotein corresponding to said amino acid sequence. [2] The peptide according to [1] above, which is a peptide consisting of the amino acid sequence set forth in SEQ ID NO:9, or a peptide consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:9. [3] The peptide according to [2] above, wherein the amino acid sequence corresponding to SEQ ID NO:9 has one base added, inserted, substituted, or deleted relative to the amino acid sequence set forth in SEQ ID NO:9. [4] The peptide according to [2] or [3] above, wherein the amino acid sequence corresponding to SEQ ID NO: 9 is a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 7. [5] A multiple antigen peptide comprising the peptide according to any one of [1] to [4] above. [6] The multiple antigen peptide according to [5] above, comprising eight or more peptides according to any one of [1] to [4] above. [7] A vaccine against Flaviviruses, comprising the multiple antigen peptides described in [8] above. [8] The vaccine described in [7] above, which does not contain an adjuvant. [9] The vaccine according to [6] or [7] above, used in combination with α-galactosylceramide without using any other adjuvant.

[10] The vaccine described in any of [7] to [9] above, wherein the Flavivirus virus is a virus selected from the group consisting of Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, St. Louis encephalitis virus, and tick-borne encephalitis virus and mutant viruses thereof.

[11] A method for activating immunity against Flavivirus in a subject in need thereof, comprising administering to the subject an effective amount of the multiple antigen peptide described in [5] or [6] above.

[0008] The multiple antigen peptide of the present invention is advantageous in that it induces antibody production against Flavivirus viruses. The multiple antigen peptide of the present invention can be advantageous in that memory immunity is established after administration, and neutralizing antibody production is further enhanced by viral infection. Furthermore, the multiple antigen peptide of the present invention is also suitable for producing IgM antibodies having infection neutralizing ability. IgM antibodies are not an antibody subclass that causes antibody-dependent enhancement (ADE) while exerting infection neutralizing ability. Furthermore, as for IgG antibodies, even if they are produced, they are not produced by helper T cells or follicular B cells, but by stimulating marginal zone B cells and B1B cells in a T cell-independent manner, and the produced antibodies are thought to lead to the development of a vaccine that can avoid antibody-dependent enhancement (ADE) while inducing antigen-specific immunity in vivo. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 shows a scheme for an experiment of administering multiple antigen peptides of the present invention to mice and for blood sampling. [Diagram 2] FIG. 2 shows the antibody titers in the serum of mice administered various multiple antigen peptides of the present invention. [Diagram 3] FIG. 3 shows the IgM antibody titers in the serum of mice administered various multiple antigen peptides of the present invention. [Figure 4] FIG. 4 shows antibody titers of IgG subclasses in the serum of mice administered various multiple antigen peptides of the present invention. [Diagram 5] FIG. 5 shows the time course of IgG antibody titer in sera (sera from mice administered the multiple antigen peptides of the present invention) used in experiments measuring virus neutralizing activity. [Figure 6] FIG. 6 shows a scheme of an experiment in which multiple antigen peptides of the present invention are administered to mice. [Figure 7] 7 shows the results of an experiment to measure virus neutralizing ability. Plaques are caused by cell death due to viruses, and a reduction in the number of plaques observed indicates the presence of virus neutralizing ability of the test serum. Detailed Description of the Invention

[0010] As used herein, a "subject" refers to an animal having an immune system, e.g., a vertebrate, e.g., an animal that is infected with a Flavivirus virus, and may be, for example, a mammal, a fish, a bird, an amphibian, a reptile, e.g., a primate such as a human, a chimpanzee, a gorilla, an orangutan, a monkey, a marmoset, and a bonobo, a quadruped (e.g., a carnivore, an artiodactyl, a perissodactyl, and a rodent) such as a pig, a rat, a mouse, a cow, a sheep, a goat, a horse, a cat, and a dog, or an animal of the order Chiroptera, e.g., a bat.

[0011] In this specification, "flavivirus" refers to a virus belonging to the Flaviviridae family. The Flaviviridae family includes the Flavivirus genus. The Flavivirus genus of the Flaviviridae family includes many pathogenic viruses such as Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, St. Louis encephalitis virus, and tick-borne encephalitis virus. These viruses are carried by mammals and birds other than humans, which amplify the viruses and transmit them to humans via mosquitoes and ticks. For this reason, it is considered impossible to completely eradicate flaviviruses from the natural world. Attempts have been made to create vaccines against them. However, there is concern that vaccines cause antibody-dependent enhancement (ADE) in flavivirus viruses, and that vaccines may worsen the infection of flavivirus viruses. This is thought to be because Flaviviruses can infect macrophages and aggravate symptoms, but the IgG receptors of macrophages promote the uptake of Flaviviruses into macrophages, and as a result, antibodies promote the infection of macrophages with Flaviviruses. In order to minimize the effect on the uptake of viruses into macrophages, it is desirable to suppress the production of IgG, and in particular, to suppress the production of IgG2a and IgG2b among the IgG subclasses. For this purpose, it is beneficial to induce antibodies independent of T cells, more specifically, to stimulate marginal zone B cells and B1B cells to produce antibodies.

[0012] As used herein, the term "adjuvant" refers to a substance that activates the immune system. Aluminum hydroxide and aluminum phosphate are commonly used as adjuvants.

[0013] As used herein, "α-galactosylceramide" is a sphingoglycolipid that can be isolated from the marine sponge Agelas mauritianus. α-galactosylceramide has the following structure:

[0014] [ka]

[0015] α-Galactosylceramide is a ligand that binds to CD1d and activates NKT cells. α-Galactosylceramide can be isolated, purified, or synthetic α-Galactosylceramide. Kain L., et al., Mol. Immunol., 68(2 Pt A):94-97,2015, has shown that α-glycosylceramide (α-GlyCer) has an in vivo effect equivalent to α-GalCer as a CD1d ligand. Therefore, α-GlyCer can be used instead of α-GalCer. Other known CD1d ligands can be used similarly.

[0016] As used herein, a "multiple antigen peptide" (MAP) is a molecule that allows multiple presentation of peptides having a specific amino acid sequence. A multiple antigen peptide may have a structure in which peptides are linked to the side chains of a backbone having a repeating structure. The backbone and peptides may be linked via a linker. An example of a multiple antigen peptide is one having a dendritic polymer backbone. An example of a multiple antigen peptide having a dendritic polymer backbone is a molecule that contains lysine (Lys) as a core (nucleus) and has an increased number of branches by linking additional lysines to the core via peptide bonds, and the number of branches n is 2 amino groups and peptides linked thereto. n (Francis, JP, et al., Immunology, 1991: 73; 249; Schott, ME, et al., Cell. Immuno. 1996: 174: 199-209; Tam, JP Proc. Natl. Acad. Sci. 1988: 85; 5409-5413).

[0017] In this specification, the term "dendritic polymer" (dendrimer) refers to a molecule having a structure that regularly branches from a center (core). A dendritic polymer is composed of a central molecule called a core and a side chain portion called a dendron. A dendritic polymer has multiple arms, and each arm of the core molecule that forms branches is connected to a molecule that has multiple arms and forms branches. A dendrimer is formed by linking such molecules that form branches with multiple arms. A dendritic polymer is expressed in generations by the number of consecutive branches from the core in the dendron portion. The central molecule provides the 0th generation branch (G0), the branch at the base of the dendron provides the 1st generation branch (G1), and further branching from that branch provides the 2nd generation branch (G2), and this is repeated to provide the nth generation branch (Gn).

[0018] In the present specification, "a partial peptide of a polyprotein of a Flavivirus virus having an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: n" {where n is a natural number} means a partial peptide of a polyprotein of a Flavivirus virus, and a peptide having a sequence corresponding to the amino acid sequence of SEQ ID NO: n by alignment. For example, SEQ ID NO: 1 has an amino acid sequence corresponding to amino acid numbers 392 to 413 of GenBank registration number: AAA81554.1, and is an amino acid sequence common to polyproteins (especially envelope proteins) of Flavivirus viruses (see Table 1). In Table 1, the amino acid numbers indicate the amino acid numbers in the amino acid sequence of the polyprotein, and the amino acid sequence shown in bold is a site suggested in Yi-Chin Fan, et al. PLoS Negl.Trop. Dis., 2015, that when mutated, a neutralizing antibody loses its neutralizing ability.

[0019] According to the present invention, (A) a peptide having a length of 11 to 30 amino acids which is a part of the amino acid sequence of a polyprotein of a Flavivirus virus, the partial peptide having the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 10, These peptides are sometimes referred to herein as the peptides of the present invention.

[0020] According to the present invention, the peptide of the present invention comprises (B) A peptide having a length of 11 to 30 amino acids that is a part of the amino acid sequence of a polyprotein of a Flavivirus virus, the peptide being a partial peptide of a polyprotein having an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 13 or an amino acid sequence corresponding to the amino acid sequence. It could be.

[0021] According to the present invention, in (B1) the peptide of the present invention, the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 13 may be the amino acid sequence set forth in SEQ ID NO: 11. According to the present invention, in (B2) the peptide of the present invention, the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 13 may be the amino acid sequence set forth in SEQ ID NO: 12. According to the present invention, in (B3) the peptide of the present invention, the amino acid sequence set forth in any one of SEQ ID NOs: 11 to 13 may be the amino acid sequence set forth in SEQ ID NO: 13.

[0022] According to the present invention, the peptides of the present invention described in (A), (B) and (B1) to (B3) above can be 11 amino acids long, 12 amino acids long, 13 amino acids long, 14 amino acids long, 15 amino acids long, 16 amino acids long, 17 amino acids long, 18 amino acids long, 19 amino acids long, 20 amino acids long, 21 amino acids long, 22 amino acids long, 23 amino acids long, 24 amino acids long, 25 amino acids long, 26 amino acids long, 27 amino acids long, 28 amino acids long, 29 amino acids long, or 30 amino acids long or longer. The multiple antigen peptide effectively exhibits the ability to induce antibody production when the length of the antigen peptide is 11 amino acids or more. The multiple antigen peptide effectively exhibits the ability to induce antibody production when the number of antigens is 4 or more.

[0023] According to the present invention, (C) the peptide of the present invention may comprise the amino acid sequence set forth in SEQ ID NO: 9. That is, according to the present invention, A peptide consisting of the amino acid sequence set forth in SEQ ID NO:9, or a partial peptide of the amino acid sequence of a polyprotein of a Flavivirus virus consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:9. is provided.

[0024] According to the present invention, (D) the peptide of the present invention may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 7. According to the present invention, (D') the peptide of the present invention may consist of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 7.

[0025] The peptides of the present invention may be isolated or purified. The peptides of the present invention may be artificially synthesized.

[0026] The amino acid sequences shown in SEQ ID NOs: 9 to 13 can induce antibodies specific to flaviviruses. The amino acid sequences shown in SEQ ID NOs: 9 to 13 are widely conserved among all flaviviruses. In contrast, longer peptides containing the amino acid sequences and amino acid sequences contiguous thereto can have high specificity for a particular flavivirus.

[0027] Each of the peptides consisting of the amino acid sequences set forth in SEQ ID NOs: 1 to 7 is 22 amino acids long. When the peptides consisting of the amino acid sequences set forth in SEQ ID NOs: 1 to 7 are incorporated into a multiple antigen peptide, they can induce antibodies specific to a flavivirus. The amino acid sequences set forth in SEQ ID NOs: 1 to 7 are widely conserved among all flaviviruses. In contrast, longer peptides containing the amino acid sequences and amino acid sequences contiguous thereto may have high specificity for a specific flavivirus.

[0028] A partial peptide of a polyprotein of a virus of the genus Flavivirus, which consists of an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NOs: 1 to 7, is as defined above. In one embodiment, the partial peptide may have an amino acid sequence corresponding to any of SEQ ID NOs: 1 to 7 that has one base added, inserted, substituted, or deleted relative to the amino acid sequence set forth in any of SEQ ID NOs: 1 to 7. The substitution may be a conservative substitution {e.g., substitution between basic amino acids, substitution between acidic amino acids, substitution between hydrophobic amino acids, and substitution between amino acids having uncharged hydrophilic side chains}. Examples of the substitution include conservative substitutions (e.g., substitution between acidic amino acids, substitution between basic amino acids, and substitution between hydrophobic amino acids). Also, in one embodiment, the partial peptide may have the amino acid sequence set forth in SEQ ID NO: 9: DRGWGNX1CGX2FGKGX3IX4X5CX6KX7 {wherein X1 is G or H, X2 is F or L, X3 is G or S, X4 is D, V or L, X5 is T or A, X6 is A or V, and X7 is F or A}. Also, in one preferred subject, (D1) the partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 1. In one preferred subject, (D2) the partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 2. In one preferred subject, (D3) the partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 3. In one preferred subject, (D4) the partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4. In one preferred subject, (D5) the partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 5. In a preferred embodiment, (D6) this partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 6. In a preferred embodiment, (D7) this partial peptide may have a sequence consisting of the amino acid sequence set forth in SEQ ID NO: 7.

[0029] According to the present invention, a multiple antigen peptide containing the peptide of the present invention (hereinafter, sometimes referred to as the "multiple antigen peptide of the present invention") is provided. In the multiple antigen peptide, the peptide of the present invention is linked to the most terminal part of the dendritic polymer backbone. The most terminal part and the peptide of the present invention may be linked via a linker. The linkage may be a covalent bond. The linker is not particularly limited as long as it does not significantly interfere with the presentation of the peptide, and linkers such as polyethylene glycol and flexible linkers (e.g., non-peptide linkers) can be used. As the linker, a peptidic linker may be used. When a peptidic linker is used, a flexible linker such as a GS linker and a linker having a secondary structure such as an α-helix structure or a β-sheet structure can be used. In the multiple antigen peptide of the present invention, one or more peptides of the present invention may be included. In a preferred embodiment, the multiple antigen peptide of the present invention may contain four or more peptides of the present invention. In a preferred embodiment, the multiple antigen peptide of the present invention may contain eight or more peptides of the present invention. The multiple antigen peptide of the present invention is preferably isolated or purified.

[0030] In one aspect of the invention, the multiple antigenic peptide of the invention comprises: A multiple antigen peptide comprising a dendritic polymer backbone and the multiple antigen peptide of the present invention, the core of the dendritic polymer being lysine, the lysine being regarded as the 0th generation, the following (requirement 1) being satisfied for k being any natural number from 1 to n, and the peptide of the present invention being linked to each of the amino groups of the lysines of the nth generation: (Requirement 1) Each amino group of a k-1 generation lysine is peptide-bonded to a k generation lysine.

[0031] In the above embodiment, n can be 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2, 3, or 4, and more preferably 2 or 3. That is, in this embodiment of the present invention, tetravalent to divalent n+1 Multiple antigenic peptides of different valences may be provided.

[0032] In one aspect of the invention, the multiple antigenic peptide of the invention comprises: The dendritic polymer may be a multiple antigen peptide (i.e., a tetravalent multiple antigen peptide) comprising a dendritic polymer backbone and the multiple antigen peptide of the present invention, in which the core of the dendritic polymer is lysine, each of the two amino groups of the lysine is peptide-bonded to a lysine forming a first-generation branch, and a peptide of the present invention is linked to each of the amino groups of the lysine forming the first-generation branch, thereby comprising four of the above peptides.

[0033] In one aspect of the invention, the multiple antigenic peptide of the invention comprises: A multiple antigen peptide (i.e., a tetravalent to octavalent multiple antigen peptide) comprising a dendritic polymer backbone and the multiple antigen peptide of the present invention, the dendritic polymer core being lysine, with lysines forming first-generation branches peptide-bonded to each of the two amino groups of the lysine, with lysines forming second-generation branches peptide-bonded to each of the amino groups of the lysines forming the first-generation branches, with a peptide of the present invention linked to each of the amino groups of the lysines forming the second-generation branches, and comprising 4 to 8 of the peptides.

[0034] In a preferred embodiment, the multiple antigen peptide of the present invention may be a tetravalent multiple antigen peptide (MAP-4) having the structure shown in the following formula (I) or an octavalent multiple antigen peptide (MAP-8) having the structure shown in the following formula (II). n+1 Multivalent antigen peptide (MAP-2 n+1 ) is provided.

[0035] [ka]

[0036] [ka]

[0037] These multiple antigen peptides can be synthesized by introducing the peptides into a dendritic skeleton immobilized on a resin. More specifically, for MAP-4, the peptides can be introduced into a resin having a dendritic skeleton having the structure of the following formula (III).

[0038] [ka]

[0039] In addition, MAP-8 introduced a peptide to a resin having the structure of the following formula (IV).

[0040] [ka]

[0041] In a preferred embodiment, the multiple antigenic peptide of the invention may have the structure of formula (III) or (IV), where the peptide is a peptide of the invention.

[0042] In a preferred embodiment, the multiple antigenic peptide of the present invention may have the structure of formula (VI): [ka]

[0043] {wherein R is -linker-peptide or -peptide, and the peptide may contain the amino acid sequence of the peptide of the present invention.} This structure was selected because lysine has two amino groups and is structurally suitable as a branching point. Note that MAP-8 may have a structure in which two compounds having the structure of formula (VI) are linked to the amino groups of further lysines via the carboxyl groups of the 0th generation lysine in formula (VI), respectively. Similarly, MAP-2 having an nth generation branch may be prepared. n+1 The structure of each can be specified.

[0044] In the above, the linker may be polyethylene glycol (PEG). In a preferred embodiment, the PEG may be, for example, a PEG having a degree of polymerization of 2 to 30, a PEG having a degree of polymerization of 5 to 20, or a PEG having a degree of polymerization of 10 to 15.

[0045] The compound having the structure of the above formula (VI) can be obtained by reacting the acetylene group (-C≡CH) of a compound having the structure of the following formula (VII) with a peptide or a peptide-linker compound by click chemistry. More specifically, the compound having the structure of the following formula (VII) can be obtained by reacting a peptide-azide group or a peptide-linker-azide group having an azide group at its terminal in the presence of a monovalent copper ion. [ka]

[0046] The compound of formula (VII) is a synthetic intermediate of the compound of formula (III), and for the convenience of reaction, an artificial amino acid having an acetylene group in the side chain is linked by a peptide bond to the amino group of the lysine that provides the first generation branch, thereby providing a reactive acetylene group at the end (see, for example, WO2015 / 190555A). Note that the synthetic intermediate of MAP-8 may have a structure in which two compounds having the structure of formula (VII) are linked to the amino groups of further lysines via the carboxyl group of the 0th generation lysine in formula (VII), respectively. Similarly, MAP-2 having an nth generation branch can be obtained. n+1 The structure of each of the synthetic intermediates can be defined.

[0047] The multiple antigen peptides of the present invention can be produced, for example, according to the method described in WO2018 / 084247A.

[0048] In a preferred embodiment, the multiple antigenic peptide of the present invention may be a multiple allogeneic antigenic peptide, in which all antigenic peptides have the same amino acid sequence.

[0049] In another preferred embodiment, the multiple antigenic peptide of the present invention may contain peptides having two or more different amino acid sequences as antigenic peptides.

[0050] In the present invention, the multiple antigenic peptide of the present invention can be administered to a subject and used to induce immunity (particularly antigen-specific immunity) in the subject. In the present invention, the multiple antigenic peptide of the present invention can be administered to a subject and used to induce antigen-specific antibody production. Also, in the present invention, the multiple antigenic peptide of the present invention can be administered to a subject and used to induce antibodies against coronavirus. The antigen-specific antibody can be a neutralizing antibody (e.g., an antibody that can neutralize infection). The antigen-specific antibody can be IgM. The antigen-specific antibody can be an antibody subclass that does not bind to the IgG receptor possessed by macrophages produced from marginal zone B cells and B1B cells.

[0051] The present invention provides an immunogenic composition comprising multiple antigenic peptides of the present invention (sometimes referred to as the "immunogenic composition of the present invention"). The immunogenic composition of the present invention can be used as a vaccine.

[0052] The vaccine of the present invention may be broadly effective against flaviviruses, and may be effective against a flavivirus selected from the group consisting of Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, St. Louis encephalitis virus, and tick-borne encephalitis virus and mutant viruses thereof.

[0053] The immunogenic composition or vaccine of the present invention may not be used in combination with an adjuvant that stimulates helper T cells (or T cell-dependent immunity) (e.g., aluminum salts, e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, liquid paraffin, lanolin, precipitating adjuvant), and is preferably not used in combination. The immunogenic composition or vaccine of the present invention may not be used in combination with an adjuvant (e.g., aluminum salts, e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, liquid paraffin, lanolin, precipitating adjuvant), and is preferably not used in combination. However, the immunogenic composition or vaccine of the present invention may be used in combination with an agent that does not stimulate helper T cells (T cell-dependent immunity), such as α-galactosylceramide. The immunogenic compositions or vaccines of the invention may further comprise agents that do not stimulate helper T cells (T cell dependent immunity), such as α-galactosylceramide.

[0054] The immunogenic composition or vaccine of the present invention may contain the multiple antigenic peptide of the present invention and a pharma- ceutically acceptable carrier and / or excipient. The immunogenic composition or vaccine of the present invention may be administered parenterally (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, nasal, mucosal, or inhalation). Thus, the pharma- ceutically acceptable carrier and / or excipient may be a carrier and / or excipient suitable for parenteral administration (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, nasal, mucosal, or inhalation). The pharma- ceutical acceptable carrier and / or excipient may, for example, be a salt, a buffer, a pH adjuster, an isotonic agent, a preservative, or water.

[0055] According to the invention, an immunogenic composition or vaccine can be a composition for use in raising antibodies against a flavivirus in a subject or in establishing immunity against a flavivirus.

[0056] According to the present invention, there is provided a multiple antigenic peptide of the present invention for use in raising antibodies against a flavivirus in a subject. According to the present invention, there is provided a multiple antigenic peptide of the present invention for use in raising antibodies against a flavivirus in a subject.

[0057] According to the invention there is provided the use of a peptide of the invention in the manufacture of an immunogenic composition or vaccine. According to the invention there is provided the use of a multiple antigenic peptide of the invention in the manufacture of an immunogenic composition or vaccine. The immunogenic composition or vaccine may be an immunogenic composition or vaccine for use in raising antibodies against a flavivirus in a subject or for establishing immunity against a flavivirus.

[0058] According to the present invention, there is provided a method for introducing an antigenic peptide into a subject, the method comprising administering to the subject a multiple antigenic peptide of the present invention. According to the present invention, there is provided a method for stimulating antigen-specific immunity in a subject, the method comprising administering to the subject an effective amount of a multiple antigenic peptide of the present invention. According to the present invention, there is provided a method for stimulating immunity against flavivirus in a subject, the method comprising administering to the subject an effective amount of a multiple antigenic peptide of the present invention. EXAMPLES

[0059] Example 1: Synthesis of antigenic peptides A partial peptide consisting of 21 amino acid residues was selected, which contains a highly homologous region of the amino acid sequences of the surface proteins of Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, St. Louis encephalitis virus, and tick-borne encephalitis virus, and also contains the amino acids of the binding site of the neutralizing antibody (bold part in the table below) reported in the paper (Yi-Chin Fan, et al. PLoS Negl.Trop. Dis.. 2015). A partial sequence of Japanese encephalitis virus was used as the sequence of the peptide portion of MAP. When this is compared with the sequences of yellow fever virus, West Nile virus, dengue virus, Zika virus, St. Louis encephalitis virus, and tick-borne encephalitis virus, the underlined amino acids differ.

[0060] [Table 1] *The numbers in the amino acid sequence represent the amino acid numbers of the terminal amino acids in the protein. *The bold letters in the amino acid sequence indicate the sequences predicted to bind to neutralizing antibodies. *The underlined amino acids in the amino acid sequence are different from the amino acid sequence of SEQ ID NO:1.

[0061] As shown in Table 1, the selected 22 consecutive amino acid sequence segments corresponding to partial peptides of the surface protein of Japanese encephalitis virus showed high homology in flaviviruses.

[0062] A peptide having the amino acid sequence of SEQ ID NO:1 was artificially synthesized according to a conventional method to prepare a tetravalent multiple antigen peptide (MAP-4) having the structure shown in the following formula (I) and an octavalent multiple antigen peptide (MAP-8) having the structure shown in the following formula (II).

[0063] [ka]

[0064] [ka]

[0065] These multiple antigen peptides were synthesized by introducing the peptides into a dendritic skeleton immobilized on a resin for efficient synthesis on the resin. More specifically, for MAP-4, the peptide was introduced into a resin having a dendritic skeleton having the structure of the following formula (III).

[0066] [ka]

[0067] In addition, MAP-8 introduced a peptide to a resin having the structure of the following formula (IV).

[0068] [ka]

[0069] In the above formula, lysine (Lys) is an amino acid having two amino groups. When the carboxyl groups of two lysines are linked to each amino group by peptide bonds, the two terminal lysines form a dendritic skeleton having a total of four amino groups. By introducing a peptide to the amino group of the lysine via a linker, a tetravalent multiple antigen peptide (MAP-4) can be prepared.

[0070] In addition, in the case of MAP-8, the terminal lysine (Lys) is further branched into two, and a peptide is introduced to a resin having Lys residues with eight Fmoc protecting groups. When the carboxyl groups of the four lysines are linked to the four amino groups of the lysine residues of the dendritic skeleton having a total of four amino groups by peptide bonds, the four terminal lysines become a dendritic skeleton having a total of eight amino groups. By introducing a peptide to the amino groups of the lysines via a linker, a tetravalent or octavalent multiple antigen peptide can be prepared.

[0071] When the above peptide was introduced, a linker was introduced to the C-terminus of the peptide. In this example, polyethylene glycol (PEG) was used as the linker. The polyethylene glycol was PEG with a polymerization degree of 12 (also referred to as "PEG(12)").

[0072] A more specific structure of MAP-4 was shown below.

[0073] [ka]

[0074] {wherein R is -PEG(12)-peptide.}

[0075] The above-mentioned MAP-4 and MAP-8 can be prepared, for example, by referring to WO2018 / 062217A.

[0076] The resulting peptides were then cleaved from the resin using 0.1% 2,2,2-trifluoroacetic acid and acetone, and the released MAP-4 and MAP-8 were then purified by high-performance liquid chromatography to obtain MAP-4 and MAP-8 (referred to as Flav-mMAP-4 and Flav-mMAP-8, respectively).

[0077] Example 2: Administration Experiments The administration experiment was performed on mice (Balb / c, female, 8 weeks old) according to the scheme shown in Figure 1. Specifically, Flav-mMAP-4 or Flav-mMAP-8 obtained in the Examples was administered to the mice at weeks 0, 1, and 5, as shown in Figure 1. Furthermore, blood was collected from the mice at weeks 0, 3, 5, 6, and 7, as shown in Figure 1, to confirm the presence or absence of antibody production and changes over time.

[0078] The dose of Flav-mMAP4 was 100 μg / administration, and the dose of Flav-mMAP8 was 100 μg / administration or 200 μg / administration. In addition, α-galactosylceramide (KRN7000, Funakoshi) was used at 0.1 μg / mouse during the second administration. Details are shown in Table 2.

[0079] [Table 2]

[0080] The antibody titer in the collected serum was measured as follows. A FLAG tag sequence (DYKDDDDK; SEQ ID NO: 8) was linked to a 22 consecutive amino acid sequence portion corresponding to a partial peptide of the selected surface protein of the Japanese encephalitis virus. The resulting peptide (JEV peptide) was further bound to bovine serum albumin (BSA) to prepare a conjugate protein of the JEV peptide and BSA. The resulting conjugate protein was immobilized on the plate surface and ELISA was performed. Specifically, the conjugate protein was immobilized on an ELISA plate by incubation at 37°C for 1 hour at a concentration of 5 μg / ml, and then blocking (SuperBlock, Thermo Fisher) was performed at room temperature for 30 minutes. After washing the plate, mouse serum diluted 5000 times with PBS was added as a test sample and reacted. The plate was then washed, and HRP-labeled anti-mouse IgM goat antibody (Southern Biotrch), HRP-labeled anti-mouse IgG goat antibody (Southern Biotrch), HRP-labeled anti-mouse IgG1 goat antibody (Southern Biotrch), HRP-labeled anti-mouse IgG2a goat antibody (Southern Biotrch), HRP-labeled anti-mouse IgG2b goat antibody (Southern Biotrch), and HRP-labeled anti-mouse IgG3 goat antibody (Southern Biotrch) were used at 5000-fold dilution to detect mouse IgM and IgG, and IgG subclasses, respectively. 3,3',5,5'-tetramethylbenzidine (TMB) was added as a color-developing substrate, and the absorbance was finally measured using an absorbance plate reader.

[0081] As a result, as shown in Figure 2, weak antibody production was observed in Group 1, which received Flav-mMAP-4. In contrast, Groups 2 and 3, which received Flav-mMAP-8, had higher antibody titers than Group 1, which received Flav-mMAP-4. Furthermore, the difference in the intensity of antibody production between individuals in Group 3, which received 200 μg, was smaller, indicating that stable antibody production could be induced.

[0082] The induction of IgM in serum in Group 3 was as shown in Figure 3. As shown in Figure 3, IgM increased after the second administration of Flav-mMAP-8, suggesting the establishment of antibody-producing memory B cells. Furthermore, the antibody titer also increased after the third administration of Flav-mMAP-8, demonstrating that the induced IgM-producing cells became memory cells and established memory immunity. Thus, it was revealed that Flav-mMAP can induce IgM and maintain that memory in immune cells. Therefore, it is believed that Flav-mMAP can be used as a vaccine.

[0083] Next, the subclasses of IgG antibodies in the serum collected from the third group were examined using ELISA. The results are shown in Figure 4. As shown in Figure 4, it was revealed that the subclasses induced by Flav-mMAP in mice were mainly IgG1, and the others were produced in small amounts or only temporarily, even if they were produced at all. In addition, since IgG1 in mice has weak binding to Fc receptors on the surface of effector cells such as macrophages, it was thought that the possibility of antibody-dependent enhancement (ADE), which is considered to be the biggest side effect of vaccines against Flaviviruses, being induced by Flav-mMAP could be reduced. Thus, it was thought that the Flav-mMAP technology would be useful as a vaccine against Flaviviruses.

[0084] Example 3: Measurement of virus neutralizing activity In Example 2, it was found that administration of Flav-mMAP induces antibody production, mainly IgM, in animals, induces memory immunity, and maintains antibody production ability for a long period of time. In this Example, the virus-neutralizing ability of the produced antibodies was evaluated.

[0085] Blood was collected from mice immunized with Flav-mMAP-8 to obtain serum. The obtained serum was used to measure the neutralizing activity of antibodies in a system in which Vero cells were infected with Japanese encephalitis virus. For this measurement, serum collected 9 weeks after administration of Flav-mMAP was used. The blood IgG concentration was measured using the same ELISA system as above. Since it was difficult to obtain antibodies against the peptide antigen used in Flav-mMAP-8, in this ELASA system, the peptide antigen was linked to Flag-tagged BSA and a calibration curve was drawn using an anti-FLAG tag antibody (Sigma) as the detection antibody to estimate the blood antibody concentration. The results are shown in Figure 5.

[0086] Next, the neutralizing activity of the virus was performed by an 80% plaque reduction neutralization test. Using the virus strain JEV / sw / Chiba / 88 / 2002, the diluted test serum was mixed with 100 PFU of the virus, and then incubated at 37°C for 1.5 hours. Then, Vero9013 cells were seeded on a 6-well plate, and 24 hours after seeding, the mixture of the virus and serum was inoculated into the cells and incubated at 37°C for 1.5 hours. After overlaying with a 0.8% agarose gel-containing culture solution, the cells were further cultured at 37°C for 4 days, fixed with formalin, stained with crystal violet, and the number of plaques was counted. Plaques are formed when cells are detached by the virus. If an antibody has virus neutralizing activity, the number of plaques formed is reduced. Therefore, in this example, the reduction rate of this plaque in the presence of the antibody at the indicated concentration was obtained, and the neutralizing ability of the antibody was evaluated. The experiment was repeated twice. The results are shown in Table 3.

[0087] [Table 3]

[0088] As shown in Table 3, in the average values ​​of two experiments, higher neutralization results were obtained in the immunized mice than in the experiment using the pooled serum before administration. In mouse 5, high antibody production was induced, and high neutralizing ability was induced. In particular, when the anti-peptide antibody concentration exceeded 20 μg / ml, a significantly high neutralizing ability was observed.

[0089] Next, the virus neutralizing ability of Flav-mMAP was examined using an animal model. The experimental system employed was the scheme shown in Figure 6. Specifically, female mice were administered Flav-mMAP-8, and after immunization, the mice were allowed to become pregnant and give birth to baby mice. The baby mice were inoculated with the virus, and the mother and baby mice were raised together. Protection against infection was confirmed for the baby mice and mother mice nursing from the mother mice. Flav-mMAP-8 was administered at 200 μg / mouse / time, and 0.1 μg of α-GalCer was co-administered at the second time. PBS was administered to the control group instead of Flav-mMAP-8. Note that the toxicity of the Japanese encephalitis virus is high, and if the virus is directly infected, the mice will die. Therefore, this experimental system was constructed as a gradual natural infection system in which the virus is infected to baby mice and the parent mice are infected from the baby mice, and a system was constructed to verify whether neutralizing antibodies are produced and induced early after infection.

[0090] As a result, the pups died of infection on the fifth day after infection (14th day after birth). This suggests that the pups may not have had sufficient protection against viral infection. On the other hand, serum was collected from the mother mice on the 14th day after birth, and the neutralizing activity of the serum against Japanese encephalitis virus was measured using the above-mentioned plaque assay. Furthermore, as shown in Figure 7, in the Flav-mMAP-8 administration group, a comparison of the pre-infection and 5 days after infection revealed that the viral infection-preventing ability of the mother mice was improved by the viral infection from the pups as a trigger. In other words, this suggests that the administration of Flav-mMAP-8 in advance enhanced the responsiveness to viral infection, and it was revealed that Flav-mMAP-8 provided memory immunity to the mother mice, and immunity was induced following viral infection.

[0091] [Consideration] Antibody-dependent enhancement (ADE) is well known for viruses such as dengue, Zika, and Ebola that infect macrophages, and cannot be avoided with conventional inactivated vaccines that use viral proteins. This is because viral proteins stimulate immune responses via T cells to produce antibodies (T cell-dependent), which induces high-affinity IgG to IgG receptors and induces ADE. Therefore, to avoid ADE, it is possible to induce antibodies that do not bind to IgG receptors, that is, to directly stimulate B cells without the intervention of T cells (T cell-independent) (Saravanan, P., et. al., Acta Virol., (48) 39-45, 2004), and technically, a vaccine using multiple antigen peptides (MAP) corresponds to this specific method.

[0092] This example shows that multiple antigen peptides presenting eight antigen peptides (i.e., octavalent) directly stimulate marginal zone B cells and B1B cells without the intervention of helper T cells to produce IgM and IgG subclasses that do not bind to IgG receptors (IgG1 and IgG3 in the case of mice). It is believed that the multiple antigen peptides directly stimulate marginal zone B cells and B1B cells to form clusters on the B cell receptors expressed on their surfaces, thereby stimulating B cells to stimulate antigen-specific antibody production.

[0093] When serum antibody levels were measured for each IgG subtype, IgG1 and IgG3, which are subtypes that do not bind to IgG receptors or have weak affinity (weak binding power), were induced, but IgG2a, which binds to IgG receptors, was not induced. This suggests that in animals immunostimulated with MAP, marginal zone B cells and B1B cells are directly stimulated, which induces only antibodies that do not bind to IgG receptors, and that the induction of antibody production is T cell-independent.

[0094] This result indicates that the B cells stimulated by MAP are marginal zone B cells and B1B cells present in the mucosa and serous membrane, and are different from the follicular B cells that produce antibodies in a T cell-dependent manner. The antibodies produced by marginal zone B cells and B1B cells are mainly antibodies that do not bind to IgG receptors (IgM and IgG3 in mice), or IgG subclasses with low binding ability (IgG1 in mice) (Bennett, KM., et. al., BMC Biotechnol, (15)71, 2015).

[0095] In addition, while IgM produced by T cell-dependent stimulation disappears from the blood for about 7 to 10 days, IgM produced in a T cell-independent manner remains in the blood for a long time (about 90 days in mice) (Dintzis, HM, et. al., Proc. Natl. Acad. Sci.USA, (73) 3671-5, 1974), and is expected to provide virus protection for a relatively long period of time.

[0096] According to the above results, ADE can be avoided by inducing antibodies that do not bind to IgG receptors, that is, by directly stimulating B cells without the intervention of T cells (T cell-independent) (Saravanan, P., et. al., Acta Virol., (48) 39-45, 2004), and it is expected that ADE can technically be avoided by using a vaccine with multiple antigen peptides.

[0097] The peptide of the present invention has an amino acid sequence that is widely conserved throughout the Flavivirus genus. Therefore, the peptide of the present invention can produce an antibody that is effective against the Flavivirus genus in general. Furthermore, the fact that the peptide is widely conserved throughout the Flavivirus genus suggests that the sequence has been conserved despite repeated natural mutations. In other words, the multiple antigen peptide of the present invention is a mutation-compatible MAP (mMAP), which is resistant to mutations, and is therefore suggested to be effective against any Flavivirus that may be created in the future.

[0098] Furthermore, MAPs can be produced by chemical synthesis, and unlike vaccines that use recombinant proteins or vectors, which require a cultivation process, they can be mass-produced quickly. This makes them an ideal method for vaccines against infectious diseases that require urgent measures, such as flaviviruses.

[0099] It is also noteworthy that the present example demonstrates that the multiple antigen peptides establish memory immunity and confer long-term immunity against flaviviruses.

[0100] Description of sequence listing SEQ ID NO:1: Part of the amino acid sequence of the Japanese encephalitis virus polyprotein as a candidate antigenic peptide; SEQ ID NO:2: Part of the amino acid sequence of the yellow fever virus polyprotein as a potential antigenic peptide; SEQ ID NO:3: Part of the amino acid sequence of the West Nile virus polyprotein as a potential antigenic peptide; SEQ ID NO:4: Part of the amino acid sequence of the dengue virus polyprotein as a potential antigenic peptide; SEQ ID NO:5: Part of the amino acid sequence of the Zika virus polyprotein as a potential antigenic peptide; SEQ ID NO:6: Part of the amino acid sequence of the polyprotein of St. Louis encephalitis virus as a potential antigenic peptide; SEQ ID NO:7: Part of the amino acid sequence of the polyprotein of tick-borne encephalitis virus as a candidate antigenic peptide; SEQ ID NO:8: Amino acid sequence of the FLAG tag; SEQ ID NO:9: Amino acid sequence of the antigen peptide candidate (22 amino acids long); SEQ ID NO:10: The amino acid sequence of a partial sequence (7 amino acids long) in the antigen peptide to which the neutralizing antibody binds; SEQ ID NO:11: An example of an amino acid sequence of a partial sequence (11 amino acids long) in an antigen peptide to which a neutralizing antibody binds; SEQ ID NO:12: An example of an amino acid sequence of a partial sequence (11 amino acids long) in an antigen peptide to which a neutralizing antibody binds; SEQ ID NO:13: An example of the amino acid sequence of a partial sequence (11 amino acids long) in an antigen peptide to which a neutralizing antibody binds

Claims

1. The multiple antigen peptides include a dendritic polymer backbone that presents four or more antigen peptides having the same amino acid sequence, and the four or more antigen peptides are linked to the most terminal arms of the dendritic polymer, The antigen peptide is a partial peptide of a polyprotein of a Flavivirus virus having a length of 11 to 30 amino acids, which is a part of the amino acid sequence of the polyprotein of a Flavivirus virus, and includes a partial peptide having an amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 7; Induce T cell-independent antibody production from marginal zone B cells and / or B1 B cells; Multiple antigenic peptides.

2. The multiple antigen peptide described in claim 1, comprising eight or more antigen peptides having the same amino acid sequence.

3. The partial peptide is linked to the extreme end of a dendritic polymer backbone, the core of which is lysine, and the following (requirement 1) is satisfied when k is any natural number from 1 to n, with the lysine being the 0th generation, and the partial peptide is linked to each amino group of the nth generation lysine {wherein n is 2, 3, 4, 5, 6, 7, 8, 9, or 10}. ： (Requirement 1) A k-th generation lysine is peptide-bonded to each of the amino groups of the k-1th generation lysines. A multiple antigen peptide according to claim 1 or 2.

4. A multiple antigen peptide, The following formula (III) or (IV): 【Chemistry 1】 or 【Chemistry 2】 The structure shown in {The peptide in the formula is a partial peptide of a polyprotein of a Flavivirus virus having a length of 11 to 30 amino acids, which is a part of the amino acid sequence of the polyprotein of a Flavivirus virus, and is a partial peptide containing an amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 7.} A multiple antigen peptide having the following structure:

5. The multiple antigen peptide described in claim 4, wherein the peptide consists of a partial peptide of a polyprotein of a Flavivirus virus having an amino acid sequence corresponding to any of the amino acid sequences set forth in SEQ ID NOs: 1 to 7.

6. A composition for use in inducing the production of T cell-independent antibodies from marginal zone B cells and / or B1 B cells, comprising a multiple antigen peptide described in any one of claims 1 to 5, against Flavivirus viruses.

7. A vaccine against Flavivirus genus viruses, comprising a multiple antigen peptide described in any one of claims 1 to 5.

8. 8. The vaccine of claim 7 which does not contain an adjuvant.

9. The vaccine according to claim 7 or 8, which is used in combination with α-galactosylceramide without using any other adjuvant.

10. The vaccine according to any one of claims 7 to 9, wherein the Flavivirus virus is a virus selected from the group consisting of Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, St. Louis encephalitis virus, and tick-borne encephalitis virus and mutant viruses thereof.

11. An immunogenic composition comprising the multiple antigenic peptide of any one of claims 1 to 5 for use in a method for inducing T cell-independent antibody production from marginal zone B cells and / or B1 B cells against Flavivirus in a subject in need thereof, the method comprising administering to the subject an effective amount of the multiple antigenic peptide defined in any one of claims 1 to 5.

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